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Calcium-activated chloride channels in vascular endothelial cells
CHAPTER 15 Calcium-Activated Chloride Channels in Vascular Endothelial Cells Bernd Nilius and Guy Droogmans KU Leuven, Laboratorium voor Fysiologie, Campus Gasthuisberg, B-3000 Leuven, Belgium
I. II. III. IV. V. VI. VII. VIII.
Introduction Biophysical Properties of C1Ca in Endothelium Mechanism of Activation Pharmacology Activation by Calmodulin-Dependent Protein Kinase Molecular Nature of C1Ca in Endothelium Discussion Summary References
!. INTRODUCTION The permeability of the membrane for K + and CI- is modulated in many cell types by changes in the concentration of free intracellular Ca 2+, [Ca2+]i. Various types of chloride channels have been described in nonexcitable cells, including cAMP-dependent, voltage-dependent C1C channels, swelling- or volumeactivated, and Ca2+-activated C1- channels (Valverde et al., 1995). Endothelial cells express Ca2+-activated K + channels that are functionally important for the control of membrane potential and driving force for Ca2+ influx during agonist or mechanical stimulation (Nilius, 1991; Daut et al., 1994). An increase in [Ca2+]i during stimulation of endothelial cells by vasoactive agonists such as acetylcholine, histamine, bradykinin, thrombin, adenosine triphosphate (ATP), and other Ca2+-releasing agonists affects their membrane potential. This stimulation evokes a pronounced membrane hyperpolarization in several types of endothelial cells (EC) due to activation of Ca2+-dependent K + channels, but in others it causes only Current Topics in Membranes, Volume 53
Copyright2002, ElsevierScience(USA).All fightsreserved, 1063-5823/02 $35.00
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small changes in membrane potential. This stabilization of the membrane potential is mainly due to activation of Ca2+-dependent C1- channels (Groschner et al., 1992, 1994; Himmel et al., 1994; Watanabe et al., 1994; Yumoto et al., 1994; Nilius and Droogmans, 2001). In this chapter we describe the salient features of Ca2+-activated C1- channels in vascular endothelial cells, and briefly discuss their possible functional impact on endothelial cell function.
I!. BIOPHYSICAL PROPERTIES OF CICa IN ENDOTHELIUM Ca2+-activated C1- channels (C1Ca) have been described in various types of EC, among which pulmonary artery EC and freshly isolated mouse aorta EC (Nilius et al., 1997b,c; Sub et al., 1999). In most cells, the current through C1Ca overlaps currents through K + channels, such as the "big" conductance Ca2+-activated K + channel, BKca, as in mouse aorta endothelium (MAEC), or the inwardly rectifying K + current through Kir 2.1 channels as in pulmonary aorta endothelium (CPAE). To suppress BKca and Kit 2.1 channels, extra- and intracellular K + is substituted by Cs + and charybdotoxin and tetraethylammonium (TEA) is applied to the extracellular solution. CICa is also often coactivated with volume-regulated C1- channels (VRAC, Nilius et al., 1997d). To prevent activation of these usually large contaminating VRAC currents, the cells wereshrunken by adding 50-100 mM mannitol to the bath solution (Nilius et al., 1997b,d). Various protocols have been used to activate C1Ca. Application of Ca2+-releasing vasoactive agonists, such as acetylcholine, histamine, bradykinin, thrombin, uridine triphosphate (UTP), and ATE activate a current that reverses close to the C1-equilibrium potential and that is closely correlated with the concomitant changes in [Ca2+]i. To bypass the inositol 1,4,5-triphosphate [Ins(1,4,5)P3] cascade cells can be dialyzed via the patch pipette with an ethyleneglycoltetraacetic acid (EGTA)-buffered Ca z+ solution to equilibrate [Ca2+]i at suitable constant values or by using the Ca 2+ ionophore ionomycin to increase [Ca2+]i. All these protocols increase [Ca2+]i and activate identical currents, which is consistent with an increase in [Ca2+]i representing the trigger for activation of these currents. Figure 1 shows typical current traces in a cell loaded with 500 nM Ca 2+ via the patch pipette. All protocols, as described above, activate strongly outwardly rectifying currents, with current densities ranging from 15 to 40 pA/pF at +100 mV. They inactivate rapidly at negative potentials and contain a slowly activating component at positive potentials (Fig. 1A-C). Outward tail currents are slowly decaying, whereas inward tail currents decay much faster (Fig. 1D). Instantaneous current-voltage relationships, as measured from the amplitude of the tail currents after stepping back to negative potentials, are linear (Fig. 1D) (Nilius et al., 1997b,c). The time course of activation at positive potentials, between +40 and + 100 mV, and that of deactivation between +80 and - 140 mV are monoexponential. The time constant
329
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FIGURE 1 Activation of C1Cain CPAE cells. (A) Intracellular [Ca2+]iwas elevatedby breaking into a CPAE cell (indicated by the arrow) with a pipette solution of 500 nM Ca2+ (bufferedwith 5 mM EGTA). (B) Current-voltage relationship immediately after whole cell access (a) and after reaching a stationary [Ca2+]ilevel (b). l-Vcurves were obtained from voltageramps. Note the accentuatedoutward rectificationand the reversalpotential at -28 mV (Ec1 = -32mV, EK = -82mV). (C) Current traces recorded at a stationary [Ca2+]iobtainedfrom voltage steps ranging from - 150 to + 150 mV (increment +25 mV). Note the slow activationat positivepotentials and deactivationat negativepotentials.Holding potential Vtt is -50 mV. (D) Tail currents during voltage steps ranging from +80 to -140 mV (decrement 20 mV) following a prepulse from -50 to + 100 mV. (E) I-V relationshipobtained from the initial amplitude of the tail currents,
of activation is clearly Ca 2+ dependent, and becomes smaller at higher [Ca2+]i. This time constant also decreases with stronger depolarization at a constant intracellular Ca 2+ concentration. The time constant of deactivation, in contrast to that o f activation, is independent o f [Ca2+]i. It is, however, clearly voltage dependent, deactivation being much faster at more negative potentials (Fig. 2). The reversal potential of the CaE+-activated current suggests that it is mainly c a r d e d by C I - . Changes in the exlracellular C1- concentration induced a Nernstian shift o f the reversal potential, which is consistent with a rather C1--selective channel. Substituting C1- by I - , F - , or gluconate also induced shifts o f the reversal potential, from which a permeation sequence Pi:Pcl:PF:Pgluconate= 1.7:1:0.7:0.4 was calculated. This permeation pattern is similar to that in Xenopus oocytes
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FIGURE 2 Kinetic properties of CICa in CPAE cells. (A) Time constants of activation (left) and deactivation (right) obtained from monoexponential fits at various voltages and [Ca2+]i. Note the decreased time constant of activation at more positive potentials and at higher [Ca2+] i. (B) Deactivation time constants from traces during voltage protocols similar to that of Fig. 1D at different [Ca2+] i. Deactivation is clearly faster at more negative potentials, but is largely independent of [Ca2+] i.
(Qu and Hartzell, 2000). In these studies, C1- is bound to a site in the channel with a Kd value of 73 raM, which is smaller than that in endothelial cells of approximately 140 mM, as estimated from single-channel data. The pore diameter, as estimated from the Xenopus experiments (Qu and Hartzell, 2000), was approximately 0.72 nm. These values are very close to the pore diameter of volume-regulated anion channels (VRAC, 1.1 nm), which show an identical permeation pattern (Nilius et al., 1999). The different modes of activation have also been used to characterize the Ca 2+activated C1- current at the single-channel level. Application of ionomycin, which elevates [Ca2+]i transiently, induced channel activity that was identified as C1Ca (Fig. 3). The single-channel conductance is approximately 7-8 pS at 300 mM extracellular C1-. Similar single-channel activity could be evoked by stimulating endothelial cells with a vasoactive agonist (Fig. 4). At high extracellular C1- concentrations, single-channel conductance is 7 pS (Figs. 4 and 5A and B) but only about 3 pS at a "physiological" CI- concentration (140 raM, Fig. 4C).
15. Ca2+-Activated C1- Channels in Endothelial Cells
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The open probability of the channel in cell-attached patches is high at positive but small at negative potentials (Fig. 5C and D). Exposing the cytoplasmic side of the membrane to various concentrations of [Ca2+]i also activated this channel in excised inside-out patches. Finally, single-channel activity could be measured in an outside-out patch configuration, if the current was activated before excision in whole cell mode by dialyzing the cell via the pipette with elevated [Ca2+]i. Single-channel current activity in the cell-attached mode was also decreased by membrane-permeable inhibitors, as will be discussed later.
i11. M E C H A N I S M OF ACTIVATION Obviously, changes in [Ca2+]i per se rather than receptor activation activate C1Ca. The current can be clearly correlated with the concomitant changes in [Ca2+]i during stimulation of endothelial cells (CPAE) with an agonist (10/zM ATP, Fig. 6A and B) or during the slow loading of endothelial cells with calcium via the patch pipette (Fig. 6D). Figure 6B shows current traces recorded during agonist application and Fig. 6D during loading the cell with the indicated [C~+]i concentrations. It is obvious that [Ca2+]i affects the time course of current
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activation. The steady-state current-[Ca2+] i relationship was fitted with the Hill equation
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15. CaZ+-Activated CI- Channels in Endothelial Cells
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voltage-dependent b i n d i n g of Ca 2+ to a site in the c h a n n e l that has a higher affinity for Ca 2+ at positive potentials. The Hill coefficient n n increased also from 1.2 at - 8 0 m V to 2.1 at + 1 0 0 inV. The value of n n > 1 suggests b i n d i n g of more than one Ca 2+ ion to the channel. The voltage dependence o f / ( c a can be described by (Woodhull, 1973)
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0.0 0.5 1.0 FIGURE 6 [Ca2+]i-current relationship during agonist stimulation and direct Ca e+ loading. (A) Calcium transient evoked by administration of 10/zM ATP to the bath. (B) Currents measured at + 100 and - 8 0 mV during the transient changes in [Ca2+]i following the administration of ATP to the bath. Current amplitudes were obtained from voltage ramps applied every 5 s. (C) Current densities at +100 and - 8 0 mV were plotted as a function of the corresponding [Ca2+]i values and fitted with Equation (2). Parameters of the fits are included, nH is 1.8 and 1.0 at +100 and - 8 0 mV, respectively. (D) Current traces during voltage step protocols in cells loaded via the patch pipette with a Ca 2+ concentration buffered at 100, 500, or 1000 aM. (E) Current densities at +100 mV and - 8 0 mV as a function of pipette Ca 2+ concentration, [Ca2+] i. Data were fitted by using Equation (2) (nil values are 2.1 and 1.2 at +100 and - 8 0 mV, respectively),
0.0
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with a Kca (0), the dissociation constant at 0 mV, of 430 nM and a value of 0.12 for 8, the fraction of the voltage sensed by the Ca2+-binding site. The Hill coefficient, nrt, was larger than I at all potentials, and increased at more positive potentials. Similar properties have been described for the Ca2+-activated C1- channel in epithelial acinar cells from parotis gland (Arreola et aL, 1996), and they might therefore reflect a more general kinetic fingerprint of Ca 2+ -activated C1- channels. Steady-state and kinetic behavior of Icl(ca) has been described with a model that assumes activation of the channel by two identical, independent, sequential Ca2+-binding steps preceding a final Ca2+-independent transition from the closed to the open state of the channel. The voltage dependence of the Hill coefficient
15. CaZ+-ActivatedC1- Channels in Endothelial Cells
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predicts a model that requires binding of more than one Ca 2+ ion to activate the channel. A minimal model for activation assumes sequential binding of Ca 2+ to two independent identical sites, whereby the last binding step describes the last transition before opening of the channel: closedl ,
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In this model ct t and/~ 1represent voltage- and Ca2+-dependent rate coefficients, o/2 and/~2 might be only voltage dependent. This model has been used to describe epithelial CICa channels (Arreola et al., 1996) that are very similar to the endothelial channels. A putative binding site for Ca 2+ is approximately 10-15% within the membrane electric field from the cytoplasmic side (Arreola et al., 1996; Nilius et al., 1997c).
IV. PHARMACOLOGY The classic stilbene chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), the antiestrogen tamoxifen, and NPPB 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) are all potent blockers of the endothelial Ca 2+activated C1- current. Inhibition by DIDS was clearly voltage dependent, as inward currents were only modestly affected compared to outward currents. Tamoxifen has been reported to be a potent inhibitor of volume-activated C1currents, which is ineffective on the CaE+-activated C1- current in epithelial cells (Valverde et al., 1993). In contrast, tamoxifen (10/zM) induced a fast, complete, and reversible inhibition of the Ca2+-activated current in endothelial cells, and even partially blocked the background current that is present before loading the cell with Ca 2+. NPPB (100/zM) also caused a complete inhibition of CICa. Both compounds induced in contrast to DIDS a voltage-independent block, and affected inward as well outward currents. Niflumic acid, a potent and reversible blocker of CaE+-activated C1- channels in Xenopus oocytes (White and Aylwin, 1990), exerts more complex actions in endothelial cells: at 100/zM it induced a fast, complete, and reversible block of the Ca2+-activated current, but the current after washout of the drug was often larger than the preceding control current. The block is strongly voltage dependent, as inward currents are much less affected than outward currents. N-Phenylanthracilic acid (NPA, 200/zM) reduced the endothelial Ca2+-activated C1- current at +100 mV by 66%. The pharmacological profile for these typical chloride channel blockers is characterized by the following sequence of sensitivity: tamoxifen > niflumic acid > NPPB > NPA ~ DIDS. Inhibition of C1Ca by DIDS, NPA, Zn 2÷, and niflumic acid has also been reported in other endothelial cell types (Groschner et al., 1994; Yumoto et al., 1994).
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Surprisingly, the antimalarial drug mefloquine and the antidepressant fluoxefine (Prozac), which is considered as a selective 5-hydroxytryptamine reuptake inhibitor, blocked C1Ca with ICs0 values of 3 (Maertens et al., 2000) and 10/zM (Maertens et aL, 1999), respectively. GTPFS, which activates Ca2+-activated C1- currents in some epithelial cells (Kibble et al., 1996) and VRAC in endothelial cells (Nilius et aL, 1994a, 1997a; Voets et al., 1998), does not activate the endothelial Iclcca). An increase in extracellular pH significantly decreased Icl,ca, whereas a reduction of pile from 7.3 to 6 did not affect Io~ca) (Nilius, unpublished).
V. ACTIVATION BY CALMODULIN-DEPENDENT PROTEIN KINASE It has also been proposed that Ca2+-activated C1- channels in epithelial cells might be regulated by the calmodulin-dependent protein kinase II (McGill et aL, 1995). This might reflect a requirement of intracellular ATP for C1Ca activation (Watanabe et aL, 1994). The calmodulin-antagonist trifluoperazine (TFP)reduced Icl(Ca) but did not affect its kinetic or rectification properties. Half-maximal inhibition occurred at a concentration of 5.7/zM. TFP often induced irreversible leakage currents at concentrations higher than 10/zM. Calmidazolium, another calmodulin antagonist, inhibited the current to the same extent as TFP. Inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] has been shown to be a messenger that modulates C1Ca channels (Ho et al., 1997) that acts at least in some cases via Ca2+-calmodulin-dependent protein kinase II (Ismailov et aL, 1996; Xie et al., 1996). It has also been shown that Ins(3,4,5,6)P4 inhibits CaE+-stimulated C1- secretion in several epithelial cells via a block of Ca2+-activated C1- channels, but none of the following alternative isomers inhibited C1- channels: Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, and Ins(1,3,4,5,6)P5 [the latter being the immediate precursor of Ins(3,4,5,6)P4](Ismailov et al., 1996; Xie et aL, 1996; Ho et al., 1997). In CPAE cells, Ins(1,4,5,6)P4 and Ins(3,4,5,6)P4 inhibited Iatca), activated by intracellular loading of the cells with Ca 2+, without significant changes in its kinetic properties. This inhibition is rather specific because half-maximal blocks appear to be between 2 and 4/zM, which is comparable with data obtained from T84colonic epithelial cells for a calmodulin-dependent protein kinase H-activated C1- conductance (Xie et al., 1996). The observed block of endothelial lcl(Ca)by the two tetrakisphosphates is at variance with the data from epithelial cells (Xie et al., 1996). However, the calmodulin-dependent protein kinase H-activated C1- current (IcI(PKII)) is clearly different from the endothelial small conductance Ca2+-activated C1- current, la~ca): (1) EC CICa is a small conductance channel, whereas ICI(PKII) is probably a 25-30 pS channel (Cunningham et al., 1995). (2) Icl~ca) is strongly outwardly rectifying, Icl(pr,n) is only weakly (Arreola et al., 1996; Xie et al., 1996;
15. Ca2+-ActivatedC1- Channels in Endothelial Cells
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Nilius et al., 1997b,c). (3) lcl(ca) is slowly activated at positive potentials, rapidly inactivated at negative potentials, and shows long tails currents (Arreola et al., 1996; Nilius et al., 1997b,c). lcl0,raI) is rather voltage independent (Xie et al., 1996). These channels are therefore probably different and might have a different sensitivity to the various tetrakisphosphates. However, Ins(3,4,5,6)P4 also inhibited the slowly activating and strongly outwardly rectifying Ca2+-activated C1- current (Ho et al., 1997; Carew et al., 2000). Neither Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, nor the pentakisphosphate Ins(1,3,4,5,6)P5 was effective (Nilius et al., 1998). These data are in contrast with endothelial CICa.
VI. MOLECULAR NATURE OF CICa IN ENDOTHELIUM The molecular nature of the CICa channel is not yet resolved. Putative candidates are the recently cloned and related membrane proteins, including the endothelial adhesion protein Lu-ECAM, the bovine bCLCA 1, murine mCLCA1, and human hCLCA1, 2, and 3 proteins. Currents, showing some similarity with CICa, have been observed in HEK cells expressing these proteins. These Ca2+-sensitive C1- currents, activated by extremely high, nonphysiological concentrations of [Ca2+]i, are outwardly rectifying and inhibited by DIDS, dithiothreitol, and niflumic acid. Cell-attached patch recordings of transfected cells reveal single channels with a slope conductance of 13.4 pS. These findings suggest that members of the CLCA family represent a Ca2+-activated C1- conductance. Proteins of this family are characterized by a precursor of approximately 130 kDa consisting of between 900 and 940 AA residues. This precursor is cleaved to form heterodimers of approximately 90 and 35 kDa. The most likely topology is 5 TM with an extracellular glycosylated N-terminus, containing a number of conserved cysteine residues and an intracellular C-terminus. The above-mentioned cleavage site is located in the intracellular loop between TM3 and TM4. The proteins contain several consensus sites for PKC phosphorylation (Gandhi et al., 1998; Gruber et aL, 1998, 1999; Gruber and Pauli, 1999; Fuller, 2000; Fuller and Benos, 2000a,b). Reverse transcription polymerase chain reaction (RT-PCR) shows a high expression of mCLCA1 in mouse aorta EC. However, so far we failed to record C1Ca-like currents in cells expressing mCLCA1 and bCLCA1 (Nilius etal., 1997c; Papassotiriou et al., 2001).
VII. DISCUSSION The Ca2+-activated C1- current, ICI,Ca, has been described in a variety of excitable and nonexcitable cells. Functional significance of this current includes the setting of the resting potential, control of excitation via generation of afterpotentials, shaping
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the action potential, modulation of oscillatory changes in intracellular calcium, [Ca2+]i, and of agonist responses (Korn et al., 1991; K16ckner, 1993; Collier et al., 1996). In nonexcitable cells, such as epithelial cells, Icl(ca) is primarily involved in the control of salt and fluid secretion, the maintenance of the pH balance, as well as osmoregulation and volume regulation, setting the driving force for Ca 2+ influx, and it might be directly linked to vectorial transport via a "push-pull mechanism" (Matthews et aL, 1989; Frizzell and Halm, 1990; Kasai and Augustine, 1990). Importantly, Ca2+-activated C1- channels in epithelial cells might substitute for defective epithelial cAMP-activated CFTR- C1- channels (Rugolo et al., 1993; Fuller, 2000; Fuller and Benos, 2000b). Ca2+-activated C1- channels have been described in several endothelial cell types (Nilius, 1991; Revest and Abbott, 1992; Groschner et al., 1994; Hosoki and fijima, 1994; Watanabe et al., 1994; Yumoto et al., 1994; Nilius etal., 1997b,c,d). Typically, these channels show a small conductance, are strongly outwardly rectifying, are activated in a voltage-dependent way, and inactivate at negative potentials. Clearly, the kinetic properties are influenced by the degree of the intracellular Ca 2+ elevation. At higher concentrations activation is fastened. Interestingly, Ca2+-activated Ca 2+ channels in Xenopus oocytes show a bimodal kinetic behavior dependent on [Ca2+]i: at concentrations lower than 1/zM, the activation pattern in Xenopus C1Ca is similar to that of endothelial channels, whereas the current did no longer rectify and was largely time independent at concentrations higher than 1 #M (Kuruma and Hartzell, 2000). This bimodal behavior was not observed for the endothelial channel. Single-channel analysis provides the first evidence for an endothelial small conductance C1- channel that is efficiently activated by Ca 2+. This channel is very similar to a Ca2+-activated C1- channel in hepatic cells (Koumi et al., 1994): both have a similar single-channel conductance and are voltage dependently blocked by DIDS and their open channel probability is voltage dependent. This may hint to a family of C1- channels that is both voltage and Ca 2+ sensitive. The permeation sequence of C1Ca is Eisenmann type 1 with eI > eBr > PC1 > PF > eglucontae,a sequence that is consistent with a relatively wide pore diameter in the range of 0.7 nm (Nilius et al., 1999; Qu and Hartzell,
2000). The single-channel conductance of Ca2+-activated CI- channels reported in the literature ranges from ~ 1 to ~380 pS (Groschner and Kukovetz, 1992; Nilius et al., 1994b; Valverde et al., 1995; Collier et al., 1996). Some of these CI- channels are modulated by Ca 2+, but are not completely activated by high concentrations of [Ca2+]i (Matthews et al., 1989), suggesting that [Ca2+]i modulates rather than directly activates them. A ~380-pS C1- channel activated by [Ca2+]i and inhibited by PKC has been described (Groschner et aL, 1992), which can be blocked by DIDS and Zn 2+, and maybe similar to the high-conductance (--,400 pS) PKC- and PKA-modulated C1- channels described in bovine aortic endothelial cells (Vaca and Kunze, 1993).
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A characteristic property of Icl~ca)is the strong outward rectification of the whole cell current, which is due to a drastic increase of the open probability of the channel if the potential is stepped from negative to positive potentials. Endothelial chloride channels, including the Ca2+-activated channel, might be functionally important in endothelium. Speculations on their functional role include the following (Revest and Abbott, 1992; Nilius et al., 1996, 1997a,b, 1999; Nilius and Droogmans, 1995, 2001): 1. Control of cell membrane potential. The slow activation of these channels as well as their strong outward rectification may suggest that their significance under physiological conditions should be small. Taking into account the rather high density of these channels, they might effectively shift the membrane potential of stimulated endothelial cells toward the C1- equilibrium potential, and possibly induce a negative feedback during agonist stimulation. 2. Modulation of agonist-induced or store-depletion-dependent intracellular [Ca2+]i signals, including the regulation of the Ca 2+ influx by controlling the membrane potential (Hosoki and Iijima, 1994, 1995; Yumoto et al., 1995). Their role in electrogenesis and membrane potential oscillations (Korn et al., 1991; Hazama et al., 1996) implies a functional role in the control of the driving force for Ca 2+ entry, as Ca 2+ oscillations were interrupted in mouse pituitary cells by blockers of ICI(Ca), 3. Control of volume and cell shape (Ueda et al., 1990, 1993; Nilius et a l., 1996, 1997a, 1999). 4. Role in cell proliferation (Voets et al., 1995; Nilius et al., 1996, 1997a). In T-lymphocytes as well as in endothelial cells, blockers of a small-conductance C1channel inhibited cell proliferation, which may suggest a physiological function of these channels in the control of mitogenic activity (Schumacber et aL, 1995). Icl~ca) might be involved in this control as well. 5. Role in vectorial transport. CaZ+-activated C1- channels have been described as essential tools to stimulate secretion in epithelial cells (Kasai and Augustine, 1990). It has also been shown that block of lcl~Ca)results in a dramatic inhibition of secretion in tumor cells (Heisler, 1991). In endothelial cells, little is known about the regulation of such transport activity, but it can be speculated that C1channels play a similar role for transcellular traffic as in epithelial cells. Another important role of CaZ+-activated C1- channels has been discussed recently: they might be upregulated under conditions of cystic fibrosis and may substitute for nonfunctional cystic fibrosis transmembrane regulator (CFTR) channels in cystic fibrosis (Rugolo et al., 1993; Fuller, 2000; Fuller et al., 2000a,b). The molecular identification of C1Ca, combined with advanced functional studies mad the advent of transgenic animals to study C1Ca function in endothelium, will solve this enigma of the functional role of small-conductance CaZ+-activated C1- channels in endothelium.
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VIIi. SUMMARY
Various procedures that increase [Ca2+]i, such as stimulation with vasoactive agonists (acetylcholine, histamine, bradykinin, ATP, UTP), but also ionomycin or loading the cells with Ca 2+ via the patch pipette, activate Ca2+-activated C1currents (Icl(Ca)) in a variety of vascular endothelial cells. This current is strongly outwardly rectifying and has a reversal potential close to the C1- equilibrium potential. Current kinetics at positive potentials is characterized by a slowly activating component and a rapid deactivation at negative potentials. Activation is faster at more positive membrane potentials and higher intracellular Ca 2+ concentrations ([Ca2+]i). Deactivation is Ca 2+ independent and faster at more negative potentials. Outward tail currents are slowly decaying, whereas inward tail currents decay much faster. Steady-state currents show strong outward rectification, but the instantaneous current-voltage relationship is nearly ohmic. The halide permeability sequence of the Ca2+-activated conductance is Eisenmann I with PI > Pc1 > PF > Pgluconat~The single channel conductance is approximately 7 pS at 300 mM extracellular C1- and less than 3 pS at 140 mM C1-. The open probability of the channel is high at positive potentials, but very small at negative potentials. DIDS and niflumic acid inhibit Icl(ca) in a voltage-dependent manner, i.e., they exert a more potent block at positive potentials. The block by NPA, NPPB, and tamoxifen is voltage independent. Niflumic acid and tamoxifen are the most potent blockers. The calmodulin antagonists trifluoperazine (TFP) and calmidazolium inhibit lcl(ca). The current is inhibited by intracellularly applied Ins(1,4,5,6)P4 and Ins(3,4,5,6)P4 with a concentration for half-maximal inhibition of approximately 10/zM. Inhibition by tetrakisphosphates occurred without significant changes in kinetic properties. Gating can be described by a two-step binding of Ca 2+ on a high-affinity site inside the channel. [Ca2+]i for half-maximal activation of Icl(ca) is voltage dependent, and suggests that the apparent binding constant for Ca 2+ decreases with depolarization. Its value at 0 mV is 430 nM, and the putative binding site is 12% within the electrical field from the cytoplasmic side. The Hill coefficient, nn, of binding is larger than 1 and increases with depolarization. The maximal C1- conductance at saturating [Ca2+]i does not depend on membrane potential. Ca2+-activated C1- currents coexist in vascular endothelial cells with at least two other C1- channels, i.e., the volume-regulated anion channels VRAC and CFTR. Their molecular identity as well as their functional role are still uncertain. Likely, coactivation with agonist-induced Ca 2+ release and Ca 2+ entry points to a possible role in regulation of Ca 2+ homeostasis in endothelial cells. In addition, they might be involved in the regulation of more complex cell functions in endothelial cells, such as transcellular transport, exocytosis, and cell proliferation.
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References Arreola, J., Melvin, J. E., and Begenisich, T. (1996). Activation of calcium-dependent chloride channels in rat parotid acinar cells. J, Gen. Physiol. 108, 35-47. Carew, M. A., Yang, X., Schultz, C., and Shears, S. B. (2000). Myo-inositol 3,4,5,6-tetrakisphosphate inhibits an apical calcinm-activated chloride conductance in polarized monolayers of a cystic fibrosis cell line. J. Biol. Chem. 275, 26906-26913. Collier,, M. L., Levesque, E C., Kenyon, J. L., and Hume, J. R. (1996). Unitary C1- channels activated by cytoplasmic Ca2+ in canine ventricular myocytes. Circ. Res. 78, 936-944. Cunningham, S. A., Awayda, M. S., Bubien, J. K., Ismailov, II, Arrate, M. E, 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. Dant, J., Standen, N. B., and Nelson, M. T. (1994). The role of the membrane potential of endothelial and smooth muscle cells in the regulation of coronary blood flow. J. Cardiovasc. Electrophysiol. 5, 154-181. Frizzell, R. A., and Halm, D. R. (1990). Chloride channels in epithelial cells. Curr. Top. Membr. Transport 37, 247-282. Fuller, C. M. (2000). Molecular and functional diversity of epithelial chloride channels. Clin. Exp. Pharmacol. Physiol. 27, 891. • Fuller, C. M., and Benos, D. J. (2000a). Ca2+-actavated C1- channels: A newly emerging anion transport family. News Physiol. Sci. 15, 165-171. Fuller, C. M., and Benos, D. J. (2000b). Elec~ophysiological characteristics of the Ca2+-activated C1channel family of anion transport proteins. Clin. Exp. Pharmacol. Physiol. 27, 906-910. 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. Groschner, K., and Kukovetz, W. R. (1992). Voltage-sensitive chloride channels of large conductance in the membrane of pig aortic endothelial cells. Pfliigers Arch. 421, 209-217. Groschner, K., Graier, W. E, and Kukovetz, W. R. (1992). Activation of a small-conductance Ca2+dependent K + channel contributes to bradykinin-induced stimulation of nitric oxide synthesis in fig aortic endothelial cells. Biochim. Biophys. Acta 1137, 162-170. Groschner, K., Graier, W. E, and Kukovetz, W. R. (1994). Histamine induces K +, Ca 2+, and C1currents in human vascular endothelial cells--role of ionic currents in stimulation of nitric oxide biosynthesis. C/rc. Res. 75, 304-314. Gmber, A. D., and Pauli, B. U. (1999). Molecular cloning and biochemical characterization of a truncated, secreted member of the human family of Ca2+-activated C1- channels. Biochim. Biophys. Acta 1444, 418-423. 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 54, 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, C1261-1270. Hazama, H., Nakajima, T., Hamada, E., Omata, M., and Kurachi, Y. (1996). Neurokinin A and Ca2+ current induce Ca2+-activated C1- currents in guinea-pig tracheal myocytes. J. Physiol. 492, 377-393. Heisler, S. (1991). Chloride channel blockers inhibit ACTH secretion from mouse pituitary tumor cells. Am. J. Physiol. 260, E505-E512. Himrnel, H. M., Rasmusson, R. L., and Strauss, H. C. (1994). Agonist-induced changes of [Ca2+]i and membrane currents in single bovine aortic endothelial cells. Am. J. Physiol. 267, C13381350.
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Ho, M. W., Shears, S. B., Bruzik, K. S., Duszyk, M., and French, A. S. (1997). Ins(3,4,5,6)P4 specifically inhibits a receptor-mediated Ca2+-dependent C1- current in CFPAC-1 cells. Am. J. Physiol. 272, C1160-1168. Hosoki, E., and Iijima, T. (1994). Chloride-sensitive Ca2+ entry by histamine and ATP in human aortic endothelial cells. Eur. J. Pharmacol. 266, 213-218. Hosoki, E., and Iijima, T. (1995). Modulation of cytosolic Ca2+ concentration by thapsigargin and cyclopiazonic acid in human aortic endothelial cells. Eur. J. Pharmacol. 288, 131-137. Ismailov, I. I., Fuller, C. M., Berdiev, B. K., Shlyonsky, V. G., Benos, D. J., and Barrett, K. E. (1996). A biologic function for an "orphan" messenger: D-myo-inositol 3,4,5,6-tetrakisphosphate selectively blocks epithelial calcium-activated chloride channels. Proc. Natl. Acad. Sci. USA 93, 10505-10509. Kasai, H., and Augustine, G. E (1990). Cytosolic Ca2+-gradients triggering unidirectional fluid secretion from exocrine pancreas. Nature 348, 735-738. Kibble, J. D., Greenwood, S. L., Clarson, L. H., and Sibley, C. E (1996). A CaE+-activated whole-cell C1- conductance in human placental cytotrophoblast cells activated via a G protein. J. Membr. Biol. 151, 131-138. Kitckner, U. (1993). Intracellular calcium ions activate a low-conductance chloride channel in smoothmuscle cells isolated from human mesenteric artery. Pfliigers Arch. 424, 231-237. Korn, S. J., Bolden, A., and Horn, R. (1991). Control of action potentials and Ca2+ influx by Ca2+dependent chloride current in mouse pituitary cells. J. Physio1439, 423-437. Koumi, S. I., 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, C. (2000). Bimodal control of a Ca2÷ activated C1- current by different Ca2+ signals. J. Gen. Physiol. 115, 59-80. Maertens, C., Wei, L., Voets, T., Droogmans, G., and Nilius, B. (1999). Block by fluoxetine of volumeregulated anion channels. Br. J. Pharmacol. 126, 508-514. Maertens, C., Wei, L., Droogmans, G., and Nilius, B. (2000). Inhibition of volume-regulated and calcium-activated chloride channels by the antimalarial mefloqnine. J. Pharmacol. Exp. Ther. 295, 29-36. Matthews, G., Neher, E., and Penner, R. (1989). Chloride conductance activated by external agonists and internal messengers in rat peritoneal mast cells. J. Physiol. 418, 131-144. McGill, J. M., Yen, M. S., Basavappa, S., Mangel, A. W., and Kwiatkowski, A. P. (1995). ATPactivated chloride permeability in biliary epithelial cells is regulated by calmodulin-dependent protein kinase II. Biochem. Biophys. Res. Commun. 208, 457-462. Nilius, B. (1991). Regulation of transmembrane calcium fluxes in endothelium. News Physiol. Sci. 6, 110-114. Nilius, B., and Droogmans, G. (1995). Ion channels of Endothelial cells. In "Physiology and Pathophysiology of the Heart" (N. Sperelakis, Ed.), pp. 961-973. Kluwer Academic Publishers, New York. Nilius, B., and Droogmans, G. (2001). Functional role of ionic channels in vascular endothelium. Physiol. Rev. 81, 1415-1459. Nilius, B., Oike, M., Zahradnik, I., and Droogmans, G. (1994a). Activation of a C1- current by hypotonic volume increase in human endothelial cells. J. Gen. Physiol. 103, 787-805. Nilius, B., Sehrer, J., and Droogmans, G. (1994b). Permeation properties and modulation of volumeactivated C1- currents in human endothelial cells. Br. J. Pharmacol. 112, 1049-1056. Nilius, B., Eggermont, J., Voets, T., and Droogmans, G. (1996). Volume-activated C1--channels. Gen. Pharmacol. 27, 67-77. Nilius, B., Eggermont, J., Voets, T., Buyse, G., Manolopoulos, V. G., and Droogmans, G. (1997a). Properties of volume-regulated anion channels in mammalian cells. Prog. Biophys. Mol. Biol. 68, 69-119.
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Nilius, B., Prenen, J., Sziics, G., Wei, L., Tanzi, E, Voets, T., and Droogmans, G. (1997b). Calciumactivated chloride channels in bovine pulmonary artery endothelial cells. J. Physiol. 498, 381396. Nilius, B., Prenen, J., Voets, T., Vandenbremt, K., Eggermont, J., and Droogmans, G. (1997c). Kinetic and pharmacological properties of the calcium activated chloride current in macrovascular endothelial cells. Cell Calcium 22, 53-63. Nilius, B., Sziics, G., Heinke, S., Voets, T., and Droogmans, G. (1997d). Multiple types of chloride channels in bovine pulmonary artery endothelial cells. J. Vasc. Res. 34, 220-228. Nilius, B., Prenen, J., Voets, T., Eggermont, J., Bruzik, K. S., Shears, S. B., and Droogmans, G. (1998). Inhibition by inositoltetrakisphosphates of calcium- and volume-activated C1- currents in macrovascular endothelial cells. Pfliigers Arch. 435, 637-644. Nilius, B., Voets, T., Eggermont, J., and Droogmans, G. (1999). VRAC: A multifunctional volumeregulated anion channel in vascular endothelium. In "Chloride Channels" (R. Kozlowski, Ed.), pp. 47-63. Isis Medical Media Ltd, Oxford. Papassotiriou, J., Eggermont, J., Droogmans, G., and Nilius, B. (2001). Lack of correlation between mCLCA expression and Ca2+ activated C1- currents. Pfliigers Arch. 442, 295-300. Qu, Z., and Hartzell, C. (2000). Anion permeation in Ca2+ activated C1- channels. J. Gen. Physiol. 116, 825-844. Revest, P. A., and Abbott, N. J. (1992). Membrane ion channels of endothelial cells. Trends Pharmacol. Sci. 13, 404-407. Rugolo, M., Mastroeola, T., Whorle, C., Rasola, A., Gruenert, D. C., Romeo, G., and Galietta, L. J. (1993). ATP and A1 adenosine receptor agonists mobilize intracellular calcium and activate K + and C1- currents in normal and cystic fibrosis airway epithelial cells. J. Biol. Chem. 268, 24779-24784. Schumacher, P. A., Sakellaropoulos, G., Phipps, D. J., and Schlichter, L. C. (1995). Small-conductance chloride channels in human peripheral T lymphoeytes. J. Membr Biol. 145, 217-232. Suh, S. H., Vennekens, R., Manolopoulos, V. G., Fmichel, M., Schweig, U., Prenen, J., Flockerzi, V., Droogmans, G., and Nilius, B. (1999). Characterisation of explanted endothelial cells from mouse aorta: Eleetrophysiology and Ca2+ signalling. Pfliigers Arch. 438, 612-620. Ueda, S., Lee, S. L., and Fanburg, B. L. (1990). Chloride efflux in cyclic AMP-induced eonfigurational change of bovine pulmonary artery endothelial cells. Circ. Res. 66, 957-967. Ueda, S., Arima, M., Matsushita, S., and Kuramoto, K. (1993). C1- channel regulation of vascular endothelial cell spreading. Jpn. Circ. J. 4, 1175-1179. Vaea, L., and Kunze, D. L. (1993). cAMP-dependent phosphorylation modulates voltage gating in an endothelial C1- channel. Am. J. Physiol. 264, C370-C375. Valverde, M. A., Mintenig, G. M., and Sepulveda, E V. (1993). Differential effects of tamoxifen and I- on three distinguishable chloride currents activated in T84 intestinal cells. Pfliigers Arch. 425, 55:2-554. Valverde, M. A., Hardy, S. P., and Sepulveda, E V. (1995). Chloride channels: A state of flux. FASEB J. 9, 509-515. Voets, T., Sziics, G., Droogmans, G., and Nilius, B. (1995). Blockers of volume-activated Cl- currents inhibit endothelial cell proliferation. Pfliigers Arch. 431, 132-134. Voets, T., Manolopoulos, V., Eggermont, J., Ellory, C., Droogmans, G., and Nilius, B. (1998). Regulation of a swelling-activated Cl-current in bovine endothelium by protein tyrosine phosphorylation and G-proteins. J. Physiol. 506, 341-352. Watanabe, M., Yumoto, K., and Ochi, R. (1994). Indirect activation by internal calcium of chloride channels in endothelial cells. Jpn. J. Physiol. 44, $233~q236. White, M. M., and Aylwin, M. (1990). Niflumic and flufenamic acids are potent reversible blockers of Ca2+-activated C1- channels in Xenopus oocytes. Mol. Pharmacol. 37, 720-724. Woodhull, A. M. (1973). Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61, 687-708.
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Xie, W., Kaetzel, M. A., Bruzik, K. S., Dedman, J. R., Shears, S. B., and Nelson, D. J. (1996). Inositol 3,4,5,6-tetrakisphosphate inhibits the calmodulin-dependent protein kinase II-activated chloride conductance in T84 colonic epithelial cells. J. Biol. Chem. 271, 14092-14097. Yumoto, K., Watanabe, M., Yamaguchi, H., and Ochi, R. (1994). ATP-induced chloride current and tonic increase of internal Ca 2+ concentration in vascular endothelial cells. Jpn. J. Physiol. 44, $241-$243. • Yumoto, K., Yamaguchi, H., and Ochi, R. (1995). Depression of ATP-induced Ca 2+ signalling by high K + and low C1- media in human aortic endothelial cells. Jpn. J. PhysioL 45, 111-122.
I. II. III. IV. V. VI. VII. VIII.
Introduction Biophysical Properties of C1Ca in Endothelium Mechanism of Activation Pharmacology Activation by Calmodulin-Dependent Protein Kinase Molecular Nature of C1Ca in Endothelium Discussion Summary References
!. INTRODUCTION The permeability of the membrane for K + and CI- is modulated in many cell types by changes in the concentration of free intracellular Ca 2+, [Ca2+]i. Various types of chloride channels have been described in nonexcitable cells, including cAMP-dependent, voltage-dependent C1C channels, swelling- or volumeactivated, and Ca2+-activated C1- channels (Valverde et al., 1995). Endothelial cells express Ca2+-activated K + channels that are functionally important for the control of membrane potential and driving force for Ca2+ influx during agonist or mechanical stimulation (Nilius, 1991; Daut et al., 1994). An increase in [Ca2+]i during stimulation of endothelial cells by vasoactive agonists such as acetylcholine, histamine, bradykinin, thrombin, adenosine triphosphate (ATP), and other Ca2+-releasing agonists affects their membrane potential. This stimulation evokes a pronounced membrane hyperpolarization in several types of endothelial cells (EC) due to activation of Ca2+-dependent K + channels, but in others it causes only Current Topics in Membranes, Volume 53
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small changes in membrane potential. This stabilization of the membrane potential is mainly due to activation of Ca2+-dependent C1- channels (Groschner et al., 1992, 1994; Himmel et al., 1994; Watanabe et al., 1994; Yumoto et al., 1994; Nilius and Droogmans, 2001). In this chapter we describe the salient features of Ca2+-activated C1- channels in vascular endothelial cells, and briefly discuss their possible functional impact on endothelial cell function.
I!. BIOPHYSICAL PROPERTIES OF CICa IN ENDOTHELIUM Ca2+-activated C1- channels (C1Ca) have been described in various types of EC, among which pulmonary artery EC and freshly isolated mouse aorta EC (Nilius et al., 1997b,c; Sub et al., 1999). In most cells, the current through C1Ca overlaps currents through K + channels, such as the "big" conductance Ca2+-activated K + channel, BKca, as in mouse aorta endothelium (MAEC), or the inwardly rectifying K + current through Kir 2.1 channels as in pulmonary aorta endothelium (CPAE). To suppress BKca and Kit 2.1 channels, extra- and intracellular K + is substituted by Cs + and charybdotoxin and tetraethylammonium (TEA) is applied to the extracellular solution. CICa is also often coactivated with volume-regulated C1- channels (VRAC, Nilius et al., 1997d). To prevent activation of these usually large contaminating VRAC currents, the cells wereshrunken by adding 50-100 mM mannitol to the bath solution (Nilius et al., 1997b,d). Various protocols have been used to activate C1Ca. Application of Ca2+-releasing vasoactive agonists, such as acetylcholine, histamine, bradykinin, thrombin, uridine triphosphate (UTP), and ATE activate a current that reverses close to the C1-equilibrium potential and that is closely correlated with the concomitant changes in [Ca2+]i. To bypass the inositol 1,4,5-triphosphate [Ins(1,4,5)P3] cascade cells can be dialyzed via the patch pipette with an ethyleneglycoltetraacetic acid (EGTA)-buffered Ca z+ solution to equilibrate [Ca2+]i at suitable constant values or by using the Ca 2+ ionophore ionomycin to increase [Ca2+]i. All these protocols increase [Ca2+]i and activate identical currents, which is consistent with an increase in [Ca2+]i representing the trigger for activation of these currents. Figure 1 shows typical current traces in a cell loaded with 500 nM Ca 2+ via the patch pipette. All protocols, as described above, activate strongly outwardly rectifying currents, with current densities ranging from 15 to 40 pA/pF at +100 mV. They inactivate rapidly at negative potentials and contain a slowly activating component at positive potentials (Fig. 1A-C). Outward tail currents are slowly decaying, whereas inward tail currents decay much faster (Fig. 1D). Instantaneous current-voltage relationships, as measured from the amplitude of the tail currents after stepping back to negative potentials, are linear (Fig. 1D) (Nilius et al., 1997b,c). The time course of activation at positive potentials, between +40 and + 100 mV, and that of deactivation between +80 and - 140 mV are monoexponential. The time constant
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15. Ca2+-Activated C1- Channels in Endothelial Cells A break
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FIGURE 1 Activation of C1Cain CPAE cells. (A) Intracellular [Ca2+]iwas elevatedby breaking into a CPAE cell (indicated by the arrow) with a pipette solution of 500 nM Ca2+ (bufferedwith 5 mM EGTA). (B) Current-voltage relationship immediately after whole cell access (a) and after reaching a stationary [Ca2+]ilevel (b). l-Vcurves were obtained from voltageramps. Note the accentuatedoutward rectificationand the reversalpotential at -28 mV (Ec1 = -32mV, EK = -82mV). (C) Current traces recorded at a stationary [Ca2+]iobtainedfrom voltage steps ranging from - 150 to + 150 mV (increment +25 mV). Note the slow activationat positivepotentials and deactivationat negativepotentials.Holding potential Vtt is -50 mV. (D) Tail currents during voltage steps ranging from +80 to -140 mV (decrement 20 mV) following a prepulse from -50 to + 100 mV. (E) I-V relationshipobtained from the initial amplitude of the tail currents,
of activation is clearly Ca 2+ dependent, and becomes smaller at higher [Ca2+]i. This time constant also decreases with stronger depolarization at a constant intracellular Ca 2+ concentration. The time constant of deactivation, in contrast to that o f activation, is independent o f [Ca2+]i. It is, however, clearly voltage dependent, deactivation being much faster at more negative potentials (Fig. 2). The reversal potential of the CaE+-activated current suggests that it is mainly c a r d e d by C I - . Changes in the exlracellular C1- concentration induced a Nernstian shift o f the reversal potential, which is consistent with a rather C1--selective channel. Substituting C1- by I - , F - , or gluconate also induced shifts o f the reversal potential, from which a permeation sequence Pi:Pcl:PF:Pgluconate= 1.7:1:0.7:0.4 was calculated. This permeation pattern is similar to that in Xenopus oocytes
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Vtai, [mV]
FIGURE 2 Kinetic properties of CICa in CPAE cells. (A) Time constants of activation (left) and deactivation (right) obtained from monoexponential fits at various voltages and [Ca2+]i. Note the decreased time constant of activation at more positive potentials and at higher [Ca2+] i. (B) Deactivation time constants from traces during voltage protocols similar to that of Fig. 1D at different [Ca2+] i. Deactivation is clearly faster at more negative potentials, but is largely independent of [Ca2+] i.
(Qu and Hartzell, 2000). In these studies, C1- is bound to a site in the channel with a Kd value of 73 raM, which is smaller than that in endothelial cells of approximately 140 mM, as estimated from single-channel data. The pore diameter, as estimated from the Xenopus experiments (Qu and Hartzell, 2000), was approximately 0.72 nm. These values are very close to the pore diameter of volume-regulated anion channels (VRAC, 1.1 nm), which show an identical permeation pattern (Nilius et al., 1999). The different modes of activation have also been used to characterize the Ca 2+activated C1- current at the single-channel level. Application of ionomycin, which elevates [Ca2+]i transiently, induced channel activity that was identified as C1Ca (Fig. 3). The single-channel conductance is approximately 7-8 pS at 300 mM extracellular C1-. Similar single-channel activity could be evoked by stimulating endothelial cells with a vasoactive agonist (Fig. 4). At high extracellular C1- concentrations, single-channel conductance is 7 pS (Figs. 4 and 5A and B) but only about 3 pS at a "physiological" CI- concentration (140 raM, Fig. 4C).
15. Ca2+-Activated C1- Channels in Endothelial Cells
331
ionomycin
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FIGUlilg 3 Activationof single C1Cachannels by applicationof 0.1 mM ionomycin.(A) Fourteen consecutivesweepsfroma cell-attachedpatchafterionomycinapplication(cellsare in an isotonic CsC1 solution, holding potential +120 mV). (B) Ca2+ elevationby ionomycinsimultaneouslymeasured with single-cliannelactivity.(C) Probabilityof the channelbeing openduring [Ca2+]ielevation. (D) Amplitudehistogramfrom data sampledduring ionomycinapplication(open peak at +0.88 pA, estimated single-channelconductance7.4 pS with 300 mM CI- in the patchpipette).
The open probability of the channel in cell-attached patches is high at positive but small at negative potentials (Fig. 5C and D). Exposing the cytoplasmic side of the membrane to various concentrations of [Ca2+]i also activated this channel in excised inside-out patches. Finally, single-channel activity could be measured in an outside-out patch configuration, if the current was activated before excision in whole cell mode by dialyzing the cell via the pipette with elevated [Ca2+]i. Single-channel current activity in the cell-attached mode was also decreased by membrane-permeable inhibitors, as will be discussed later.
i11. M E C H A N I S M OF ACTIVATION Obviously, changes in [Ca2+]i per se rather than receptor activation activate C1Ca. The current can be clearly correlated with the concomitant changes in [Ca2+]i during stimulation of endothelial cells (CPAE) with an agonist (10/zM ATP, Fig. 6A and B) or during the slow loading of endothelial cells with calcium via the patch pipette (Fig. 6D). Figure 6B shows current traces recorded during agonist application and Fig. 6D during loading the cell with the indicated [C~+]i concentrations. It is obvious that [Ca2+]i affects the time course of current
332
Nilius and Droogmans ATP
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FIGURE 4 Activation of single-channel C1Ca by the vasoactive agonist ATE (A) Elevation of [Ca2+]i dndng administration of 10/zM ATP (CPAE cell). (B) Time course of the open probability during [Ca2+] i. (C) Single-channel traces (cell-attached patch, traces are numbered in B, single-channel current amplitude is 0.6 pA as measured from amplitude histogram, 300 mMC1- in the pipette, holding potential is +80 mV). (D) Current in cell-attached patch mode from an endothelial cell incubated in K+-Krebs ' solution before and during application of 0.1/zM ionomycin (holding potential is - 100 mV, corresponding to a driving force of 136 mV for Cl- ions, 140 mM C1- in the pipette). The current was continuously sampled at a rate of 2 kHz and divided in consecutive stretches of 2 s. For each of these sweeps we calculated off-line mean current and current variance. The solid line represents the fit of the data points to equation (r2 = i. I - (12/N). The estimated number of channels in the patch is three and the single-channel current amplitude is 0.5 pA. Taking into account the driving force for C1- we obtained a single-channel conductance of 3.6 pS.
activation. The steady-state current-[Ca2+] i relationship was fitted with the Hill equation
I&(m%
(1)
Ica(ca) = 1 + (Kca/[Ca2+]i)"" where Kca is the Ca z+ concentration for half-maximal activation, I CI,Ca max is the current at saturating Ca 2+ concentrations, and nr] is the Hill coefficient. The concentration for half-maximal activation is clearly voltage dependent, and decreased from approximately 650 nM at - 8 0 mV to 300 nM at +100 mV. This suggests a
15. CaZ+-Activated CI- Channels in Endothelial Cells
333
-50mV
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FIGURE 5 C1Ca single-channel conductance and voltage dependence of open probability. (A) Channel activity recorded in cell-attached patches at three different potentials. Elevation of [Ca2+]i was evoked by bath application of 1/zM ionomycin (pipette solution is 150 mM BaC12). (B) Singlechannel current-voltage relationships. The single-channel conductance and the reversal potential, as calculated from a linear fit, were 7.8 pS and -13.3 mV (Ecl= -16mV), respectively. (C) Amplitude histograms from the same cell at two membrane potentials. Channel activity was evoked by ionomycin (cell-attached patches, pipette solution 150 mM BaCI2, bath K+-Krebs). Single-channel amplitudes and open probabilities were obtained from Gaussian fits to the histograms. (D) Voltage dependence of the open probability. Depolarization of the patch increases the open probability of the Ca2+-activated channel. The solid line represents a fit of the data to the Boltzman equation p = {1 + exp[-(V - V1/z)/s]}-1.
voltage-dependent b i n d i n g of Ca 2+ to a site in the c h a n n e l that has a higher affinity for Ca 2+ at positive potentials. The Hill coefficient n n increased also from 1.2 at - 8 0 m V to 2.1 at + 1 0 0 inV. The value of n n > 1 suggests b i n d i n g of more than one Ca 2+ ion to the channel. The voltage dependence o f / ( c a can be described by (Woodhull, 1973)
{-zFSV\
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1 /
(2)
I
15(
334
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0.0 0.5 1.0 FIGURE 6 [Ca2+]i-current relationship during agonist stimulation and direct Ca e+ loading. (A) Calcium transient evoked by administration of 10/zM ATP to the bath. (B) Currents measured at + 100 and - 8 0 mV during the transient changes in [Ca2+]i following the administration of ATP to the bath. Current amplitudes were obtained from voltage ramps applied every 5 s. (C) Current densities at +100 and - 8 0 mV were plotted as a function of the corresponding [Ca2+]i values and fitted with Equation (2). Parameters of the fits are included, nH is 1.8 and 1.0 at +100 and - 8 0 mV, respectively. (D) Current traces during voltage step protocols in cells loaded via the patch pipette with a Ca 2+ concentration buffered at 100, 500, or 1000 aM. (E) Current densities at +100 mV and - 8 0 mV as a function of pipette Ca 2+ concentration, [Ca2+] i. Data were fitted by using Equation (2) (nil values are 2.1 and 1.2 at +100 and - 8 0 mV, respectively),
0.0
0•5
1.0
1.5
with a Kca (0), the dissociation constant at 0 mV, of 430 nM and a value of 0.12 for 8, the fraction of the voltage sensed by the Ca2+-binding site. The Hill coefficient, nrt, was larger than I at all potentials, and increased at more positive potentials. Similar properties have been described for the Ca2+-activated C1- channel in epithelial acinar cells from parotis gland (Arreola et aL, 1996), and they might therefore reflect a more general kinetic fingerprint of Ca 2+ -activated C1- channels. Steady-state and kinetic behavior of Icl(ca) has been described with a model that assumes activation of the channel by two identical, independent, sequential Ca2+-binding steps preceding a final Ca2+-independent transition from the closed to the open state of the channel. The voltage dependence of the Hill coefficient
15. CaZ+-ActivatedC1- Channels in Endothelial Cells
335
predicts a model that requires binding of more than one Ca 2+ ion to activate the channel. A minimal model for activation assumes sequential binding of Ca 2+ to two independent identical sites, whereby the last binding step describes the last transition before opening of the channel: closedl ,
Ca
fll
closed2ca ,
Ca
fll
closedca , ' open ]~2
In this model ct t and/~ 1represent voltage- and Ca2+-dependent rate coefficients, o/2 and/~2 might be only voltage dependent. This model has been used to describe epithelial CICa channels (Arreola et al., 1996) that are very similar to the endothelial channels. A putative binding site for Ca 2+ is approximately 10-15% within the membrane electric field from the cytoplasmic side (Arreola et al., 1996; Nilius et al., 1997c).
IV. PHARMACOLOGY The classic stilbene chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid (DIDS), the antiestrogen tamoxifen, and NPPB 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) are all potent blockers of the endothelial Ca 2+activated C1- current. Inhibition by DIDS was clearly voltage dependent, as inward currents were only modestly affected compared to outward currents. Tamoxifen has been reported to be a potent inhibitor of volume-activated C1currents, which is ineffective on the CaE+-activated C1- current in epithelial cells (Valverde et al., 1993). In contrast, tamoxifen (10/zM) induced a fast, complete, and reversible inhibition of the Ca2+-activated current in endothelial cells, and even partially blocked the background current that is present before loading the cell with Ca 2+. NPPB (100/zM) also caused a complete inhibition of CICa. Both compounds induced in contrast to DIDS a voltage-independent block, and affected inward as well outward currents. Niflumic acid, a potent and reversible blocker of CaE+-activated C1- channels in Xenopus oocytes (White and Aylwin, 1990), exerts more complex actions in endothelial cells: at 100/zM it induced a fast, complete, and reversible block of the Ca2+-activated current, but the current after washout of the drug was often larger than the preceding control current. The block is strongly voltage dependent, as inward currents are much less affected than outward currents. N-Phenylanthracilic acid (NPA, 200/zM) reduced the endothelial Ca2+-activated C1- current at +100 mV by 66%. The pharmacological profile for these typical chloride channel blockers is characterized by the following sequence of sensitivity: tamoxifen > niflumic acid > NPPB > NPA ~ DIDS. Inhibition of C1Ca by DIDS, NPA, Zn 2÷, and niflumic acid has also been reported in other endothelial cell types (Groschner et al., 1994; Yumoto et al., 1994).
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Nilius and Droogmans
Surprisingly, the antimalarial drug mefloquine and the antidepressant fluoxefine (Prozac), which is considered as a selective 5-hydroxytryptamine reuptake inhibitor, blocked C1Ca with ICs0 values of 3 (Maertens et al., 2000) and 10/zM (Maertens et aL, 1999), respectively. GTPFS, which activates Ca2+-activated C1- currents in some epithelial cells (Kibble et al., 1996) and VRAC in endothelial cells (Nilius et aL, 1994a, 1997a; Voets et al., 1998), does not activate the endothelial Iclcca). An increase in extracellular pH significantly decreased Icl,ca, whereas a reduction of pile from 7.3 to 6 did not affect Io~ca) (Nilius, unpublished).
V. ACTIVATION BY CALMODULIN-DEPENDENT PROTEIN KINASE It has also been proposed that Ca2+-activated C1- channels in epithelial cells might be regulated by the calmodulin-dependent protein kinase II (McGill et aL, 1995). This might reflect a requirement of intracellular ATP for C1Ca activation (Watanabe et aL, 1994). The calmodulin-antagonist trifluoperazine (TFP)reduced Icl(Ca) but did not affect its kinetic or rectification properties. Half-maximal inhibition occurred at a concentration of 5.7/zM. TFP often induced irreversible leakage currents at concentrations higher than 10/zM. Calmidazolium, another calmodulin antagonist, inhibited the current to the same extent as TFP. Inositol 3,4,5,6-tetrakisphosphate [Ins(3,4,5,6)P4] has been shown to be a messenger that modulates C1Ca channels (Ho et al., 1997) that acts at least in some cases via Ca2+-calmodulin-dependent protein kinase II (Ismailov et aL, 1996; Xie et al., 1996). It has also been shown that Ins(3,4,5,6)P4 inhibits CaE+-stimulated C1- secretion in several epithelial cells via a block of Ca2+-activated C1- channels, but none of the following alternative isomers inhibited C1- channels: Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, and Ins(1,3,4,5,6)P5 [the latter being the immediate precursor of Ins(3,4,5,6)P4](Ismailov et al., 1996; Xie et aL, 1996; Ho et al., 1997). In CPAE cells, Ins(1,4,5,6)P4 and Ins(3,4,5,6)P4 inhibited Iatca), activated by intracellular loading of the cells with Ca 2+, without significant changes in its kinetic properties. This inhibition is rather specific because half-maximal blocks appear to be between 2 and 4/zM, which is comparable with data obtained from T84colonic epithelial cells for a calmodulin-dependent protein kinase H-activated C1- conductance (Xie et al., 1996). The observed block of endothelial lcl(Ca)by the two tetrakisphosphates is at variance with the data from epithelial cells (Xie et al., 1996). However, the calmodulin-dependent protein kinase H-activated C1- current (IcI(PKII)) is clearly different from the endothelial small conductance Ca2+-activated C1- current, la~ca): (1) EC CICa is a small conductance channel, whereas ICI(PKII) is probably a 25-30 pS channel (Cunningham et al., 1995). (2) Icl~ca) is strongly outwardly rectifying, Icl(pr,n) is only weakly (Arreola et al., 1996; Xie et al., 1996;
15. Ca2+-ActivatedC1- Channels in Endothelial Cells
337
Nilius et al., 1997b,c). (3) lcl(ca) is slowly activated at positive potentials, rapidly inactivated at negative potentials, and shows long tails currents (Arreola et al., 1996; Nilius et al., 1997b,c). lcl0,raI) is rather voltage independent (Xie et al., 1996). These channels are therefore probably different and might have a different sensitivity to the various tetrakisphosphates. However, Ins(3,4,5,6)P4 also inhibited the slowly activating and strongly outwardly rectifying Ca2+-activated C1- current (Ho et al., 1997; Carew et al., 2000). Neither Ins(1,4,5,6)P4, Ins(1,3,4,5)P4, Ins(1,3,4,6)P4, nor the pentakisphosphate Ins(1,3,4,5,6)P5 was effective (Nilius et al., 1998). These data are in contrast with endothelial CICa.
VI. MOLECULAR NATURE OF CICa IN ENDOTHELIUM The molecular nature of the CICa channel is not yet resolved. Putative candidates are the recently cloned and related membrane proteins, including the endothelial adhesion protein Lu-ECAM, the bovine bCLCA 1, murine mCLCA1, and human hCLCA1, 2, and 3 proteins. Currents, showing some similarity with CICa, have been observed in HEK cells expressing these proteins. These Ca2+-sensitive C1- currents, activated by extremely high, nonphysiological concentrations of [Ca2+]i, are outwardly rectifying and inhibited by DIDS, dithiothreitol, and niflumic acid. Cell-attached patch recordings of transfected cells reveal single channels with a slope conductance of 13.4 pS. These findings suggest that members of the CLCA family represent a Ca2+-activated C1- conductance. Proteins of this family are characterized by a precursor of approximately 130 kDa consisting of between 900 and 940 AA residues. This precursor is cleaved to form heterodimers of approximately 90 and 35 kDa. The most likely topology is 5 TM with an extracellular glycosylated N-terminus, containing a number of conserved cysteine residues and an intracellular C-terminus. The above-mentioned cleavage site is located in the intracellular loop between TM3 and TM4. The proteins contain several consensus sites for PKC phosphorylation (Gandhi et al., 1998; Gruber et aL, 1998, 1999; Gruber and Pauli, 1999; Fuller, 2000; Fuller and Benos, 2000a,b). Reverse transcription polymerase chain reaction (RT-PCR) shows a high expression of mCLCA1 in mouse aorta EC. However, so far we failed to record C1Ca-like currents in cells expressing mCLCA1 and bCLCA1 (Nilius etal., 1997c; Papassotiriou et al., 2001).
VII. DISCUSSION The Ca2+-activated C1- current, ICI,Ca, has been described in a variety of excitable and nonexcitable cells. Functional significance of this current includes the setting of the resting potential, control of excitation via generation of afterpotentials, shaping
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Nilius and Droogmans
the action potential, modulation of oscillatory changes in intracellular calcium, [Ca2+]i, and of agonist responses (Korn et al., 1991; K16ckner, 1993; Collier et al., 1996). In nonexcitable cells, such as epithelial cells, Icl(ca) is primarily involved in the control of salt and fluid secretion, the maintenance of the pH balance, as well as osmoregulation and volume regulation, setting the driving force for Ca 2+ influx, and it might be directly linked to vectorial transport via a "push-pull mechanism" (Matthews et aL, 1989; Frizzell and Halm, 1990; Kasai and Augustine, 1990). Importantly, Ca2+-activated C1- channels in epithelial cells might substitute for defective epithelial cAMP-activated CFTR- C1- channels (Rugolo et al., 1993; Fuller, 2000; Fuller and Benos, 2000b). Ca2+-activated C1- channels have been described in several endothelial cell types (Nilius, 1991; Revest and Abbott, 1992; Groschner et al., 1994; Hosoki and fijima, 1994; Watanabe et al., 1994; Yumoto et al., 1994; Nilius etal., 1997b,c,d). Typically, these channels show a small conductance, are strongly outwardly rectifying, are activated in a voltage-dependent way, and inactivate at negative potentials. Clearly, the kinetic properties are influenced by the degree of the intracellular Ca 2+ elevation. At higher concentrations activation is fastened. Interestingly, Ca2+-activated Ca 2+ channels in Xenopus oocytes show a bimodal kinetic behavior dependent on [Ca2+]i: at concentrations lower than 1/zM, the activation pattern in Xenopus C1Ca is similar to that of endothelial channels, whereas the current did no longer rectify and was largely time independent at concentrations higher than 1 #M (Kuruma and Hartzell, 2000). This bimodal behavior was not observed for the endothelial channel. Single-channel analysis provides the first evidence for an endothelial small conductance C1- channel that is efficiently activated by Ca 2+. This channel is very similar to a Ca2+-activated C1- channel in hepatic cells (Koumi et al., 1994): both have a similar single-channel conductance and are voltage dependently blocked by DIDS and their open channel probability is voltage dependent. This may hint to a family of C1- channels that is both voltage and Ca 2+ sensitive. The permeation sequence of C1Ca is Eisenmann type 1 with eI > eBr > PC1 > PF > eglucontae,a sequence that is consistent with a relatively wide pore diameter in the range of 0.7 nm (Nilius et al., 1999; Qu and Hartzell,
2000). The single-channel conductance of Ca2+-activated CI- channels reported in the literature ranges from ~ 1 to ~380 pS (Groschner and Kukovetz, 1992; Nilius et al., 1994b; Valverde et al., 1995; Collier et al., 1996). Some of these CI- channels are modulated by Ca 2+, but are not completely activated by high concentrations of [Ca2+]i (Matthews et al., 1989), suggesting that [Ca2+]i modulates rather than directly activates them. A ~380-pS C1- channel activated by [Ca2+]i and inhibited by PKC has been described (Groschner et aL, 1992), which can be blocked by DIDS and Zn 2+, and maybe similar to the high-conductance (--,400 pS) PKC- and PKA-modulated C1- channels described in bovine aortic endothelial cells (Vaca and Kunze, 1993).
15. CaZ+-ActivatedC1- Channels in Endothelial Cells
339
A characteristic property of Icl~ca)is the strong outward rectification of the whole cell current, which is due to a drastic increase of the open probability of the channel if the potential is stepped from negative to positive potentials. Endothelial chloride channels, including the Ca2+-activated channel, might be functionally important in endothelium. Speculations on their functional role include the following (Revest and Abbott, 1992; Nilius et al., 1996, 1997a,b, 1999; Nilius and Droogmans, 1995, 2001): 1. Control of cell membrane potential. The slow activation of these channels as well as their strong outward rectification may suggest that their significance under physiological conditions should be small. Taking into account the rather high density of these channels, they might effectively shift the membrane potential of stimulated endothelial cells toward the C1- equilibrium potential, and possibly induce a negative feedback during agonist stimulation. 2. Modulation of agonist-induced or store-depletion-dependent intracellular [Ca2+]i signals, including the regulation of the Ca 2+ influx by controlling the membrane potential (Hosoki and Iijima, 1994, 1995; Yumoto et al., 1995). Their role in electrogenesis and membrane potential oscillations (Korn et al., 1991; Hazama et al., 1996) implies a functional role in the control of the driving force for Ca 2+ entry, as Ca 2+ oscillations were interrupted in mouse pituitary cells by blockers of ICI(Ca), 3. Control of volume and cell shape (Ueda et al., 1990, 1993; Nilius et a l., 1996, 1997a, 1999). 4. Role in cell proliferation (Voets et al., 1995; Nilius et al., 1996, 1997a). In T-lymphocytes as well as in endothelial cells, blockers of a small-conductance C1channel inhibited cell proliferation, which may suggest a physiological function of these channels in the control of mitogenic activity (Schumacber et aL, 1995). Icl~ca) might be involved in this control as well. 5. Role in vectorial transport. CaZ+-activated C1- channels have been described as essential tools to stimulate secretion in epithelial cells (Kasai and Augustine, 1990). It has also been shown that block of lcl~Ca)results in a dramatic inhibition of secretion in tumor cells (Heisler, 1991). In endothelial cells, little is known about the regulation of such transport activity, but it can be speculated that C1channels play a similar role for transcellular traffic as in epithelial cells. Another important role of CaZ+-activated C1- channels has been discussed recently: they might be upregulated under conditions of cystic fibrosis and may substitute for nonfunctional cystic fibrosis transmembrane regulator (CFTR) channels in cystic fibrosis (Rugolo et al., 1993; Fuller, 2000; Fuller et al., 2000a,b). The molecular identification of C1Ca, combined with advanced functional studies mad the advent of transgenic animals to study C1Ca function in endothelium, will solve this enigma of the functional role of small-conductance CaZ+-activated C1- channels in endothelium.
340
Nilius and Droogmans
VIIi. SUMMARY
Various procedures that increase [Ca2+]i, such as stimulation with vasoactive agonists (acetylcholine, histamine, bradykinin, ATP, UTP), but also ionomycin or loading the cells with Ca 2+ via the patch pipette, activate Ca2+-activated C1currents (Icl(Ca)) in a variety of vascular endothelial cells. This current is strongly outwardly rectifying and has a reversal potential close to the C1- equilibrium potential. Current kinetics at positive potentials is characterized by a slowly activating component and a rapid deactivation at negative potentials. Activation is faster at more positive membrane potentials and higher intracellular Ca 2+ concentrations ([Ca2+]i). Deactivation is Ca 2+ independent and faster at more negative potentials. Outward tail currents are slowly decaying, whereas inward tail currents decay much faster. Steady-state currents show strong outward rectification, but the instantaneous current-voltage relationship is nearly ohmic. The halide permeability sequence of the Ca2+-activated conductance is Eisenmann I with PI > Pc1 > PF > Pgluconat~The single channel conductance is approximately 7 pS at 300 mM extracellular C1- and less than 3 pS at 140 mM C1-. The open probability of the channel is high at positive potentials, but very small at negative potentials. DIDS and niflumic acid inhibit Icl(ca) in a voltage-dependent manner, i.e., they exert a more potent block at positive potentials. The block by NPA, NPPB, and tamoxifen is voltage independent. Niflumic acid and tamoxifen are the most potent blockers. The calmodulin antagonists trifluoperazine (TFP) and calmidazolium inhibit lcl(ca). The current is inhibited by intracellularly applied Ins(1,4,5,6)P4 and Ins(3,4,5,6)P4 with a concentration for half-maximal inhibition of approximately 10/zM. Inhibition by tetrakisphosphates occurred without significant changes in kinetic properties. Gating can be described by a two-step binding of Ca 2+ on a high-affinity site inside the channel. [Ca2+]i for half-maximal activation of Icl(ca) is voltage dependent, and suggests that the apparent binding constant for Ca 2+ decreases with depolarization. Its value at 0 mV is 430 nM, and the putative binding site is 12% within the electrical field from the cytoplasmic side. The Hill coefficient, nn, of binding is larger than 1 and increases with depolarization. The maximal C1- conductance at saturating [Ca2+]i does not depend on membrane potential. Ca2+-activated C1- currents coexist in vascular endothelial cells with at least two other C1- channels, i.e., the volume-regulated anion channels VRAC and CFTR. Their molecular identity as well as their functional role are still uncertain. Likely, coactivation with agonist-induced Ca 2+ release and Ca 2+ entry points to a possible role in regulation of Ca 2+ homeostasis in endothelial cells. In addition, they might be involved in the regulation of more complex cell functions in endothelial cells, such as transcellular transport, exocytosis, and cell proliferation.
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