The endogenous calcium-activated Cl channel in Xenopus oocytes: A physiologically and biophysically rich model system

CHAPTER 1 The Endogenous Calcium-Activated C! Channel in Xenopus Oocytes: A Physiologically and Biophysically Rich Model System Khaled Machaca,* Zhiqiang Qu, t Akinori Kuruma, ~ H. Criss Hartzell, t and Nael McCarty §'* *Department of Physiology and Biophysics, University of Arkansas Medical Science, Little Rock, Arkansas; tDepartment of Call Biology, Emory University School of Medicine, Atlanta, Georgia 30322, ~Lahnratory for Developmental Neurobiology, RIKEN Brain Science Institute, Wako-shi, Saltama 351-0198, Japan; and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322

I. Inffoductiun II. Physiological Roles of Ca2+-Activated C1- Channels in Xenopus Eggs A. Oocyte Maturation B. Fertilization and Ca2+ Signals C. Block to Polyspermy D. Role of Icl.ca HI. Mechanisms of Gating of CI(Ca) Channels in Oocytes A. Voltage-Dependent Ca Affinity B. Model of CI(Ca) Channel Gating C. Comparison of Xenopus CI(Ca) Channels with Those in Mammalian Cells D. Mechanisms of Channel Activation by Ca IV. Anion Permeation in CI(Ca) Channels A. Selectivity B. Pharmacology of the CI(Ca) Channel C. Model of the Xenopus Ca-Activated C1 Channel Pore D. Toward a Biophysical Definition of Ca-Activated C1 Channels E. Do Cloned CLCA Channels Fit These Criteria? V. Toward a Definition of C1 Selectivity A. Structural Comparisons: Four Ways to Build a Chloride Channel B. Functional Comparisons: Commonalities and Distinguishing Features in Permeation VI. Summary and Conclusions References *Present address: School of Biology, Georgia Institute of Technology, Atlanta, GA 30332 Current Topics in Membranes, Volume 53

Copyright2002, ElsevierScience(USA). All fightsreserved. 1063-5823/02$35.00

4

Machaca et al.

i. INTRODUCTION Ca2+-activated chloride currents (IcLca) have been identified in a wide range of organisms from mammals (Kidd and Thorn, 2000a) to invertebrates (Robertson and Martin, 1996), and in many tissues such as neurons (Frings et al., 2000), muscle (Large and Wang, 1996; Klockner, 1993; Lamb et al., 1994), hepatocytes (Koumi et al., 1994), and secretory epithelia (Begenisich and Melvin, 1998). Icl,ca has been implicated in volume regulation, secretion, and membrane excitability (Begenisich and Melvin, 1998; Lamb et al., 1994; Frings et al., 2000), but the precise role of ICLCain physiological processes often remains elusive. This is because in most instances lo,ca is present in conjunction with other Ca2+-dependent cation currents, as well as other C1 currents that complicate the determination of the exact functional contribution of IcLca. In contrast to other preparations, Xenopus laevis oocytes have provided an exceptional model to address the physiological significance of la,ca. This is partly due to the fact that IcLc~ is the predominant current in these cells. It is clear in this system that Icl.Ca is essential for the prevention of polyspermy during fertilization. In this chapter, we will first describe the role of Icl,ca in the fast block to polyspermy, and then describe the biophysical characteristics of CI(Ca) channel gating and anion permeation.

II. PHYSIOLOGICAL ROLES OF CaZ+-ACTIVATED CI- CHANNELS IN XENOPUS EGGS A. Oocyte Maturation

Stage VI Xenopus oocytes are arrested at the G2/M transition of the cell cycle. These fully grown oocytes must undergo a maturation period before becoming competent to support embryonic development. During this so-called "meiotic maturation," the oocyte enters meiosis, progresses through the first meiotic division resulting in the extrusion of a polar body, and arrests at metaphase of the second meiotic division until fertilization (Bement and Capco, 1990). We will refer to mature oocytes arrested at metaphase II as "eggs," to differentiate them from immature oocytes arrested at G2]M. Fertilization relieves the metaphase U block and allows the egg to complete meiosis before beginning zygotic development. Meiotic maturation is naturally induced by the hormone progesterone released from follicular cells that surround the oocyte. Through a complex cascade of kinases that is beyond the scope of this discussion, progesterone induces the entry of oocytes into meiosis following the activation of maturation promoting factor (MPF) (Coleman and Dunphy, 1994).

1. Calcium-Activated CI Channel in Xenopus

5

B. Fertilization and Ca 2+ Signals In Xenopus as in all other species investigated, fertilization results in a rise in cytoplasmic Ca 2+ levels. This Ca 2+ rise is necessary and sufficient for egg activation (Stricker, 2000). The cytoplasmic Ca 2+ rise at fertilization takes the form of a single, or multiple Ca 2+ transients depending on the species (see Stricker, 2000 for a review). For example, in jellyfish, sea urchin, and Xenopus (Fontanilla and Nuccitelli, 1998), a single Ca 2+ wave is observed at fertilization. In contrast, in annelids, ascidians, and mammals, multiple Ca 2+ transients can be detected. Presumably the detailed spatial and temporal characteristics of the fertilization-induced Ca 2+ signals are important for the normal initiation of embryonic development. The rise in cytoplasmic Ca 2+ levels upon fertilization in Xenopus is responsible for the block to polyspermy at two levels. First it activates lo.ca, which leads to membrane depolarization ("fertilization potential") and a block to sperm entry. Second it induces the fusion of cortical granules ("fertilization envelope"), which provides the long-term block to sperm fusion.

C. Block to Polyspermy All animal species have developed mechanisms to ensure that only a single male nucleus fuses with the egg nucleus. This is accomplished by different mechanisms depending on the species, as illustrated by the different orders of amphibia. In anura (frogs, toads) such as Xenopus, only a single sperm is allowed to enter the egg (Herlant, 1911), indicating that this order must have a cell membranemediated block to polyspermy. Whereas in the urodela order (newts, salamanders) polyspermy is physiological, where several sperm penetrate the egg, but only one sperm nucleus fuses with the egg nucleus, and additional sperm nuclei disintegrate (Fankhauser and Moore, 1941). One obvious candidate mechanism to ensure monospermic fertilization in anurans is the "fertilization envelope," which is generated following cortical granule fusion (Wolf, 1974; Grey et aL, 1974). Cortical granules are large vesicles containing various proteases and glycoproteins located in the egg cortex. Upon fertilization, cortical granules fuse with the cell membrane, releasing their content and creating around the egg an envelope that has been shown to be impermeant to sperm (Grey et al., 1976). Although the fertilization envelope is clearly sufficient to block sperm entry, it develops too slowly (--~5 min) to account for a complete block to polyspermy. This conclusion is supported by experiments showing that polyspermy can be induced despite the formation of the fertilization envelope (Bataillon, 1919). Therefore, some other mechanism must act at a faster time scale to block polyspermy in these eggs.

6

Machaca et al.

We now know that the so-called "fast block to polyspermy" in anurans is due to membrane depolarization following the activation of Icl,ca- An electrical block is ideally suited for such large eggs because it will quickly spread across the entire cell membrane. The concept of electrical activity playing a role at fertilization began with the suggestion that changes in egg membrane potential could be associated with the initiation of development (Hagiwara and Jaffe, 1979). Such changes were successfully detected in several species including echinoderms, amphibia, and fish (Hagiwara and Jaffe, 1979). At fertilization the membrane potential is depolarized from a negative resting potential. Because of the timing of these potential changes it was proposed that they play a role in polyspermy block. Indeed Jaffe (1976) showed that membrane depolarization is sufficient to block sperm entry in sea urchin, therefore demonstrating the existence of an electrically mediated block to polyspermy. Changes in membrane potential were also observed in anurans during artificial "egg activation" (Maeno, 1959; Ito, 1972). Egg activation is the process, usually induced by pricking the egg with a glass needle, whereby eggs undergo morphological changes similar to fertilized eggs but in the absence of sperm. The membrane depolarization associated with egg activation following pricking is referred to as the "activation potential." It was first shown in the late 1950s that the activation potential is associated with a decrease in membrane resistance due to the activation of a C1- selective conductance (Maeno, 1959; Ito, 1972). Studies by Cross and Elinson (1980) and Grey and co-workers (1982) showed that anuran eggs exhibit a membrane depolarization at fertilization, with characteristics and ionic requirements similar to those of the activation potential. Therefore, fertilization and pricking appear to activate the same cascade of cellular events associated with the initiation of development. Fertilization induces an initial jump in membrane potential from - 3 0 mV to +5 mV that occurs quickly (< 1 s). This membrane depolarization is associated with a dramatic decrease in membrane resistance (,-~200-fold) due to the activation of C1- selective conductance (Cross and Elinson, 1980; Grey et al., 1982; Webb and Nuccitelli, 1985). The initial rapid depolarization was followed by a gradual upward shift in membrane potential reaching +20 mV (Cross and Elinson, 1980; Grey et al., 1982). Egg membrane depolarization was transient, lasting only several minutes. This fertilization-induced depolarization is referred to as the "fertilization potential." Several pieces of evidence, from these and previous studies, show that membrane depolarization at fertilization is responsible for the fast block to polyspermy in anurans. (1) Fertilization results in membrane depolarization (Cross and Elinson, 1980; Grey et al., 1982; Webb and Nuccitelli, 1985), which temporally correlates with the fast block to polyspermy. (2) Holding the membrane potential at positive values prevents sperm entry but does not affect cortical granule fusion or cleavage (Cross and Elinson, 1980; Grey et al., 1982; Webb and Nuccitelli, 1985). (3) Blocking membrane depolarization results in polyspermy without blocking cortical granule fusion (Cross and Elinson, 1980).

1. Calcium-Activated C1 Channel in Xenopus

7

The fact that membrane depolarization is sufficient to block sperm entry argues for the presence of a voltage-sensitive effector involved in sperm egg fusion. Taking advantage of cross-species fertilization between salamander and Xenopus, Jaffe et aL (1983) elegantly showed that this voltage-sensitive effector localizes to Xenopus sperm. Whereas membrane depolarization blocks the ability of Xenopus sperm to fertilize Xenopus eggs, the ability of salamander sperm to fertilize Xenopus eggs was unaffected by membrane voltage.

D. Rote of lcl,ca By the mid-1980s it was established that the fast electrical block to polyspermy in anurans is due to membrane depolarization, following the activation of a Ca 2+dependent C1- conductance. Interestingly, hints of a role for anions in anuran fertilization have been lurking in the literature well before the idea of an electrical block to polyspermy was conceived. In 1919 Bataillon showed that fertilization in high C1- media, or following substitution of C1- with I- or Br-, results in polyspermy. He further showed that polyspermy, in these ion substitution experiments, occurred despite the normal formation of the fertilization envelope. These simple experiments argue that C1- is involved in polyspermy block at a stage before formation of the fertilization envelope. It took about four decades for the significance of these findings to be appreciated in the context of membrane potential changes. In 1959 Maeno showed that egg activation is associated with membrane depolarization due to the activation of a C1- selective conductance. These results were confirmed (Ito, 1972) and later expanded to fertilization (Cross and Elinson, 1980; Grey et al., 1982). Cross and Ellinson (1980) showed that in Rana, anions induce polyspermy with different efficiencies (I- > Br- > C1-). The efficiency of polyspermy block by different anions correlates well with the level of depolarization induced. I - was the most effective anion at inducing polyspermy and resulted in the smallest membrane depolarization. Identical results were obtained in Xenopus, where Grey et al. (1982) showed that I- and Br-, but not F-, induced polyspermy without inhibiting the generation of the fertilization envelope. The effects of different anions on polyspermy and the fertilization potential correlate nicely with the recent biophysical characterization of la,ca in Xenopus. Qu and Hartzell (2000) showed the following anion permeability sequence for Xenopus la,ca: I- > Br- > CI- > F-. This sequence is identical to the potency of these anions in inducing polyspermy. This is expected because membrane depolarization is induced by C1- efflux out of the egg. Fertilizing eggs in high C1media decreases the driving force for C1- efflux and diminishes the extent of membrane depolarization. In addition, fertilizing eggs in solutions containing I or Br-, which are more permeant than C1- through the CI(Ca) channel, would be

8

Machaca et al.

expected to shift the reversal potential of/CI(Ca) t o more negative potentials and therefore decrease the level of the depolarization. F-, which is less permeant than CI-, would not be expected to have this effect and, indeed, it does not. Therefore, almost a century after the first observation of the effectiveness of different anions to induce polyspermy in anurans, these findings can now be explained in terms of the permeability sequence of Icl,ca to different anions. Therefore, in anurans fertilization activates a C1- conductance resulting in membrane depolarization and a fast block to polyspermy. How is this C1- conductance activated? There were hints for a role for Ca 2+ in this process. Egg activation in response to pricking depends on the presence of Ca 2+ in the extracelhilar solution (Wolf, 1974). Knowing this and knowing that membrane depolarization is due to C1- efflux, Cross (1981) tested the hypothesis that the fertilization-induced C1conductance is due to a rise in cytoplasmic Ca 2+ levels. Indeed he showed that direct injection of Ca 2+ into the egg induces electrical changes with properties and C1- dependence similar to those observed upon fertilization. Interestingly, injection of Ca 2+ into the animal hemisphere was more effective than injection into the vegetal hemisphere. We and others (Kline and Nuccitelli, 1985; Gomez-Hernandez et al., 1997; Machaca and Hartzell, 1998) have shown that this polarization is due to a great enrichment of Icl.ca channels in the animal versus vegetal hemisphere. This is consistent with the fact that sperm entry localizes to the animal hemisphere. Furthermore, treating cells with the Ca 2+ ionophore A23187 also results in membrane depolarization, consistent with a CaZ+-dependent activation of the C1- conductance. At fertilization the activation of this C1- conductance occurs in the form of a wave of inward current that travels across the egg membrane (Kline and Nuccitelli, 1985). These studies argued for a rise in cytoplasmic Ca 2+ levels at fertilization. Nuccitelli and co-workers have shown that this Ca 2+ rise at fertilization is due to Ca 2+ release from intracellular stores, in response to increased levels of inositol 1,4,5-trisphosphate (IP3). IP3 binds to the IP3 receptor, which is localized to the endoplasmic reticulum membrane and induces the mobilization of Ca 2+ stored in this organelle. Injection of IP3 into X e n o p u s eggs induces egg activation, including membrane depolarization and cortical granule fusion, as observed at fertilization (Kline and Nuccitelli, 1985; Busa et al., 1985). Furthermore, an increase in cytoplasmic Ca 2+ levels was detected following fertilization, using Ca2+-selective electrodes (Busa and Nnccitelli, 1985). Interestingly, this Ca 2+ rise travels as a wave across the egg cytoplasm (Fontanilla and Nuccitelli, 1998), with characteristics similar to the fertilization-induced wave of inward current (C1efflux) (Kline and Nuccitelli, 1985). The correlation between a rise in cytoplasmic Ca 2+ at fertilization and the activation of Icl,ca was confirmed by studies showing that fertilization induces an increase in IP3 levels in the egg, which is required for the generation of the Ca 2+ wave (Larabell and Nuecitelli, 1992; Nuccitelli et al., 1993; Snow et al., 1996). In turn, injection of IP3 or Ca 2+ into oocytes activates

1. Calcium-Activated C1 Channel in Xenopus

9

a Ca2+-activated C1- c o n d u c t a n c e (lcl,ca) (Miledi and Parker, 1984; Parker and Miledi, 1986, 1987; F e r g u s o n et al., 1991) previously characterized biophysically (Barish, 1983). Both the fertilization and activation potentials do not require extracellular Ca. This, along with the recent finding that store-operated Ca 2+ influx is not present in eggs (Machaca and Haun, 2000), argues that d u r i n g fertilization the physiological Ca 2+ signal is m a i n l y due to Ca 2+ release from internal stores. The correlation b e t w e e n Ca 2+ signals a n d Icl,Ca has not b e e n investigated in eggs, but studies in oocytes offer important insights. Figure 1 shows the effect

A 10

IP3 i n j e c t i o n

/

A

s eID

l.-"

0

-5

sdo

1obo Time (sec)

B ~¢,)dur~ ca~ht(

10

. . . . . . . . . . . te~c,) d u r ; ~ r d ~ s e from s l o ~

.-"'" ¢.= t.= .=,i

0

/

...................................j

(

c) -5

o

I'

i

i

Time (msec)

FIGURE 1 Development of Ca2+-activated C1- currents in Xenopus oocytes. Oocyte was voltage clamped with two microelectrodes and injected with IP3 at the time indicated. The oocyte was voltage clamped with two microelectrodes and stimulated once every 10 s with a voltage-clamp episode consisting of three 1-s pulses to -t-60, -120, and +60 mV. (A) Development of currents with time after IP3 injection. Squares: current at the end of the first +60-mV pulse. Circles: current at the end of the - 120-mV pulse. Triangles: current 100 ms after onset of second +60-mV pulse. (B) Current traces: current during Ca release from stores (30 s after 11)3injection) solid line and C! current during Ca influx through store-operated Ca channels (10 min after IP3 injection) dashed line. These data were originally published in the American Journal of Physiology (Kuruma and HartzeU, 1999). Copyright American Physiological Society.

10

Machaca et aL

of injection of IP3 into a voltage-clamped stage VI Xenopus oocyte (Hartzell, 1996; Kuruma and Hartzell, 1999). Injection of IP3 first stimulates a massive but transient release of Ca 2+ from internal stores that begins within seconds after injection. Ca 2+ release continues for 1-2 min and ceases when the stores become depleted. Ca 2+ release is accompanied by a Ca-activated C1- current that appears to be voltage and time dependent. The current is activated slowly by depolarization and is deactivated by hyperpolarization (note the tail currents at - 1 2 0 mV). After the stores have become depleted, store-operated Ca 2+ entry slowly develops over the next 10-20 min. Store-operated Ca 2+ entry (SOCE), also called "capacitative Ca z+ entry," refers to Ca 2+ influx that occurs through plasma membrane Ca 2+ channels that are controlled by the level of Ca 2+ in the lumen of Ca 2+ stores (Putney, 2001). SOCE stimulates Ca-activated C1- current having a distinctly different waveform: the current is time and voltage dependent at steady state. Furthermore, the outward current is observed when the depolarizing pulse occurs after a strong hyperpolarizing pulse (compare the current during the first and second +60-mV pulses in Fig. 1). The outward current inactivates in about a second. As discussed below, we believe that the Ca-activated C1- currents activated by Ca 2+ release from stores and Ca z+ influx are mediated by a single type of C1 channel. The different waveforms of the current during Ca 2+ release and Ca 2+ influx are a consequence of the differing voltage dependencies of Ca 2+ release and influx and the voltage-dependent Ca 2+ affinity of the Ca-activated Cl-channel (see Section Ill.A). Parker and Yao found no direct correlation between Ca z+ signals measured using a cytoplasmic Ca 2+ dye and Icl,Ca in oocytes (Parker and Yao, 1994). They concluded from this study that I¢1,Caresponds to the rate of rise of cytoplasmic Ca 2+ rather than absolute Ca 2+ levels. We have confirmed the discrepancy between cytoplasmic Ca 2+ levels and lc~,Cabut reached a different conclusion regarding the activation of Icl(Ca) by Ca 2+. Using a membrane-bound Ca 2+ dye we have shown that Icl,Cakinetics correlate nicely at a millisecond time scale with changes in Ca z+ levels immediately below the plasma membrane (Machaca and Hartzell, 1999). This argues that I¢1,Cain oocytes is activated directly by the levels of Ca z+ immediately below the cell membrane. Therefore, during Ca 2+ signaling in oocytes two Ca z+ subdomains are generated: one below the cell membrane with fast dynamics and one in the bulk cytosol with significantly slower dynamics (Machaca and Hartzell, 1999). The complex waveforms of CI(Ca) currents in oocytes as shown in Fig. 1 are a consequence of the fact that Ca 2+ release from stores in oocytes provides a smaller Ca 2+ signal to the CI(Ca) channel than does Ca 2+ influx, partly because of the relative proximity of the Ca z+ influx channel to the CI(Ca) channel. Because the apparent affinity of the CI(Ca) channel for Ca is voltage dependent (see Section HI.A) small Ca 2+ signals preferentially activate outward current. This

1. Calcium-Activated C1 Channel in Xenopus

11

means that small amounts of Ca 2+ release from stores will not be very effective at activating the CI(Ca) channels at the typical negative resting potentials of eggs. This provides a mechanism whereby "maverick" Ca 2+ signals or depolarizations that might be initiated by environmental changes such as temperature, mechanical disturbance, or changes in salinity would not trigger fast block to polyspermy. Indeed, because low Ca 2+ concentrations permit only outward currents, CI(Ca) channels would tend to maintain the oocyte in a hyperpolarized state when bulk cytosolic [Ca] is less than ~500 nM. Only fertilization, which raises bulk cytosolic Ca 2+ to levels above 1/zM, would be effective in depolarizing the oocyte and triggering fast block to polyspermy.

III. MECHANISMS OF GATING OF CI(Ca) CHANNELS IN OOCYTES

A. Voltage-Dependent Ca Affinity The voltage-dependent Ca 2+ affinity of CI(Ca) channels has been demonstrated using excised patches, where the cytosolic face of the membrane can be exposed to different buffered Ca 2+ concentrations (Kuruma and Hartzell, 2000). Such an experiment is shown in Fig. 2. At [Ca2+] below --q00 nM, very little C1- current was measurable, but at [Ca 2+] between 100 nM and 1 /zM Ca, time-dependent outward currents were stimulated at positive voltages. These currents resemble those seen in response to Ca 2+ release from stores in intact oocytes (Fig. 1). At [Ca2+] higher than 1 /zM, both inward and outward steady-state currents were observed. These currents exhibited little time dependence (Fig. 2D). The steadystate current-voltage (I-V) curves of the currents are shown in Fig. 3. The current was strongly outwardly rectifying at low Ca 2+ but was linear at high [Ca2+]. This change in the rectification properties with differing [Ca2+] is a consequence of the voltage-dependent Ca 2+ affinity of the channel (Fig. 4). In this figure, the conductances (Gm) were determined from the instantaneous amplitude of the tail currents (as shown in Fig. 2). In Fig. 4A, conductance is plotted as a function of Vm for different [Ca2+]. In Fig. 4B these data are replotted as a function of [Ca2+] for various membrane potentials (Vm). As Vm was made more positive, the relationship between Gm and [Ca 2+] shifted to the left. The apparent affinity of the channel could be estimated for each potential as the [Ca2+] that produced a half-maximal increase in current. This value (apparent Kd) became 4-fold larger between +120 and - 1 2 0 mV (Fig. 4C). The Hill coefficients, estimated from the slopes of the curves, were approximately 3 at all voltages. This value can be used as an estimate of the minimum number of Ca 2+ molecules required to open a channel. These data show that the apparent affinity of the channel for Ca 2+ depends on voltage.

12

Machaca et al.

120 mV 0 mV

-120 mV

A
B

~ n M Ca ~

5O

~

o

O -50

C

0i -501 •

D

.

.

,

,

,

1.4

1,5

.

121~MCa

0.0

0,5

1.0 1.3

Time (msee) FIGURE 2 Ca2+-activated C1- currents in excised patches. Inside-out patches were excised from a Xenopus oocyte. The cytosolic face of the membrane was exposed to different free Ca2+ concentrations (A-D) [buffered with ethyleneglycoltetraacetic acid (EGTA)] as indicated. The membrane was voltage clamped by the protocol shown above (A). The patch was exposed on the cytosolic side to solutions containing different free [Ca2+] as indicated. Reproduced from Kuruma and Hartzell (2000), Journal of General Physiology, 2000, 115, 59-80, by copyright permission of Rockefeller University Press.

1. Calcium-Activated C1 Channel in Xenopus

13

80 60 40 20 'D

[3

~'

[]

-100 u ~

_ •

,~

,~------',-o 0

} -40

50 100 Voltage (mY) [] 600 nM • 2 ~M

-60

FIGURE 3 Steady-state current voltage relationship of Ca-activated C! currents in inside-out excised patches. The current at the end of a 1-s duration voltage pulse was plotted vs. the membrane potential for low and high free [Ca]. Squares: 600 nM Ca; triangles: 2/xM Ca. Reproduced from Kuruma and Hartzell (2000), Journal of General Physiology, 2000, 15, 59-80, by copyright permission of Rockefeller University Press.

Other data also support this interpretation. The time course of activation of the current is not consistently voltage dependent, whereas the deactivation of the current occurs with an exponential time course that is voltage dependent. These data suggest that channel closing is voltage sensitive but that channel opening is Ca dependent. This is supported by experiments using rapid Ca 2+ concentration jumps. The activation of the current is Ca dependent but not significantly dependent on the potential at which the patch is held. In contrast, the deactivation of the current upon Ca 2+ washout is strongly dependent on the membrane potential. The current decays faster at more negative potentials (Kuruma and Hartzell, 2000). When we first observed the complex waveforms of CI(Ca) currents in Xenopus oocytes (Fig. 1), we thought that two or more species of CI(Ca) channels could be responsible for the macroscopic currents. This idea was driven largely by the very different waveforms of the currents elicited by Ca 2+ release from stores and Ca 2+ influx through SOC channels, although there were other differences between the currents elicited by Ca 2+ release and Ca 2+ influx that fueled this speculation (Hartzell, 1996; Kuruma and Hartzell, 1999). These differences, for example in the

14

Machaca

et al.

A

0,5"

2.4 ~M

"~' 0.3.

¢-

l

p

M

0.2.

460nM

o

0.0,

170 nM .

.

.

-100 B

.

.

.

-50

.

.

.

.

0 Vm (mV)

.

.

50

100

1,0 0.8 0.6

o

0.4 0.2 -120 mV

0.0 . . . . . . .

1

t

10

,

,'

,"'

[Cal (p.M)

2

-150 -100

•

-50

0

50

100

150

vm (mv) FIGURE 4 Voltage-dependent Caz÷ sensitivity of Cl(Ca) channels. (A) Conductance (Gin) was measured from the instantaneous tail currents at - 1 2 0 mV after depolarizations to different potentials (see traees in Fig. 2). (B) Gm in (A) was normalized to 1 for the maximum conductance measured and plotted vs. [Ca]. Curves are superimposed for seven different voltages from left to right: +120, +80, +40, 0, -40, - 8 0 , and - 120 inV. 03, C) The data points in (A) were fit to the Hill equation and the ECs0s and Hill coefficients (n14)of the fits were plotted. Reproduced from Kuruma and Hartzell (2000), Journal of General Physiology, 2000, 15, 59-80, by copyright permission of Rockefeller University Press.

1. Calcium-Activated CI Channel in Xenopus

15

shape of the instantaneous I-V relationships, were later realized to be due to errors introduced by our inability to measure a true instantaneous I-V curve in whole oocytes. Because oocytes have large capacitative currents, pseudoinstantaneous I-V curves were measured several milliseconds after the onset of the voltage pulse. Because ionic currents decayed significantly during this time interval, errors were introduced into the shape of the instantaneous I-V curves. It was only after we recorded CI(Ca) currents in excised patches, where we could control [Ca 2+] more rigorously and the time resolution was better, that we realized that it was possible to explain the complex waveforms in whole oocyte recordings in terms of a single type of CI(Ca) channel that exhibited voltage-dependent Ca 2+ affinity.

B. Model oJ:Cl(Ca) Channel Gating These data are consistent with a model in which Ca 2+ binding to the channel allosterically opens the channel and the conformation of the allosteric site is voltage sensitive such that the affinity of the site for Ca 2+ binding is voltage sensitive (Fig. 5). The alternative interpretation of the data that Ca 2+ is an open-channel

FIGURE 5 Model of the voltage-dependentCa2+ sensitivityof CI(Ca) channels. The model assumes that there are three Ca2+-bindingsites per channelbecauseno ~3, but only two sites are visible.Openingrate is assumedto be dependenton the numberof Ca2+ ions bound.0 Ca: the channel is closed in the absence of Ca. High Ca: at [Ca] > Ko at -120 mV, Ca2+ is bound to all three sites independentof voltage and both inward and outward steady-state currents are recorded. Low Ca: at [Ca] < Kd at --120 mV, more sites are occupied at +100 mV than at -100 mV and more outward current is observed. =

16

Machaca et al.

blocker that is driven into the channel at negative transmembrane potentials is not consistent with the data. This model would not explain the observation that channel deactivation upon Ca 2+ washout in fast-perfusion experiments with excised patches is voltage sensitive and that channel activation is not dependent on voltage. Our model is supported by a detailed gating scheme (Kuruma and Hartzell, 2000). The model of Fig. 5 is also supported by the observation that the affinity of the channel for Ca 2+ is affected by the species of permeant anion (Kuruma and Hartzell, 2000). For example, SCN- has a much higher apparent affinity for the channel than does C1-. In the presence of SCN- as permeant anion, the affinity for Ca 2+ of the channel at the same voltage is about 2-fold higher than it is in the presence of C1-. This suggests that the occupancy of the pore by permeant anion affects Ca 2+ binding. This interaction between permeant anion and Ca 2+ is inconsistent with both ions acting in the permeation pathway, however, because one would expect that the affinity of the channel for Ca 2+ would be decreased, not increased, by a permeant anion having a higher affinity if Ca 2+ acted as an open channel blocker at negative potentials. As mentioned above, an alternative to the model depicted in Fig. 5 would be one in which Ca 2+ binds to a site within the permeation pathway. That is, the voltage dependence of Ca 2+ affinity would reflect Ca 2+ movement into the channel pore when the cell is depolarized and Ca 2+ movement out of the pore when the cell is hyperpolarized. An argument against this model is that the presence of a large cation in the pore might be expected to block anion movement. However, the CI(Ca) channel is slightly permeant to cations (Qu and Hartzell, 2000). Furthermore, Franciolini and Nonner (1987, 1994a,b) have shown that another species of anion channel is permeant to very large monovalent cations and have suggested that permeant anions form mixed complexes with permeant cations while traversing the channel. Although the anion permeability of the channel studied by Franciolini and Nonner is not affected by Ca 2+, the specter of cation participation in anion permeability raises the question whether Ca 2÷ might form part of the anion-binding site in the pore.

C. Comparison of Xenopus CI(Ca) Channels with Those in Mammalian Cells Xenopus CI(Ca) channels appear to be nearly identical to channels expressed in a variety of mammalian cells. For example, Fig. 6 shows CI(Ca) currents in a mouse kidney inner medullary collecting duct cell line (IMCD-K2). In the whole cell configuration, when the cell is internally dialyzed with a solution with [Ca2+] <50 nM, very small rapidly inactivating currents are observed, but when the cells are internally dialyzed with solutions containing 600 nM [Ca2+], large time-dependent outwardly-rectifying currents are recorded. These currents (Fig. 6A) look almost identical to those currents seen in excised patches fromXenopus oocytes exposed to

1. Calcium-Activated CI Channel in Xenopus

17

4-

42o:

2o

-4-2" ,

.

.

.

.

,

0

-

+

•

5OO

•

i

-

1000

"6

i

.

.

.

.

0

Time (msec) 4.

21 -I+o -ioo J

.

.

.

.

I

1000

Time ( m s ~ )

c

'

i

500

-.m.+.!

=' =~ __~...,_._

steady-state

,~,

1,oo

V m (mV)

instanta~ FIGURE 6 Ca2+-activated CI- channels in IMCD-K2 cells. Cells were patched using the whole-cell configuration. The pipet solution contained 600 nM free Ca. (A) Traces used to determine the steady-state IV curve. The cell was voltage clamped from a holding potential of -40 mV to different potentials for 750 ms followed by a pulse to -100 inV. (B) Traces used to determine the instantaneous I-V curve. The cell was voltage clamped from a -40 mV holding potential to + 100 mV followed by pulses to different potentials. (C) IV curves. Solid squares: currents at the end of the 750ms depolarization in (A) were plotted vs. membrane potential. Open circles: instantaneous currents at the start of the second pulse were plotted vs. membrane potential. Compare these currents to those we have studied in Xenopusoocyte (Kuruma and Hartzell, 1999).

the s a m e [Ca 2+] [Fig. 2B; see also K u r u m a and Hartzell (1999)]. L i k e the Xenopus Icl(ca), the I M C D current has a linear instantaneous I-V (Fig. 6B and C) with a reversal potential identical to ECl. W h e n cytosolic C a 2+ is increased to 2 / z M , t i m e - i n d e p e n d e n t n o n r e c t i f y i n g currents w e r e r e c o r d e d (not shown), as o b s e r v e d in Xenopus (Fig. 2D). T h e s e features are shared with a n u m b e r o f m a m m a l i a n CI(Ca) currents including ones r e c o r d e d in rat lacrimal gland (Evans and Marty, 1986) and rat parotid g l a n d ( A r r e o l a et al., 1996).

-

18

Machaca et al.

D. Mechanisms of Channel Activation by Ca In the above discussion we have assumed that Ca 2+ activates the channel by binding directly to it. Is there any evidence to support this assumption? We find that we can reversibly activate CI(Ca) channels in excised patches by addition of Ca 2+ in the absence of any energy source such as adenosine triphosphate (ATP). This suggests that phosphorylation is not required, although it could be argued that the channel may be phosphorylated before it is excised. The observation that the currents in excised patches run down might be consistent with such as presumption. However, in a rather extensive survey, we have found no effect of a variety of purified phosphatases (PPaselA, calcineurin, alkaline phosphatase, lamba phosphatase) on currents in excised patches. Furthermore, after rundown, we cannot reactivate currents with protein kinase A, protein kinase C, or calmodulindependent kinase II (CaMKII) and these kinases have no effect on currents before rundown. Thus, these data indicate to us that this channel, in contrast to CI(Ca) channels from some other cell types such as T84 cells (Arreola et al., 1998; Ho et al., 2001), do not require phosphorylation for activation by Ca 2+.

IV. ANION PERMEATION IN CI(Ca) CHANNELS

A. Selectioity Like most C1- channels, Ca-activated C1- channels are relatively nonselective among anions (Qu and Hartzell, 2000). Anions that are larger than C1- are considerably more permeant than CI-, as estimated from shifts in reversal potential of the currents in excised patches exposed to C1- on one side and the test ion on the other (Fig. 7, solid squares). This pattern of ion selectivity differs significantly from that found in voltage-gated K + channels (Fig. 7, open circles), which are highly selective for cations in a very narrow size range. The Xenopus CI(Ca) channel differs from C1C channels, CFTR, and the outwardly rectifying C1 channel in having a very high relative permeability for large pseudohalides, such as thiocyanate (SCN-). The Xenopus CI(Ca) channel is ~11 times more permeant to SCN- than to C1-. In this regard, the CI(Ca) channel closely resembles the GABAA and glycine receptors (see Section V). Although larger anions enter the channel pore more readily than smaller anions, larger anions also reside in the pore more tightly. Thus, anions with high relative permeabilities have relatively lower conductances. The ability of the anion to enter the pore is related to the ease with which bound water that surrounds the anion in bulk solution is replaced by "channel water." We have estimated that the apparent dielectric constant of the CI(Ca) channel is about 20, which is less than water (80). Thus, the energy requirement to move anions from bulk solution into the channel is less for lar~er anions havinz a lower energy of hydration.

1. Calcium-Activated C1 Channel in Xenopus

15-

19

oTI

Q.

/\

-5.

12.

/0,04

/

/,Br

o i..¢/c,

....................

1.0

1.5

2.0

2.5

o.ol

3.0

3.5

Ionic Radius (A) FIGURE 7 Ionicselectivityof CI(Ca)channels and voltage-gatedK+ channels. Solid squares: relativeanionicpermeabilityof CI(Ca)channels(Px/Pcl)was deten-ninedunderbiionicconditions(Qu and Hartzell,2000). Opencircles: comparisonto publisheddata on cationicselectivityof voltage-gated K channel (Hille, 1992). Once an anion is in the channel, its conductance through the channel depends at least in part on its interactions with the wall of the pore. The pore of the CI(Ca) channel, like that of other anion channels (see Section V), might best be viewed as a two-part filter. The "size filter" that determines permeability may be distributed along a significant fraction of the pore length, whereas anion-binding sites that determine anion conductance may be more discretely localized. Until the CI(Ca) pore is identified molecularly, we are bound to remain relatively ignorant of the nature of these interactions. However, it seems likely that hydrophobic interactions play a role. Hydrophobic anions are good channel blockers because they lodge in the pore and block movement of more permeant anions. For example, the large pseudohalide anion tricyanomethanide [C(CN)3] is the most permeant anion we have tested (~14 times more permeant than C1-). In mixtures of C1- and C(CN) 3 on the outside of the patch, C(CN) 3 blocks outward C1- current with an IC50 of less than 1 mM. This suggests that C(CN) 3 has an affinity for residues in the CI(Ca) pore ,-~100-fold higher than does C1-.

B. Pharmacology of the CI(Ca) Channel Many of the classic C1- channel blockers are hydrophobic anions. We have examined the blocking properties of anthracene-9-carboxylic acid (A9C), diphenylamine-2-carboxylic acid (DPC), 4,4'-diisothiocyanostilbene-2-2'-disulfonic acid (DIDS), and nifiumic acid (NFA) on CI(Ca) channels (Qu and Hartzell, 2001). All of the drugs blocked the channel from the extracellular side, although

20

Machaca et al.

because the drugs are hydrophobic they are able to gain access to the extracellular face even when applied to the intracellular side. Estimates of their blocking positions in the pore suggest that A9C blocks at a site 60% across the voltage field, DPC and DIDS block at a site 30% across, and NFA blocks at a site about 10% of the way across. The relative order of potency of block was NFA > A9C > DIDS > DPC (Kis at -1-100 mV were 10.1, 18.3, 48, and 111 /xM). Models of the equilibrium geometries of the blocking molecules suggest that the position of block is related to the geometry of the molecule. All of the molecules can be oriented in a way that their cross-sectional dimensions are less than 0.77 n m wide by 0.94 n m tall. Within this range, molecules such as NFA that are wider do not enter the pore as deeply as molecules such as A9C that are smaller in this dimension. Our model of the CI(Ca) channel pore is shown in Fig. 8. This sieve-like behavior suggests that none of the blockers interacts strongly with specific structures in the pore. Rather, the blocking behavior depends only upon size.

FIGURE $ Model of a CI(Ca) pore. The dimensionsof the pore of the Xenopus CI(Ca) channel were determinedby measuringthe ability of different C1 channelblockers applied to the inside and outside of excised patches to block Ca-activatedC1conductance(Qu and Hartzell, 2001). The voltage dependence of the block was used to estimate the distanceinto the pore where the blocker lodged. Only two blockers, DPC and NFA, and one permeant anion, C(CN)f are shown, but others were also tested.

1. Calcium-Activated C1 Channel in Xenopus

21

C. Model of the Xenopus Ca-Activated Cl Channel Pore From measurements of anion permeation and block, we have estimated the functional dimensions of the CI(Ca) pore. The data suggest that the pore narrows from dimensions that will accommodate NFA (0.77 x 0.94 nm) at the extracellular end to approximately 0.33 x 0.75 nm [the dimensions of the largest permeant anion, C(CN)~-] at some point > 6 0 % of the way across the channel to the cytosolic side. Figure 8 shows our conception of the CI(Ca) pore. In this model, we assume a constant taper of the channel from the outside to the inside, but the possibility exists that the smallest pore dimensions occur within the channel closer to its mid-point. This ambiguity results from the absence of blockers that enter the pore > 6 0 % from the outside.

D. Toward a Biophysical Definition of Ca-Activated CI Channels It remains to be answered how many different kinds of CI(Ca) channels exist. The definitive answer awaits cloning and functional characterization of each channel subtype. However, understanding the behavior of heterologously expressed channels requires knowledge of the behavior of the native channels. Xenopus CI(Ca) channels comprise one clearly delineated subtype of CI(Ca) channels. Table I lists the properties a channel should have to be included in this subtype of channel. Data in the literature are actually relatively sparse concerning which CI(Ca) currents fit these criteria. Table H shows a compilation of CI(Ca) currents from different cell types and an evaluation of how they fit the criteria set out in Table I. This compilation shows that the CI(Ca) channels in the literature differ in their

TABLE I

Signature Properties of XenopusOocyteType Ca-ActivatedC1Channels Activated directlyby Ca2+ (phosphorylationnot required) Voltage-dependentCa2+ affinityin the 1/zM range Ca2+-sensitive kinetics and apparent voltagedependence at low [Ca2+] Anion nonselective; larger halide anions are more permeant than smaller ones Highly permeant to pseudohalide anions such as SCN Voltage-dependentblock from outside by A9C with Ki ~ 18/zM Sensitive to extracellular DIDS, DPC, and NFA with affinities dependent on conditions Linear instantaneous I-V Steady-state I-V shape depends on [Ca]

Z

v

Z

rj

A

:B

e~

%

go

N

0

h

A

^

A

A

A

A

A

r..)

A ^

Z ^

A

A

^

~

A

~z~ A

A

A

A

A

o

o~

rO

v

~'

v

v

'd

g .g ,-.,

©

1. Calcium-Activated CI Channel in Xenopus

23

apparent Ca 2+ affinity by three orders of magnitude and have different patterns of anion selectivity and pharmacology. These data suggest that there are at least several subtypes of CI(Ca) channels. However, many epithelial Cl(Ca) channels have very similar characteristics and may comprise one subtype that is typified by the Xenopus oocyte channel.

E. Do Cloned CLCA Channels Fit These Criteria? Recently, a family of putative Ca-activated C1- channels has been cloned (see Chapters 17, 18, and 19, this volume). These cloned channels are relatively uncharacterized biophysically, but in any case, they do not seem to fall into the Xenopus oocyte class. The bovine CaCC channel does not require Ca for activation: the channel has a large background conductance even at low [Ca2+] (Cunningham et al., 1995; Ji et al., 1998). The bovine CaCC channel is the only member of this family to have been examined with regard to C1- selectivity (Ji et al., 1998). The Erev of this channel expressed in Xenopus oocytes changes -~30 mV with a 19-fold change in [C1-], which suggests that the channel is poorly selective for anions: a 77-mV shift is predicted for a highly selective C1- channel. However, the recombinant channel in lipid bilayers has been reported to have an anion:cation selectivity of 9:1 (Chapter 18, this volume). The relative permeability to other halide anions has been investigated in both biochemically purified and recombinant bCaCC channels in lipid bilayers. The relative permeability sequence of I (2.1) > NO3 (1.7) > Br (1.2) > C1 (1.0) was reported for the biochemically purified channel (Ran et al., 1992), which would fit our criteria for a Xenopus-oocyte type Cl(Ca) channel. The human and mouse CLCA1 and CLCA2 are blocked by 2 mM dithiothreitol (DTT), 300/zM DIDS, and 100/zM NFA (Gandhi et al., 1998; Gruber et el., 1998, 1999), but the bovine CaCC is not blocked by NFA (Cunningham et al., 1995). Thus, the human and mouse clones resemble the Xenopus oocyte Cl(Ca) with regard to DIDS and NFA sensitivity, but the Xenopus channel is not affected by DTT. The A9C sensitivities of the cloned channels have not been examined. The bovine CaCC and Xenopus oocyte channel differ significantly in their sensitivity to NFA. Finally, the CLCA currents are uniformly voltage independent, whereas the Xenopus oocyte channel exhibits time-dependent kinetics at low [Ca2+]. Recently, it has been suggested that human CLCA3 is not a channel (Gruber and Panli, 1998).

V. TOWARD A DEFINITION OF CI SELECTIVITY Four classes of cloned anion channels have been described to date. For each of these classes of channels we will now summarize what is known about the molecular ~ t r l l ~ t l l r e of the channel nroteins (Fie. 9) so that we may relate this to their

24

Machaca et al.

B

FIGURE 9 Putative structures for a single subunit of the pore-forming peptides for each class of anionchanneldescribedso far. (A) A ligand-gatedanionchannel, such as the GIyR.(B) CFrR. (C) C1Cchannel; the two topologicalrenderings are shown. (D) CLCA-typechannel. In each figure, the amino-(N) and carboxy-(C) termini are indicated. In (B), N and R indicate nucleotide-binding and regulatorydomains,respectively. respective permeation properties (Fig. 10). This, in turn, will allow us to search for commonaiities and distinguishing features among the channel types.

A. Structural Comparisons: Four Ways to Build a Chloride Channel

1. Ligand-Gated Anion Channels The neuronal chloride channels formed by the GABAA receptors, GABAc receptors, and the glycine receptors (GABAAR, GABAcR, and GlyR, respectively) are members of the superfamily of ligand-gated ion channels [LGICs; for a recent review, see Jackson (1999)]. Cousins to the cation-permeable LGICs such as nicotinic acetylcholine receptors (nAChR), anion-selective LGICs are composed of a pseudosymmetrical arrangement of five subunits, each of which contains four transmembrane domains (Fig. 9A). GABAAR and GlyR channels are heterooligomers containing a mixture of subunit types. GABAcR channels are homooligomeric, being built of five copies of a single subunit type. Within each subunit, the second transmembrane domain (M2 segment) contributes amino acids that line the pore. The M2 segment confers the binding sites for open-channel blockers such as the local anesthetic QX-222 in nAChR (Leonard et al., 1988; Charnet et al., 1990) and cyanotriphenylborate in GlyR (Rundstr6m et aL, 1994); these drugs reach their binding sites from the extracellular aspect of the pore,

25

1. Calcium-Activated C1 Channel in Xenopus

A

B

C

D

LGIC

CFTR

ClC

Cl(Ca)

FIGURE 10 The putative structure of the pore for each type of channel. The boundaries of the membrane are shown as dashed lines. Pores are drawn assuming that the voltage drop across the membrane is roughly linear and that electrical distance is physically congruent with the length of the pore; both of these are likely to be overly simplistic assumptions. The extracellular end is at the top. The numbers in circles identify approximate locations of binding sites for permeating anions and blocking molecules, as follows: (1) Cl-, SCN-, or C(CN)3; (2) A9C; (3) DPC; (4) NFA; (5) benzoate or hexanoate; and (6) cyanotriphenylborate in GlyR and local anesthetics such as QX-222 in nAChR. References to studies supporting these notions are given in the text.

indicating that the extracellular end of the pore is wide. Three rings of charge in the M2 segments of the nAChR determine conductance and ion selectivity: one ring at the extracellular end of the pore, one at the intraceUular end of the pore, and an intermediate ring near the intracellular end of the pore (Imoto et al., 1988; Lester, 1992). The cation-preferring homomeric or7 subtype of nAChR can be transformed into an anion channel by making a limited set of mutations in and around the inner and intermediate charge rings in order to match the consensus sequence of GABAAR and GlyR channels at these positions (Galzi et al., 1992). Mutation to lysine of an asparagine near the external ring in a Drosophila GABA-gated channel also resulted in a channel that is permeable to cations, showing that the main determinants of selectivity in GABAAR and GlyR channels reside at or near the determinants of selectivity in nAChR channels (Wang et al., 1999). Hence, the M2 segments of LGIC channels line the pore walls to form an inverted teepee arrangement (Fig. 10A) where the region of narrowest diameter is slightly extracellular to the intracellular end of the pore (Lester, 1992).

2. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) The CFFR protein is the locus of the primary defect in cystic fibrosis, a lethal genetic disease (Riordan et al., 1989). CFTR forms a C1- channel found in many tissues of epithelial origin, where it is involved in transepithelial secretion and absorption [for reviews, see Dawson et al. (1999) and McCarty (2000)]. A member

26

Machaca et al.

of the ABC transporter superfamily, the putative secondary structure of CFTR bears little resemblance to any other ion channel described to date (Fig. 9B). The predicted structure includes 12 putative transmembrane domains, plus three large cytoplasmic domains: two nucleotide-binding domains and a regulatory domain. The cytoplasmic domains are involved in regulation of channel activity by phosphorylation via protein kinases A and C, and subsequent gating of channel opening and closing by ATP binding and hydrolysis at the nucleotide-binding domains. Functional CFTR chloride channels are currently thought to be formed by a monomer of CFTR peptide, although a dimeric structure has been proposed (Devidas and Guggino, 1997; Zerhusen etal., 1998; Eskandari etal., 1998; but see Marshall et al., 1994). Determinants of selectivity have been localized primarily to amino acids found in the extracellular two-thirds of the sixth (TM6) and twelfth (TM12) transmembrane domains (Linsdell et al., 1997; Mansoura et al., 1998; Linsdell et al., 2000; McCarty and Zhang, 2001), although contributions also appear to be made by amino acids in TM5 and TM11 (Smith et al., 1997; Zhang et al., 2000a). Structures involved in interactions with open-channel blockers such as DPC are also found in these four TM domains (McDonough et al., 1994; Zhang et al., 2000a,b). The tertiary structure of Cb'TR is less clear than the quaternary structure of the LGICs; nonetheless, we have proposed that the CFTR pore is built in a similar manner whereby these four TM domains line the pore and approach each other most closely at a slight distance from the extracellular end of the pore (Fig. 10B). Hence, CFTR appears to be an inverted version of the anion-selective LGICs (McCarty, 2000). 3. CIC Family The cloning of the voltage-gated C1- channel from Torpedo electroplax (Jentsch et aL, 1990) brought about the recognition of a new and very widespread family of anion channels: the C1C channels. Ten C1C variants have been described thus far, with a wide variety of gating and permeation characteristics [for reviews, see Jentsch et al. (1999); Maduke et al. (2000); and Fahlke (2001)]; the most carefully studied are the C1C-0 and C1C-1 variants. C1C channels are involved in regulation of cellular excitability, transepithelial transport, and acidification of intracellular organelles; mutations in C1C channels are associated with myotonias of skeletal muscle (Fahlke et al., 1997a) as well as Dent's disease and Bartter's syndrome (Jentsch et aL, 1999), disorders of the renal tubule, as well as osteopetrosis (Kornak et al., 2001). The clear voltage dependence of C1C channel gating has been studied extensively. For C1C-0 and C1C-1 channels the gating charge is carried by the permeating anion itself, not by a canonical S4-1ike domain common to all voltage-gated cation channels (Pusch et al., 1995; Pusch, 1996). Hence, there are strong interactions between permeation and gating in C1C channels. In contrast, an intracellularly located aspartic acid was proposed to be a voltage sensor in C1C-1 (Fahlke et al., 1995; Jentsch et aL, 1999). The transmembrane topology of

1. Calcium-ActivatedCI Channelin Xenopus

27

C1C channels has been investigated using glycosylationmapping, protease protection, and cysteine modification studies (Schmidt-Rose and Jentsch, 1997; Fahlke et al., 1997c), but two alternate views remain (Fig. 9C). There are 12 predicted TM domains that cross the membrane ~10 times. Most contentious is the question of whether TM4 crosses the membrane. Regardless of the topology, it is clear that functional C1C channels are homodimers, and that each monomer in the dimer forms its own pore, although the possibility exists that the inner vestibule is a shared structural feature (Miller, 1982; Miller and White, 1984; Middleton et al., 1996; Ludewig et al., 1996; Mindell and Maduke, 2001; Fahlke et al., 1998, 2001). Mixed dimers of C1C-0 with either C1C-1 or C1C-2 showed conductances characteristic of each C1C variant, indicating the presence of two separate pores formed by each functional dimer (Weinreich and Jentsch, 2001). The low resolution structure of a prokaryotic C1C channel recently confirmed the dimeric arrangement (Mindell et al., 2001). The determinants of anion selectivity in C1C channels appear to reside predominantly in three regions: the P1 region, composed of the highly conserved GKxGPxxH sequence in TM4; the P2 region, composed of amino acids at the extracellular end of TM5; and the P3 region, composed of a cluster of amino acids at the cytoplasmic end of TM3 (Fahlke et al., 1997a,c, 2001). However, unidentified regions likely also contribute (Ludewig et al., 1997; Fahlke, 2001). Differential accessibility to extracellular vs. intracellular MTS reagents of cysteines engineered at sites in the P1 and P2 regions suggested that the pore is greater than 10/~ wide at both the extraceUular and intracellular ends, with a sharply defined narrowing between the K and P residues in the conserved P1 sequence (Fahlke et al., 1997c). Consistent with the localization of the narrow region toward the extracellular end, Palade and Barchi (1997) found that the aromatic carboxylic acid A9C blocked muscle chloride channels from the outside of the cell with little apparent voltage dependence, as if the drug was not able to enter far into the electrical field. Hence, the tertiary structure of a C1C monomer appears to resemble that of CFTR in that the narrowest region lies toward the extraceUular end (Fig. 10C) and differs from that proposed for the Xenopus oocyte CI(Ca) channels (Qu and Hartzell, 2001). 4. CLCA Family The first gene encoding a member of the calcium-activated chloride channel family of proteins, bCaCC or bCLCA1, was cloned from bovine trachea in 1995 by Fuller and colleagues (Cunningham et al., 1995). Since then, other variants from murine (mCLCA1) and human (hCLCA1, hCLCA2) libraries have also been isolated (Gandhi et al., 1998; Gruber et al., 1998, 1999). The transmembrane topology of hCLCA2 was determined, using glycosylation site mapping and protease protection (Gruber et al., 1999). The proposed topology indicates five transmembrane domains, with the peptide's amino-terminus extracellular and carboxyterminus intraceUular (Fig. 9D). The transmembrane topology of other members of the CLCA family have not been experimentally established and hydropathy

28

Machaca et aL

plots suggest that transmembrane domains may not be highly conserved. The tertiary and/or quaternary structures of the CLCAs are as yet unclear, although functional channels can apparently be formed by expression of a single construct in mammalian cells or Xenopus oocytes suggesting that the functional channel may be a monomer or a homooligomer. None of the cloned CLCA-type channels has been subjected to detailed biophysical characterization; hence, it is premature to attempt to construct a diagram describing the functional properties of these channels. However, at the risk of incorrectly linking the uncloned but wellcharacterized Xenopus endogenous CI(Ca) channel to the cloned but relatively uncharacterized CLCA channels, we will use the details of permeation in the Xenopus CI(Ca) channel as representative of the group for the sake of comparison. Future characterization of the CLCA channels will be required to resolve this issue. As described above, we have studied the geometry of the CI(Ca) pore (Qu and Hartzell, 2000, 2001). C(CN)~- permeates the pore, and is conductive, suggesting that the minimum pore diameter is > 6/~. All pore-blocking drugs, such as A9C and NFA, reach their binding sites from the extracellular end of the pore. Comparing the geometries of these drugs it is clear that small drug molecules (e.g., A9C) penetrate farther into the pore than do large drug molecules (e.g., NFA), suggesting that the CI(Ca) channel pore is wide at the extracellular end and narrows to a restriction more than halfway toward the intracellular end (Figs. 8 and 10D). Hence, the pore of the CI(Ca) channel resembles that of the anion-selective LGICs. Structure/function studies have yet to identify regions of the CI(Ca) or CLCA proteins that contribute to the pore.

B. Functional Comparisons: Commonalities and Distinguishing Features in Permeation

1. Anion/Cation Selectivity Of the four classes of anion channels, three are poorly selective between either Na + and C1- or K + and C1-. For the anion-selective LGICs, Cb'rFR, and CI(Ca) channels, the permeability to cations relative to anions (Peat~Pc1)is in the range of 0.05 to 0.2 (Bormann etaL, 1987; Tabcharani etal., 1997; Qu and Hartzell, 2000). However, C1C-1 exhibits stronger anion selectivity with PNa/Pa being close to zero (Fahlke et al., 1997a). 2. Affinity for Chloride All four classes of anion channels exhibit low affinity for C1. Kdcl ranged from -,~120 mMin LGICs (Bormann et al., 1987) to ~73 mM in CI(Ca) channels (Qu and Hartzell, 2000) and -,~38 mM in CFYR (Tabcharani et al., 1997). Kdcl in CIC channels varies according to subtype. In C1C-0 channels, which appear to have

1. Calcium-ActivatedC1 Channel in Xenopus

29

only one Cl-binding site, K Cl was 75 mM (Miller and White, 1982). In rat C1C1 channels, the calculated affinity for C1 depends upon whether C1 activity is changed in the intracellular solution or extracellular solution: the inner Cl-binding site exhibited a Kd of 33 mM while the outer site exhibited a Kd of 6 mM (Fahlke et al., 1997b; Rychkov et al., 1998). Affinity for C1 in the LGICs, CFFR, and CI(Ca) channels has been measured from only one side of the membrane. It is possible, therefore, that similar side dependence will be found in these channels if assayed from both sides. 3. Pore Block by Permeant Anions All four classes of anion channels are sensitive to pore block by I-, SCN-, and/or C10~- (Bormann et aL, 1987; Tabcharani et aL, 1993, 1997; Mansoura et al., 1998; Rychkov et al., 1998; Qu and Hartzell, 2000; McCarty and Zhang, 2001). In these experiments, current carried by C1- is reduced in the presence of these "foreign anions" due to their higher affinity binding and longer residence time at anionbinding sites in the pore. Anion binding makes important contributions to anion selectivity in all four classes of channels. 4. Anomalous Mole Fraction Effects (AMFEs) Anomalous conductance behavior in mixtures of anions is usually considered indicative of the presence of multiple binding sites in ion channel pores (Hille, 1992). AMFEs have been reported in all four classes of C1 channels, but AMFEs are not consistently observed. 5. Anion Permselectivity Three of the four classes exhibit relative permeability sequences qualitatively characteristic of the "lyotropic" series (Wright and Diamond, 1977; Smith et al., 1999): SCN > NO3 > I > Br > C1. For some channels, the placement of I- in this order is subject to protocol-dependent blockade of the pore by this anion (Linsdell et al., 1997). Lyotropic permselectivity is consistent with anion-binding sites characterized by a low field strength that may be adjacent to regions of hydrophobicity. Table HI provides a summary of experimentally determined relative permeabilities in each channel type [for reviews discussing other examples, see Dawson et al. (1999); Jackson (1999); Fahlke (2001)]. Where multiple references are given, the values supplied are averages from each study. It is evident from this summary that the patterns of selectivity are similar for the CI(Ca) channels, LGICs, and CFTR, but that the C1C channels show rather different behavior. Hence, it is likely that common mechanisms underlie anion selectivity in three of the four classes of anion channels, but that other mechanisms may be used in the C1C channels. The CI(Ca) channels and GABAcR channels are strongest in their ability to discriminate between permeant anions, as evidenced by the large values of PSCN/PCl in these studies.

30

Machaca et al. TABLE 1II

Relative Permeability Comparisons for Four Classes of Channels Relative permeability Channel CI(Ca) X e n o p u s

SCN-

NO 3

11

CI(Ca) rat

2.4

bCLCA-1

I-

Br-

C1-

4

2

1

Qu and Hartzell (2000)

2.7

1.6

1

Evans and Marty (1986b)

1

Cunningham et al. (1995b)

3

Reference

GABAAR

7.3

2.8

1.5

1

Bormann et al. (1987)

GlyR

7.0

1.8

1.4

1

Bormann et al. (1987)

GABAcR

11.5

4.7

5.8

2.3

1

Wotring et al. (1999)

CFI"R

3.1

1.5

2.0 or 0.5

1.3

1

Linsdell et al. (1997); Mansoura et aL (1998); Linsdell et al. (2000); McCarty and Zhang (2001)

rC1C-1

1.6

0.2

0.2

0.4

1

Rychkov et al. (1998)

hC1C-1

0.9

0.5

0.3

0.6

1

Fahlke et al. (2001)

6. Pore Sizes The relationship between relative permeability and anion size may be used to estimate the minimum pore diameter. Pore size is greatest for the CI(Ca) and GABAcR channels, at >6/~ (WoWinget al., 1999; Qu and Hartzell, 2000); this may be an underestimate for the CI(Ca) channels. Pore sizes for CFrR, C1Cs, GABAAR channels, and GlyR channels cluster around 5.5/~ (Bormann et aL, 1987; Linsdell et al., 1997; McCarty and Zhang, 2001; Fahlke et al., 2001; Rychkov et al., 1998). It should be recognized that the pore shape may be irregular, even flexible to some extent, and assigning pore sizes assuming a circular opening may be misleading. 7. Block by Hydrophobic Anions All four classes of anion channels appear to be blocked by hydrophobic anions, including aromatic carboxylic acids such as DPC, NFA, and A9C (Bryant and Morales-Aguilera, 1971; Evonuik and Skolnick, 1988; White and Aylwin, 1990; Wu and Hamill, 1992; McCarty et al., 1993; McDonough et al., 1994; Astill et al., 1996; Gandhi et al., 1998; Gruber et al., 1998, 1999; Zhang et al., 2000b; Rychkov et al., 2001; Qu and Hartzell, 2001). Interestingly, the bovine CLCA1 channel was insensitive to block by NFA whereas the other three CLCA variants are blocked by 100 btM NFA (Cunningham et al., 1995). Studying the voltage dependence of block by these anions has provided important information regarding pore structure. Permeant hydrophobic anions such as benzoate and hexanoate were used to identify anion-binding sites in C1Cs (Rychkov et al., 2001). Picrotoxin and

1. Calcium-ActivatedCI Channel in Xenopus

31

cyanotriphenylborate also block the LGICs (Berger et al., 1993; RundstriSm et al., 1994). CI(Ca) and C1C channels are also blocked by extracellular application of disulfonic stilbenes such as DIDS (Russel and Brodwick, 1981; Cunningham et al., 1995; Gandhi et al., 1998; Gruber et al., 1999; Qu and HartzeU, 2001), whereas CFFR and the LGICs are DIDS insensitive.

8. Gating Coupled to Permeation Links between permeation and gating have been shown for three of the four classes. This is strongest for the C1Cs, where the permeating anion appears to provide the charge for gating these voltage-dependent channels (Pusch et aL, 1995). Interactions between permeation and gating have also been shown for CFTR, although this has been demonstrated only in pore-domain mutants (Zhang et al., 2000a, 2001). CI(Ca) channels exhibit differential Ca sensitivity when the permeating anion was C1- compared to SCN- (Qu and Hartzell, 2000).

Vl. SUMMARY AND CONCLUSIONS The Xenopus oocyte Ca(C1) channel is the flagship member of the Ca-activated C1 channel family. Because this channel is so well characterized on a physiological and biophysical level, we propose that this channel should be used as a benchmark for comparing other Ca-activated C1 channels. From Table II, it is clear that some mammalian CI(Ca) channels closely resemble the Xenopus oocyte channel, whereas others are decidedly different. The CI(Ca) channel has the interesting feature that its sensitivity to Ca z+ is voltage sensitive. The channel is less sensitive to Ca 2+ at hyperpolarized potentials than at depolarized potentials. This apparent voltage-dependent affinity of the channel for Ca 2+ results from the voltage dependence of the closing reaction. In Xenopus egg, sperm entry during fertilization turns on CI(Ca) channels as a consequence of Ca 2+ release from internal stores. Opening these channels produces the fertilization potential to prevent polyspermy (Webb and Nuccitelli, 1985; Fontanilla and Nuccitelli, 1998). Our data show that increases in [Ca2+] > 1/xM would be required to produce the fertilization potential, because the strong outward rectification of the C1 current at lower [Ca2+] would severely limit the inward current required for depolarization. This prediction is supported by measurements showing that [Ca2+] at the membrane reaches a peak of ,--1.2 /zM after fertilization (Fontanilla and Nuccitelli, 1998). Because the membrane potential of the egg is determined almost entirely by C1- conductances, the voltagedependent Ca 2+ sensitivity of CI(Ca) channels in the egg may serve the rather simple function of ensuring a high [Ca2+] threshold for activation of the fast block to polyspermy. However, in excitable cells where the membrane potential regularly oscillates above and below Ecl due to other conductances, the bimodal

32

Machaca et al.

behavior of CI(Ca) channels at different [Ca2+] could have more interesting consequences. Specifically, we hypothesize that at low [Ca2+], CI(Ca) channels would carry mainly outward current and thus come into play in repolarizing the cell after an excitatory stimulus. In contrast, at high [Ca2+], CI(Ca) channels could become excitatory by carrying inward current at membrane potentials negative to Ecl (between - 3 0 and - 6 0 mV in most excitable cells). This bimodal regulation can explain the participation of CI(Ca) channels in the genesis of cardiac arrhythmias. In some species, an outward CI(Ca) current normally plays a role in phase 1 repolarization of the cardiac action potential (Zygmunt, 1994; Papp et al., 1995; Hirayama et al., 2001). However, under conditions of Ca 2+ overload, this channel produces transient inward currents that are arrhythmogenic (January and Fozzard, 1988; Zygmunt et al., 1998; Berlin et al., 1989). The complex voltage and Ca 2+ sensitivity of CI(Ca) channels place these channels in a pivotal position for regulation of cellular excitability. Another question that arises is the physiological anion transported by these channels. It seems likely that C1- is a major component. We find that HCO~- and acidic amino acids, at least under biionic conditions, exhibit low permeability through CI(Ca) channels. However, given the relatively nonselective nature of the CI(Ca) pore, one cannot help but wonder whether there are other anions that are permeant, or whether permeability of amino acids or HCO~- may be modulated by other regulatory processes, such as phosphorylation or other subunits. Clearly the Xenopus oocyte CI(Ca) channel has been a valuable model system for studying the physiology, biophysics (gating and permeation), pharmacology, and regulation of CI(Ca) channels. However, the next step clearly will require cloning and characterization of this channel to resolve questions about the number of subtypes of CI(Ca) channels and the molecular mechanisms underlying the apparent voltage-dependent Ca sensitivity and anion selectivity of the channel. References Anderson, M. P., and Welsh, M. J. (1991). Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc. Natl. Acad. Sci. USA 88, 6003-6007. Arreola, J., Melvin, J., and Begenisich, T. (1996). Activation of calcium dependent chloride channels in rat paroid acinar cells. Z Gen. Physiol. 108, 35-47. Arreola, J., Melvin, J. E., and Begenisich, T. (1998). Differences in regulation of Ca2+-activated C1channels in colonic and parotid secretory cells. Am. J. Physiol. 274, C 161-C 166. Asti11, D. St. J., Rychkov, G., Clarke, J. D., Hughes, B. E, Roberts, M. L., and Bretag, A. H. (1996). Characteristics of skeletal muscle chloride channel C1C-1 and point mutant R304E expressed in Sf-9 insect cells. Biochirr~ Biophys. Acta 1280, 178-186. Barish, M. E. (1983). A transient calcium-dependent chloride current in the immature Xenopus oocyte. J. Physiol. 342, 309-325. Bataillon, E. (1919). Analyze de l'activation par la technique des oeufs ntis et la polyspermie experimentale chez les batraciens, Ann. Sci. Nat. Zool. 10, 1-38.

1. Calcium-Activated C1 Channel in Xenopus

33

Begenisich, T., and Melvin, J. E. (1998). Regulation of chloride channels in secretory epithelia. J. Membr. BioL 163, 77-85. Bement, W. M., and Capco, D. G. (1990). Transformation of the amphibian oocyte into the egg: Structural and biochemical events. J. Electron Microsc. Tech. 16, 202-234. Berger, H. A., Travis, S. M., and Welsh, M. J. (1993). Regulation of the cystic fibrosis transmembrane conductance regulator C1- channel by specific protein kinases and protein phosphatases. J. Biol. Chem. 268, 2037-2047. Berlin, J. R., Cannell, M. B., and Lederer, W. J. (1989). Cellular origins of the transient inward current in cardiac myocytes. Role of fluctuations and waves of elevated intracellular calcium. Circ. Res. 65, 115-126. Bormann, J., Hamill, O. P., and Sakmann, B. (1987). Mechanism of anion permeation through channels gated by glycine and r -aminobutyric acid in mouse cultured spinal neurones. J. Physiol. 385, 243286. Bryant, S. H., and Morales-Aguilera, A. (1971 ). Chloride conductance in normal and myotonic muscle fibres and the action of monocarboxylic aromatic acids. J. Physiol. 219, 367-383. Busa, W. B., and Nuccitelli, R. (1985). An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis. J. Cell Biol. 100, 1325-1329. Busa, W. B., Ferguson, J. E., Joseph, S. K., WiUiamson, J. R., and Nuccitelli, R. (1985). Activation of frog (Xenopus laevis) eggs by inositol trisphosphate. I. Characterization of Ca2+ release from intracelhilar stores. J. Cell Biol. 101, 677-682. Charnet, P., Labarca, C., Leonard, R. J., Vogelaar, N. J., Czyzyk, L., Gouin, A., Davidson, N., and Lester, H. A. (1990). An open-channel blocker interacts with adjacent turns of ~-helices in the nicotinic acetylcholine receptor. Neuron 2, 87-95. Cliff, W. H., and Frizzell, R. A. (1990). Separate C1 conductances activated by cAMP and Ca in C1 secreting epithelial cells. Proc. Natl. Acad. Sci. USA 87, 4956-4960. Coleman, T. R., and Dunphy, W. G. (1994). Cdc2 regulatory factors. Curr. Opin. Cell Biol. 6, 877-882. Collier, M. L., Levesque, P. C., Kenyon, J. L., and Hume, J. R. (1996). Unitary C1- channels activated by cytoplasmic Ca2+ in canine ventficular myocytes. Circ. Res. 78, 936-944. Cross, N. L. (1981). Initiation of the activation potential by an increase in intraceUular calcium in eggs of the frog, Rana pipiens. Dev. Biol. 85, 380-384. Cross, N. L., and Elinson, R. P. (1980). A fast block to polyspermy in frogs mediated by changes in the membrane potential. Dev. Biol. 75, 187-198. Cunningham, S. A., Awayda, M. S., Bubien, J. K., Ismailov, I. I., Arrate, M. P., Berdiev, B. K., Benos, D. J., and Fuller, C. M. (1995). Cloning of an epithelial chloride channel from bovine trachea. J. Biol. Chem. 270, 31016-31026. Dawson, D. C., Smith, S. S., and Mansoura, M. K. (1999). CFTR: Mechanism of anion conduction. Physiol. Rev. 79, $47-$45. Devidas, S., and Guggino, W. B. (1997). CFTR: Domains, structure, and function. J. Bioenerg. Biomembr. 29, 443-451. Eguiguren, A. L., Sepulveda, E V., Riveros, N., and Stutzin, A. (1996). Calcium-activated chloride currents and non-selective cation channels in a novel cystic fibrosis-derived human pancreatic duct cell line. Biochem. Biophys. Res. Commun. 225, 505-513. Eskandari, S., Wright, E. M., Kreman, M., Starace, D. M., and Zampighi, G. A. (1998). Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy. Proc. Natl. Acad. Sci. USA 95, 11235-11240. Evans, M. G., and Marry, A. (1986). Calcium-dependent chloride currents in isolated cells from rat lacrimal glands. J. Physiol. 378, 437-460. Evonuik, G., and Skolnick, P. (1988). Picrate and niflumate block anion modulation of radioligand binding to the r-aminobutyric acid/benzodiazepine receptor complex. Mol. Pharmacol. 34, 837842.

34

M a c h a c a et al.

Fahlke, C. (2001). Ion permeation and selectivity in C1C-type chloride channels. Am. J. Physiol. 280, F748-F757. Fahlke, C., Rtidel, R., Mitrovic, N., Zhou, M., and George, A. L., Jr. (1995). An aspartic acid residue important for voltage-dependent gating of human muscle chloride channels. Neuron 15, 463-472. Fahlke, C., Beck, C. L., and George, A. L., Jr. (1997a). A mutation in autosomal dominant myotonia congenita affects pore properties of the muscle chloride channel. Proc. Natl. Acad. Sci. USA 94, 2729-2734. Fahlke, C., Dtirr, C., and George, A. L., Jr. (1997b). Mechanism of ion permeation in skeletal muscle chloride channels. J. Gen. Physiol. 110, 551-564. Fahlke, C., Yu, H. T., Beck, C. L., Rhodes, T. H., and George, A. L., Jr. (1997c). Pore-forming segments in voltage-gated chloride channels. Nature 390, 529-532. Fahlke, C., Rhodes, T. H., Desai, R. R., and George, A. L., Jr. (1998). Pore stoichiometry of a voltagegated chloride channel. Nature 394, 687-690. Fahlke, C., Desai, R. R., Gillani, N., and George, A. L., Jr. (2001). Residues lining the inner pore vestibule of human muscle chloride channels. J. Biol. Chem. 276, 1759-1765. Fankhauser, G., and Moore, C. (1941). Cytological and experimental studies of polyspermy in the newt, Triturus viridescens. I. Normal fertilization. J. Morphol. 68, 647-385. Ferguson, J. E., Han, J. K., Kao, J. P., and Nuccitelli, R. (1991). The effects of inositol trisphosphates and inositol tetrakisphosphate on Ca 2+ release and C1- current pattern in the Xenopus laevis oocyte. Exp. Cell Res. 192, 352-365. Fontanilla, R. A., and Nuccitelli, R. (1998). Characterization of the sperm-induced calcium wave in Xenopus eggs using confocal microscopy. Biophys. J. 75, 2079-2087. Franciolini, E, and Nonner, W. (1987). Anion and cation permeability of a chloride channel in rat hippocampal neurons. J. Gen. Physiol. 90, 453-478. Franciolini, E, and Nonner, W. (1994a). A multi-ion permeation mechanism in neuronal background chloride channels. J. Gen~ Physiol, 104, 725-746. Franciolini, F., and Nonner, W. (1994b). Anion-cation interactions in the pore of neuronal background chloride channels. J. Gen. PhysioL 104, 711-723. Frings, S., Reuter, D., and Kleene, S. J. (2000). Neuronal cae+-activated C1- channels--homing in on an elusive channel species. Prog. Neurobiol. 60, 247-289. Galzi, J.-L., Devillers-Thi6ry, A., Hussy, N., Bertrand, S., Changeux, J.-P., and Bertrand, D. (1992). Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature 359, 500-505. Gandhi, R., Elble, R. C., Gruber, A. D., Schreur, K. D., Ji, H.-L., Fuller, C. M., and Pauli, B. U. (1998). Molecular and functional characterization of a calcium-sensitive chloride channel from mouse lung. J. Biol. Chem. 273, 32096-32101. Gomez-Hernandez, J.-M., Stithmer, W., and Parekh, A. B. (1997). Calcium dependence and distribution of calcium-activated chloride channels in Xenopus oocytes. J. Physiol. 502.3, 56%574. Grey, R. D., Wolf, D. P., and Hedrick, J. L. (1974). Formation and structure of the fertilization envelop in Xenopus laevis. Dev. Biol. 36, 44-61. Grey, R. D., Working, P. K., and Hedrick, J. L. (1976). Evidence that the fertilization envelope blocks sperm entry in eggs of Xenopus laevis: Interaction of sperm with isolated envelopes. Dev. Biol. 54, 52-60. Grey, R. D., Bastiani, M. J., Webb, D. J., and Schertel, E. R. (1982). An eleclrical block is required to prevent polyspermy in eggs fertilized by natural mating of Xenopus laevis. Dev. Biol. 89, 475484. Gruber, A. D., and Pauli, B. U. (1998). Molecualr cloning and biochemical characterization ofa trucated secreted member of the human family of Ca2+-activated CI- channels. Biochim. Biophys. Acta 1444, 418-423.

1. Calcium-Activated CI Channel in Xenopus

35

Gruber, A. D., Elble, R. C., Ji, H.-L., Schreur, K. D., Fuller, C. M., and Pauli, B. U. (1998). Genomic cloning, molecular characterization, and functional analysis of human CLCA1, the first human member of the family of Ca2+-activated C1- channel proteins. Genomics 1, 200-214. Gruber, A. D., Schreur, K. D., Ji, H.-L., Fuller, C. M., and Pauli, B. U. (1999). Molecular cloning and transmembrane structure of hCLCA2 from human lung, trachea, and mammary gland. Am. J. Physiol. 276, C1261-C1270. Hagiwara, S., and Jaffe, L. A. (1979). Electrical properties of egg cell membranes. Annu. Rev. Biophys. Bioeng. 8, 385-416. Hallani, M., Lynch, J. W., and Barry, P. H. (1998). Characterization of calcium-activated chloride channels in patches excised from the dendritic knob of mammalian olfactory receptor neurons. J. Membr. Biol. 161, 163-171. Hartzell, H. C. (1996). Activation of different C1 currents in Xenopus oocytes by Ca liberated from stores and by capacitative Ca influx. J. Gen. Physiol. 108, 157-175. Herlant, M. (1911). Recherches sur les oeufs di-et-trispermiques de grenouille. Arch. Biol. 26,103-336. Hille, B. (1992). "Ion Channels of Excitable Membranes", 2nd ed. Sinauer Associates, Inc., Sunderland, MA. Hirayama, Y., Kuruma, A., Hiraoka, M., and Kawano, S. (2001). Beat dependent alteration of Ca2+activated C1- current during rapid stimulation in rabbit ventricular myocytes. Jpn. Heart J. 42, 207-219. Ho, M. W., Kaetzel, M. A., Armstxong, D. L., and Shears, S. B. (2001). Regulation of a human chloride channel, a paradigm for integrating input from calcium, type 1I calmodulin-dependent protein kinase, and inositol 3,4,5,6-tetrakis phosphate. J. Biol. Chem. 276, 18673-18680. Imoto, K., Busch, C., Sakmann, B., Mishina, M., Konno, T., Nakai, J., Bujo, H., Mori, Y., Fukuda, K., and Numa, S. (1988). Rings of negatively charged amino acids determine the acetylcholine receptor channel conductance. Nature 335, 645-648. Ishikawa, T., and Cook, D. I. (1993). A Ca2+-dependent C1- current in sheep parotid secretory cells. J. Membr. Biol. 135, 261-271. Ito, S. (1972). Effects of media of different ionic composition on the activation potential of anuran egg ceils. Dev. Growth Differ. 14, 217-227. Jackson, M. B. (1999). Ligand-gated channel: Postsynaptic receptors and drug targets. Adv. Neurol. 79, 511-524. Jaffe, L. A. (1976). Fast block to polyspermy in sea urchin eggs is electrically mediated. Nature 261, 68-71. Jaffe, L. A., Cross, N. L., and Picheral, B. (1983). Studies of the voltage-dependent polyspermy block using cross-species fertilization of amphibians. Dev. Biol. 98, 31%326. January, C. T., and Fozzard, H. A. (1988). Delayed after depolarizations in heart muscle: Mechanisms and relevance. Pharmacol. Rev. 411, 219-227. Jentsch, T. J., Steinmeyer, K., and Schwarz, G. (1990). Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348, 510-514. Jentsch, T. J., Friedrich, T., Schriever, A., and Yamada, H. (1999). The C1C chloride channel family. Pflugers Arch. 437, 783-795. Ji, H.-L., DuVall, M. D., Patton, H. K., Satterfield, C. L., Fuller, C. M., and Benos, D. J. (1998). Functional expression of a truncated Ca2+-activated C1- channel and activation by phorbol ester. Am. J. Physiol. 274, C455-C464. Kidd, J. E, and Thorn, E (2000a). lntracellular Ca2+ and C1- channel activation in secretory ceils. Annu. Rev. Physiol. 62, 493-513. Kidd, J. E, and Thorn, P. (2000b). The properties of the secretogogue-evoked chloride current in mouse pancreatic acinar cells. Pflugers Arch. 441, 489-497. Kleene, S. J. (1993). Origin of the chloride current in olfactory transduction. Neuron 11, 123-132.

36

Machaca et al.

Kline, D., and NucciteUi, R. (1985). The wave of activation current in the Xenopus egg. Dev. BioL 111, 471-487. Klockner, U. (1993). Intracellular calcium ions activate a low-conductance chloride channel in smoothmuscle ceils isolated from human mesenteric artery. Pflugers Arch. 424, 231-237. Kornak, U., Kasper, D., Bosl, M.R., Kaiser, E., Schweizer, M., Schulz, A., Friedrich, W., Delling, G., and Jentsch, T. J. (2001). Loss of the C1C-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205-215. Koumi, S., Sato, R., and Aramaki, T. (1994). Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes. J. Gen. Physiol. 104, 357-373. Kuruma, A., and Hartzell, H. C. (1999). Dynamics of calcium regulation of chloride currents in Xenopus oocytes. Am. J. Physiol. 276, C161-C175. Kuruma, A., and HartzeU, H. C. (2000). Bimodal control ofa Ca2+-activated Cl- channel by different Ca2+ signals. J. Gen. Physiol. 115, 59-80. Lamb, E S., Volk, K. A., and Shibata, E. E (1994). Calcium-activated chloride current in rabbit coronary artery myocytes. Circ. Res. 75, 742-750. Larabell, C. A., and Nuccitelli, R. (1992). Inositol lipid hydrolysis contributes to the Ca 2+ wave in the activating egg of Xenopus laevis. Dev. Biol. 153, 347-355. Large, W. A., and Wang, Q. (1996). Characteristics and physiological role of the Ca2+-activate C1conductance in smooth muscle. Am. J. Physiol. 271, C435-C454. Leonard, R. J., Labarca, C. G., Charnet, E, Davidson, N., and Lester, H. A. (1988). Evidence that the M2 membrane-spanning region lines the ion channel pore of the nicotinic receptor. Science 242, 1578-1581. Lester, H. A. (1992). The permeation pathway of neurotransmitter-gated ion channels. Annu. Rev. Biophys. Biomolecular Structure 21, 267-292. Linsdell, P., Tabcharani, J. A., Rommens, J. M., Hou, Y.-X., Chang, X.-B., Tsui, L.-C., Riordan, J. R., and Hanrahan, J. W. (1997). Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions. J. Gen. Physiol. 110, 355-364. Linsdell, P., Evagelidis, A., and Hanrahan, J. W. (2000). Molecular determinants of anion selectivity in the cystic fibrosis transmembrane conductance regulator chloride channel pore. Biophys. J. 78, 2973-2982. Ludewig, U., Pusch, M., and Jentsch, T. J. (1996). Two physically distinct pores in the dimeric C1C-0 chloride channel. Nature 383, 340-343. Ludewig, U., Jentsch, T. J., and Pusch, M. (1997). Inward rectification in C1C-0 chloride channels caused by mutations in several protein regions. J. Gen. Physiol. 110, 165-171. Machaca, K., and Hartzell, H. C. (1998). Asymmetrical distribution of Ca-activated C1 channels in Xenopus oocytes. Biophys. J. 74, 1286-1295. Machaca, K., and Hartzell, H. C. (1999). Reversible Ca gradients between the sub-plasmalemma and cytosol differentially activate Ca-dependent C1 currents. J. Gen. PhysioL 113, 249-266. Machaca, K., and Haun, S. (2000). Store-operated calcium entry inactivates at the germinal vesicle breakdown stage of Xenopus meiosis. J. Biol. Chem. 275, 38710-38715. Maduke, M., Miller, C., and Mindell, J. A. (2000). A decade of CIC chloride channels: Structure, mechanism, and many unsettled questions. Annu. Rev. Biophys. Biomol. Struct. 29, 411438. Maeno, T. (1959). Electrical characteristics and activation potential of Bufo eggs. J. Gen. Physiol. 43, 139-157. Mansoura, M. K., Smith, S. S., Choi, A. D., Richards, N. W., Strong, T. V., Drumm, M. L , Collins, E S., and Dawson, D. C. (1998). Cystic fibrosis transmembrane conductance regulator (CFTR) anion binding as a probe of the pore. Biophys. J. 74, 1320-1332. Marshall, J., Fang, S., Ostedgaard, L. S., O'Riordan, C. R., Ferrara, D., Amara, J. E, Hoppe, H. IV, Scheule, R. K., Welsh, M. J., Smith, A. E., and Cheng, S. H. (1994). Stoichlometry of

1. Calcium-Activated C1 Channel in Xenopus

37

recombinant cystic fibrosis transmembrane conductance regulator in epithelial cells and its functional reconstitution into cells in vitro. J. BioL Chem. 269, 2987-2995. McCarty, N. A. (2000). Permeation through the CFTR chloride channel. J. Exp. Biol. 203, 1947-1962. McCarty, N. A., and Zhang, Z.-R. (2001). Identification of a region of strong discrimination in the pore of CFrR. Am. J. Physiol. (in press). McCarty, N. A., McDonough, S., Cohen, B. N., Riordan, J. R., Davidson, N., and Lester, H. A. (1993). Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator C1- channel by two closely related arylaminobenzoates. J. Gen. Physiol. 102, 1-23. McDonough, S., Davidson, N., Lester, H. A., and McCarty, N. A. (1994). Novel pore-lining residues in CFrR that govern permeation and open-channel block. Neuron 13, 623-634. Middleton, R. E., Pheasant, D. J., and Miller, C. (1996). Homodimeric architecture of a ClC-type chloride ion channel. Nature 383, 337-340. Miledi, R., and Parker, I. (1984). Chloride current induced by injection of calcium into Xenopus oocytes. J. Physiol. 357, 173-183. Miller, C. (1982). Open-state substructure of single chloride channels from Torpedo electroplax. Philos. Trans. R. Soc. Lond. 299, 401-411. Miller, C., and White, M. M. (1982). Probes of the conduction pathway of a voltage-gated C1- channel from Torpedo. J. Gen. Physiol. 78, 1-18. Miller, C., and White, M. M. (1984). Dimeric structure of single chloride channels from Torpedo electroplax. Proc. Natl. Acad. Sci. USA 81, 2772-2775. Mindell, J. A., and Maduke, M. (2001). C1C chloride channels. Genome Biol. 2, 3003.1-3003.6. Mindell, J. A., Maduke, M., Miller, C., and Grigorieff, N. (2001). Projection structure of a C1C-type chloride channel at 6.5 A resolution. Nature 409, 219-223. Nilius, B., Prenen, J., Szucs, G., Wei, L., Tanzi, F., Voets, T., and Droogmans, G. (1997). Calciumactivated chloride channels in bovine pulmonary artery endothelial cells. J. Physiol. 498, 381-396. Nishimoto, I., Wagner, J., Schulman, H., and Gardner, P. (1991). Regulation of C1- channels by multifunctional CaM kinase. Neuron 6, 547-555. Nuccitelli, R., Yim, D. L., and Smart, T. (1993). The sperm-induced Ca2+ wave following fertilization of the Xenopus egg requires the production of Ins( 1,4,5)P3. Dev. Biol. 158, 200-212. Palade, P. T., and Barchi, R. L. (1997). On the inhibition of muscle membrane chloride conductance by aromatic carboxylic acids. J. Gen. PhysioL 69, 879-896. Papp, Z., Sipido, K., Callewaert, G., and Carmeliet, E. (1995). Two components of [Ca2+]i-activated C1- current during large [Ca2+]i transients in single rabbit heart Purkinje cells. J. Physiol. 483.2, 319-330. Parker, I., and Miledi, R. (1986). Changes in intracellular calcium and in membrane currents evoked by injection of inositol tris~hosphate into Xenopus oocytes. Proc. R. Soc. Lond. 228, 307-315. Parker, I., and Miledi, R. (19875. Inositol trisphosphate activates a voltage-dependent calcium influx in Xenopus oocytes. Proc. R. Soc. Lond. 231, 27-36. Parker, I., and Yao, Y. (1994). Relation between intracellular Ca2+ signals and Ca(2+)-activated CIcurrent in Xenopus oocytes. Cell Calcium 15, 276-288. Pusch, M. (1996). Knocking on channel's door: The permeating chloride ion acts as the gating charge in CIC-0. J. Gen. Physiol. 108, 233-236. Pusch, M., Ludewig, U., Rehfeldt, A., and Jentsch, T. J. (1995). Gating of the voltage-dependent chloride channel C1C-0 by the permeant anion. Nature 373, 527-531. Putuey, J. W., Jr. (2001). Channelling calcium. Nature 410, 648-649. Qu, Z., and Hartzell, H. C. (2000). Anion permeation in Ca2+-activatedCl- channels. J. Gen. Physiol. 116, 825-844. Qu, Z., and Hartzell, H. C. (2001). Functional geometry of the permeation pathway of Ca2+-activated C1- channels inferred from analysis of voltage-dependent block. J. Biol. Chem. 276, 1842318429.

38

M a c h a c a et al.

Ran, S., Fuller, C. M., Arrate, M. E, Latorre, R., and Benos, D. J. (1992). Functional reconstruction of a chloride channel protein from bovine trachea. J. Biol. Chem. 267, 20630-20637. Riordan, J. R., Rommens, J. M., Kerem, B.-S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzzi, M. C., Collins, E S., and Tsui, L.-C. (1989). Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245, 1066-1072. Robertson, A. P., and Martin, R. J. (1996). Effects of pH on a high conductance Ca-dependent chloride channel: A patch-clamp study in Ascaris suum. Parasitology 113 (Pt 2), 191-198. RundstrSm, N., Schmieden, V., Betz, H., Bormann, J., and Langosch, D. (1994). Cyanotriphenylborate: Subtype-specific blocker of glycine receptor chloride channels. Proc. Natl. Acad. Sci. 91, 89508954. Russel, J. M., and Brodwick, M. S. (1981). CyclicAMP-stimulated chloride fluxes in dialyzed barnacle muscle fibers. J. Gen. Physiol. 78, 499-520. Rychkov, G. Y., Pusch, M., Roberts, M. L., Jentsch, T. J., and Bretag, A. H. (1998). Permeation and block of the skeletal muscle chloride channel, C1C-1, by foreign anions. J. Gen. Physiol. U l , 653-665. Rychkov, G. Y., Pusch, M., Roberts, M. L., and Bretag, A. H. (2001). Interaction of hydrophobic anions with the rat skeletal muscle chloride channel CIC- 1: Effects on permeation and gating. J. Physiol. 530, 379-393. Schmidt-Rose, T., and Jentsch, T. J. (1997). Transmembrane topology of a C1C chloride channel. Proc. Natl. Acad. Sci. USA 94, 7633-7638. Smith, S. S., Mansoura, M. K., Schafer, J. A., Cooke~ C. R., Shariat-Madar, Z., Sun, E, and Dawson, D. C. (1997). The fifth putative transmembrane helix of C F r R contributes to the pore architecture. Biophys. J. 72, A365 (abstract). Smith, S. S., Steinle, E. D., Meyerhoff, M. E., and Dawson, D. C. (1999). Cystic fibrosis transmembrane conductance regulator: Physical basis for lyotropic anion selectivity patterns. J. Gen. Physiol. 114, 799-817. Snow, E, Yim, D. L., Leibow, J. D., Saini, S., and Nucciteili, R. (1996). Fertilization stimulates an increase in inositol trisphosphate and inositol lipid levels inXenopus eggs. Dev. Biol. 180,108-118. Stricker, S. A. (2000). Comparative biology of calcium signaling during fertilization and egg activation in animals. Dev. Biol. 211, 157-176. Tabcharani, J. A., Rommens, J. M., Hou, Y.-X., Chang, X.-B., Tsul, L.-C., Riordan, J. R., and Hanrahan, J. W. (1993). Multi-ion pore behavior in the CFTR chloride channel. Nature 366, 79-82. Tabeharani, J. A., Linsdell, E, and Hanrahan, J. W. (1997). Halide permeation in wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels. J. Gen. Physiol. 110, 341-351. Taleb, O., Feltz, E, Bossu, J.-L., and Felta, A. (1988). Small-conductance chloride channels activated by calcium on cultrned endocrine cells from mammalian pars intermedia. Pflugers Arch. 412, 641-646. Wang, C.-T., Zhang, H.-G., Rochelean, T. A., ffrench-Constant, R. H., and Jackson, M. B. (1999). Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila. Biophys. J. 77, 691-700. Webb, D. J., and Nuccitelli, R. (1985). Fertilization potential and electrical properties of the Xenopus laevis egg. Dev. Biol. 107, 395-406. Weinreich, E, and Jentsch, T. J. (2001). Pores formed by single subunits in mixed dimers of different C1C chloride channels. J. Biol. Chem. 276, 2347-2353. White, M. M., and Aylwin, M. (1990). Niflumic and flufenamic acids are potent reversible blockers of Ca2+-activated CI- channels in Xenopus oocytes. Mol. Pharmacol. 37, 720-724. Wolf, D. E (1974). The cortical granule reaction in living eggs of the toad, Xenopus laevis. Dev. Biol. 36, 62-71.

1. Calcium-Activated CI Channel in Xenopus

39

Wotring, V. E., Chang, Y., and Weiss, D. S. (1999). Permeability and single channel conductance of human homomeric pl GABAc receptors. J. Physiol. 521, 327-336. Wright, E. M., and Diamond, J. M. (1977). Anion selectivity in biological systems. Physiol. Rev. 57, 109-156. Wu, G., and Hamill, O. P. (1992). NPPB block of Ca++-activated CI- currents in Xenopus oocytes. Pfliigers Arch. 420, 227-229. Zerhusen, B., Zhao, J., Xie, J., Davis, P. B., and Ma, J. (1998). A single conductance pore for chloride ions formed by two cystic fibrosis transmembrane conductance regulator molecules. J. Biol. Chem. 274, 7627-7630. Zhang, G., Arreola, J., and Melvin, J. E. (1995). Inhibition by thiocyanate of muscarinic-induced cytosolic acidification and Ca2+ entry in rat sublingual acini. Arch. Oral Biol. 40, 111-118. Zhang, Z.-R., McDonough, S. I., and McCarty, N. A. (2000a). Interaction between permeation and gating in a putative pore-domain mutant in CFTR. Biophys. J. 79, 298-313. Zhang, Z.-R., Zeltwanger, S., and McCarty, N. A. (2000b). Direct comparison of NPPB and DPC as probes of CFTR expressed in Xenopus oocytes. J. Membr. Biol. 175, 35-52. Zhang, Z.-R., Zeltwanger, S., Smith, S. S., Dawson, D. C., and McCarty, N. A. (2001). Voltage-sensitive gating induced by a mutation in the fifth transmembrane domain of CFFR. Am. J. Physiol. (in press). Zygmunt, A. C. (1994). Intracellular calcium activates a chloride current in canine ventricular myocytes. Am. J. Physiol. 267, H1984-H1995. Zygmunt, A. C., Goodrow, R. J., and Weigel, C. M. (1998). 1NaCaand lcl(Ca)contribute to isoproterenolinduced afterhyperpolarizations in midmyocardial cells. Am. J. Physiol. 275, H1979-H1999.