Ionic channel rundown in excised membrane patches

BB

ELSEVIER

Biochimica et Biophysica Acta 1286 (1996) 53-63

Biochi ~mic~a et BiophysicaA~.ta

Ionic channel rundown in excised membrane patches Fr~d6ric Becq 1 Department of Physiology, McGill University, Montreal, Quebec, H3G 1Y6 Canada

Received 13 July 1995; revised 4 January 1996; accepted 12 January 1996

Contents 1. Introduction and general characteristics of rundown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

53

2. Chloride channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel . . . . . . . . . . . 2.2. Other chloride channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

55 55 56

3. Potassium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

57 57 57 58

3.1. ATP-dependent K channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Epithelial K channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Calcium-dependent K channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Calcium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59

5. Other channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

59

6. Mechanisms of ionic channel rundown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

60

7. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

61

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

62

1. Introduction and general characteristics of rundown In 1975, Kostyuk, Krishtal and Pidoplichko [32] developed a technique for p e r f o r m i n g intracellular dialysis on isolated nerve cell bodies. This method, which can be v i e w e d as the precursor of the whole-cell patch-clamp technique, was applied to snail n e u r o n e s [32] and then to sensory n e u r o n e s from rat dorsal root g a n g l i a [33]. The calcium current vanished with time in both animal species.

1 Present address: Laboratoire de Neurobiologie cellulaire, CNRS, 31 chemin J. Aiguier, 13402 Marseille, France. Fax: + 33 91 226333. 0304-4157/96/$32.00 © 1996 Elsevier Science B.V. All rights reserved PH S0304-4157(96)00002-0

A suggestion was m a d e that this effect m a y have been due to the w a s h i n g out of some of the cytoplasmic factors necessary for c a l c i u m channels to function. The calcium current decline did in fact slow d o w n w h e n the cell interior was perfused with cyclic A M P and M g A T P [33], which pointed to the i n v o l v e m e n t of protein kinase A in the channel regulation. The d e v e l o p m e n t of the patch-clamp technique [20] has m a d e it possible to study single ionic c h a n n e l events and macroscopic ionic currents in excitable, m u s c u l a r and epithelial cells. Some types of channels are intriguing because their activity spontaneously decreases in the cell-free configuration. This p h e n o m e n o n is called ' r u n d o w n ' or

54

F. Becq / Biochimica et Biophysica Acta 1286 (1996) 53-63

wash-out and occurs immediately after patch excision in studies on a single channel or during cell dialysis with the whole-cell configuration of the patch-clamp technique [14,41]. The current rundown that occurs during whole-cell recording sessions was previously thought to result from the cellular contents being diluted with the patch-pipette solution. The nystatin whole-cell technique was subsequently developed to prevent this dilution from occurring [28]. It is now generally recognized that rundown is not due to an experimental artefact. Moreover, the working hypothesis adopted in most studies is that rundown mainly reflects the tonic activity of membrane-bound enzymes that normally regulate on-cell channels. Once the membrane patch has been isolated from the cell in the inside-out configuration, the cytoplasmic face of the channel is in contact with the bath solution. Since the composition of the solution in both the bath and the pipette can be easily monitored to determine the levels of the divalent cations (Ca 2+, Mg2+), nucleotides (ATP, ADP, GTP...), enzymes (kinases, phosphatases, proteinases...) and enzyme modulators it contains, the strategies generally used to prevent rundown have consisted of changing the compositions of both solutions. Although ionic channels have various biophysical properties, functions and molecular characteristics, rundown can be said to have some general characteristics. The rundown process is not channel-specific, since it has been observed with various ionic channels including chloride, potassium and calcium channels. Rundown is time-dependent, and generally fast, so that the complete closure of the channels occurs within a few minutes (Fig. 1). The rate of rundown varies considerably, however, from one study to another (see following sections). Rundown has never been reported so far to show any voltage dependence. The loss of channel activity does not result from a lack of mem-

brane integrity (which might have been caused by the patch excision procedure). The evidence for these statements is as follows: (1) the single channel current amplitude remains stable during rundown (no changes occur in the channel unitary conductance); (2) the rundown of ionic currents in excised patches corresponds to the spontaneous drops in either the channel opening probability (Po) or the number of channels present in multi-channel patches or both; (3) within the same patch, the rundown process does not affect the activity of other channels insensitive to excision; moreover, it can be followed by the opening of previously silent channels (see Ref. [44]); (4) since inactivated channels can not be reactivated by simply passing the pipette tip through the water/air interface, the rundown does not result from the formation of closed vesicles; and (5) after rundown, channels can be reactivated in various ways, including those involving phosphorylationdependent processes. The gradual loss of channel activity involves a decrease in either the opening probability or the number of active channels (as observed in most studies). It may alternatively reflect the sudden closure of the channels without any subsequent change in the Po [53]. A two-step model is generally proposed to explain the rundown (see for example Refs. [7,22]) where the initial phase of rundown occurs immediately after patch excision and the best fit is obtained using a fast time constant. During this phase, 60% to 80% of the overall initial channel activity is lost. A second phase then occurs, which can be fitted with a slow time constant. In other systems, during long-lasting whole-cell recordings, rundown occurs in three phases, two of which are characterized by a slow rate of decay and one during which the current declines quickly [6]. Lastly, since the possibility of reversing rundown, and hence of reactivating the channels, has been found to decrease with time [23,57], either the properties or the integrity of the

0.5pAl excised NaCI +

I'

2pA I

I

/ 8s

~-""""~

0.5pA I 820ms Fig. 1. Example of the rundown of cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity recorded from a transfected Chinese hamster ovary (CHO) cell. The channels were first activated on-cell by 15 /xM forskolin and then excised (arrow) in MgATP-free and PKA-free solution. Closed state is indicated by dashed lines. The spontaneous decrease in channel activity is illustrated by the two expanded time and amplitude scale traces.

F. Becq / Biochimica et Biophysica Acta 1286 (1996) 53-63

membrane may be altered with time. Alternatively slow inactivating mechanisms may develop which affect the channel activity. Understanding the mechanisms whereby ionic channel rundown occurs is an important issue, since it may throw some light on how ionic channels are regulated in their natural membrane environment. The aim of this review is to summarize what is known so far about the biochemical processes underlying the rundown of various ionic channels. One of the main ways in which ionic channel rundown and activity are controlled seems to involve endogenous enzymes acting either on the channel itself or on closely associated components.

A

Po

55

i.o

c.a

MgATP-free and PKA-free I

0.6

0.4 0.2

0.0

B

~

,

,

,

. . . . . . . . ,. . . . . . .

,

(lllllll U

1mM MgA'I'P

Po

0.6

0.4

0.2

2. Chloride channels 0.0

2.1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel

C

lmM MgATP + 180riM PKA I

Po 0.6

CFTR is an epithelial ohmic low conductance chloride channel. Its activity is controlled by two convergent processes; the binding (or perhaps hydrolysis) of ATP to two nucleotide binding domains (NBF1 and 2), and the reversible phosphorylation at multiple sites by protein kinase A and C and by phosphatases (see Ref. [21] for a recent review). The channel is responsible for most of the cAMP-dependent chloride current produced by epithelial cells in response to hormonal stimulation [4,18,21]. Channel opening on-cell can be induced using membrane permeant cAMP agonists or kinase activators [4,13,18,22,23,51]. When CFTR channels are excised in the inside-out patch-clamp configuration, the channel activity runs down (Figs. 1 and 2A) within two minutes [4,13,18,23,51 ]. Rundown of CFTR sometimes develops slowly in cell-attached patches [18,21]. The rapid loss of activity depends very little on the presence or absence of ATP (Fig. 2B) in the bath [5,13,23] or on the presence of the non-hydrolysable ATP analogue AMP-PNP (Becq and Hanrahan, unpublished observation); whereas the poorly hydrolysable ATP analogue ATPyS slows down the loss of CFTR activity (Becq and Hanrahan, unpublished data). High or low concentrations of calcium, as well as GTP and various cation species (potassium, sodium, choline) all fail to prevent rundown [13]. These findings show that CFTR rundown is not affected by monovalent cations, GTP and C a 2 +.

Several conclusions can be deduced about ATP from the rundown experiments. First, no membrane-bound kinases are present in the vicinity of CFrR, because MgATP alone failed to prevent rundown, whereas the addition of purified catalytic subunit of protein kinase A (PKA) in the presence of MgATP prevented it. Secondly, hydrolysis of ATP is a pre-requisite for the PKA-mediated phosphorylation of CFTR that prevents rundown. Lastly, since ATP alone or AMP-PNP failed to block C F r R deactivation, it

0.4 0.2 0.0

,

0

.

,

.

,

,

,

200

.

,

400

.

,

•

,

600

time (s) Fig. 2. The rundown of CFTR channel activity in transfected CHO cells is not affected by MgATP alone but prevented by M g A T P + P K A . Time dependence of opening probability (Po) at consecutive 10-s sweeps during cell-attached (c.a) and inside-out (i.o) recordings. Excision was at time noted 0 (arrows). A - C , CFTR channels stimulated on-cell with 15 /zM forskolin and then excised in the presence of either MgATP-free and PKA-free solution (A), or 1 mM MgATP (B) or 1 mM M g A T P + 180 nM catalytic subunit of PKA (C). Channel Po was assessed during a stable period of activity lasting 600 s.

seems unlikely that rundown may involve hydrolysis of ATP molecules at NBF sites. Whether or not the binding of ATP to NBF is part of this process is not known. The presence of PKA in the bath together with MgATP maintain the activity of excised channels (Fig. 2C) [5,51]. However, the fact that PKA prevents the rundown of CFTR is not always immediately apparent. Sometimes, after excision, an initial loss of channel activity can be observed in the presence of PKA and MgATP. Stable and high Po can also be recorded in the presence of PKA immediately after the excision of the patch membrane (Fig. 2C), showing in this case that the CFTR channels are not deactivated. These data may reflect rapid dephosphorylation of excised CFTR channels or some variations in the accessibility for kinases to the multiple consensus phosphorylation sites of CFTR. Phosphorylation by kinases reverses rundown after the complete closure of

56

F. Becq / Biochimica et Biophysica Acta 1286 (1996) 53-63

excised channels [4,23,51 ], demonstrating that the rundown does not correspond to the irreversible loss of channel activity due to channel excision. Rundown therefore mainly reflects the loss or alteration of components or enzymes that appear to be essential to the functioning of the channel. Alternatively, channel rundown may also be due to the activity of membrane-associated phosphatases, which in cell-free patches are no longer counteracted by cytosolic kinases or endogenous modulators. In the absence of MgATP and PKA and with phosphatase inhibitors present in the bath, the CFTR channel activity is either maintained or its decrease in activity is considerably slowed down [3,5,51]. The phosphatase inhibitors fluoride, vanadate [5,51] and bromotetramisole [5] added to the bath prolonged the CFTR channel activity for several minutes. Moreover, the fact that adding alkaline phosphatase to the ATP-containing solution accelerated the rundown [5] suggests that dephosphorylation at some sites is critical for the normal activity of CFTR to occur. Taken as a whole, these observations show that dephosphorylation of CFTR by an endogenous phosphatase is indeed responsible for the loss of activity of the CFTR channel (see Table 1). Since the rundown of CFTR is observed in the nominal absence of calcium or calmodulin, the protein phosphatase PP2B (or calcineurin phosphatase) seems unlikely to play a role in this process [5,51]. The nature of the phosphatase may depend on the cell type. For example, okadaic acid (a membrane permeant PP1 and PP2A protein phosphatase inhibitor) inhibits = 25% of the decrease in CFTR activity after cAMP has been washed out from cardiac cells, the remaining 75% being okadaic acid insensitive [29]. In other cells, CFTR rundown is not affected by either okadaic acid or calyculin A (another PP1 and PP2A inhibitor), which suggests that channel rundown in this system may not be dependent on the presence of endogenous PP1 or PP2A [5,51]. Xanthine derivatives such as IBMX (3-iso-

Table 1 Regulation of the rundown of various chloride channels Channel type Cell type Inhibition of rundown Cystic fibrosis (CFTR)

transfected CHO cells airway epithelial cells

Ca2+-dependent Outward rectifier (ORCC)

pancreatic d u c t ceils cardiac cells smooth muscle cells HT 29 cells airway epithelial cells

phosphorylation by P K A , phosphatase inhibitors, xanthine phosphorylationby P K A , phosphatase inhibitors, xanthine phosphorylation by P K A , xanthine okadaic Ca2+-dependent process 5 mM ATP, 37°C

butyl-l-methyl xanthine) and theophylline (1,3-dimethylxanthine) prevent the rundown of excised CFTR channels [3,5]. In addition, they promoted the opening of on-cell CFTR channels [3,5], inhibited the membrane-associated phosphatase activity in some cells (see Ref. [3]), and reduced the dephosphorylation rate of CFTR proteins in an isolated membrane preparation [5].

2.2. Other chloride channels

In addition to the CFTR chloride channel, the rundown of two other chloride channels has been described (Table 1). In smooth muscle cells, a low conductance chloride channel is activated by a calcium-dependent process [31]. The channel activity recorded from excised patches showed a fast rundown, within 1 minute, which could be overcome with methods allowing calcium ions to enter the cell (prior to excision), which suggests that the loss of activity may be due to a calcium-dependent process [31]. In another study, the spontaneous inactivation of a Ca2+-dependent chloride channel identified in the human colonic epithelial cell HT29 was described [44]. The rundown of Ca2+-de pendent chloride channel in HT29 cells is rapid, but since it is not affected by ATP or calmodulin, Ca2+-dependent phosphatase is probably not involved in the loss of activity [44]. The main factors responsible for this process are still unknown. Using whole-cell recordings from airway epithelial cells, Schwiebert et al. [50] observed that the current developed by outwardly rectifying chloride channels (ORCC) declined spontaneously when studied at room temperature with 1 mM ATP in the pipette solution (Table 1). The authors successfully prevented the ORCC rundown by raising the ATP concentration in the pipette to 5 mM and the temperature to 35°C [50].

Mechanisms of rundown

Endogenous enzyme (channel-associated)

Ref.

dephosphorylation

phosphatase

[5,51]

dephosphorylation

phosphatase

[5,22,23]

dephosphorylation

phosphatase

[3,4,13] [29] [31] [44] [50]

57

F. Becq / Biochimica et Biophysica Acta 1286 (1996) 53-63

3. Potassium channels 3.1. ATP-dependent K channels

Potassium channels (Table 2) whose activity can be inhibited by high ATP concentrations applied to the internal surface of the plasma membrane (KAT p channels) have been identified in a variety of cell types [2,43]. In single channel and whole-cell studies, the activity of KATP channel runs down spontaneously within 1-10 min. Like that of the CFTR channel, the activity of the KATr, channel is tightly regulated by ATP and phosphorylation processes. KATP channels open when ATP is removed from the intracellular side of the membrane. At low concentrations, ATP alone prevents the rundown from occurring in pancreatic /3 [17] and follicular cells [27]. Since various ATP analogues, AMP-PNP, AMP-PCP and ATPTS have no effect on the rundown, hydrolysis of ATP may be necessary to keep the channel open [45]. PKA reopens KATP channels and exogenous phosphatases reverse the phosphorylation and close the channels in follicular cells [27]. In isolated insulin-secreting cells, the rundown of KATP channels is Mg2+-dependent [34], which inhibits the process in a concentration-dependent manner, while a complete blockade occurs in Mg2+-free solution. Prock and Ashcroft [46] have established that the presence of trypsin in the bathing medium during inside-out recordings prevented the rundown of KATP channel activity from occurring in pancreatic /3 cells. They therefore suggested that an endogenous, membrane-associated proteinase may be present in the patch membrane together

Table 2 Regulation of the rundown of various K channels Channel Cell type Inhibition of rundown type KATP

ROM K1

i-K and s-K IR-K

mice pancreatic /3 cells rat islet CRI-G cells

nucleotidest r y p s i n Mg 2+-free conditions

follicular cells rat CCD c e l l s

nucleotides phosphorylation by PKA MgATP, Mg2+-free, vanadate, and okadaic acid, phosphorylationby PKA MgATP, Mg2+-free, vanadate

expressedin X oocytes (cloned from rat kidney) rat CCD cells

K B~c

guinea pig chromaffin cells T84 cells

BK

rat pituitary cells smooth muscle cells

cGMP + MgATP (basal membrane) MgATP,calyculin A xanthine (IBMX) phosphorylation by PKA MgATP, okadaic a c i d

with the channel. The presence of membrane-bound phosphatases has also been postulated. As in the case of excised CFTR channels, endogenous phosphatases may dephosphorylate the channel protein itself thus causing the closure of the channel [34]. Although MgATP is able to reactivate KATe channels in excised membrane patches, its efficiency appears to decrease with time, and to be completely lost after complete rundown. It was therefore suggested that a membrane-bound kinase involved in the phosphorylation of the channel may have been lost after patch excision [57]. 3.2. Epithelial K channels

In epithelia, potassium channels are essential for the transport of salt across the cell layer to be possible and constitute a key feature of the cross-talk between the transport proteins located in apical and basolateral membranes. The K channels observed in epithelial cells include voltage-dependent K channels, ATP-dependent K channels and Ca 2+-dependent K channels. In renal rat cortical collecting duct cells (CCD cells), various populations of K channels have been described so far [26,42,57]. Although they have different biophysical properties and different regulatory mechanisms, they all run down in excised patches (Table 2). Inwardly rectifying low-conductance channels are a distinct population of ATP-sensitive K channels located in the membrane of CCD cells. The activity of these channels is also controlled by protein kinases A and C (see Ref. [57]). In excised patches, Wang and Giebisch [57] observed the

Mechanisms of rundown

Endogenous enzyme (channel-associated)

Ref.

dephosphorylation proteolysis Mg 2+-dependent dephosphorylation

proteinase,kinase

[17,46] [34]

dephosphorylation (Mg2+, vanadateand okadaic acid sensitive) dephosphorylation (vanadate and Mg2 + sensitive)

PP2A and Mg 2+_ dependent phosphatase (PP2C ?) Mg 2+ and vanadate sensitive phosphatase

dephosphorylation (PP1 or PP2A1)

cGMP-dependent protein kinase PP1 or PP2A

[27] [35,57]

[42]

[26] [30] [52]

dephosphorylation (type 1 phosphatase, PP1)

phosphatase (PP l), kinase G proteins okadaic acid-sensitive phosphatase, kinase

[7,8,12] [16,58] [9,37]

58

F. Becq / Biochimica et Biophysica Acta 1286 (1996) 53-63

rundown of inwardly rectifying low-conductance channels bathed in an ATP-free solution in the presence of Mg 2+. As mentioned above, high concentrations of ATP inhibit the channel activity, whereas low concentrations activate it. McNicholas et al. [42] have observed that the ATP-regulated ROMK1 K channel expressed in Xenopus oocytes shares some functional characteristics with the native lowconductance K channels of CCD cells [35,57]. The rundown of low-conductance K channels and ROMK1 K channels occurs progressively within 10 min of the excision, but it can be reversed with 0.1 mM ATP [35,42,57]. Since ATP is necessary for protein kinase activity to occur, these data suggest that phosphorylation processes are essential for the channel activity to be maintained. The rundown of low-conductance K channels and ROMK1 K channels is abolished when Mg 2÷ is removed from the bath or with a solution containing the broad-spectrum phosphatase inhibitor orthovanadate and Mg 2÷ [35,42]. Protein phosphatase as well as kinase activities are critically dependent on the presence of Mg 2+. The effect of Mg 2+ on the channel therefore suggests that the rundown is controlled by a Mg2÷-dependent, vanadate-sensitive phosphatase. The specific phosphatase PP1 and PP2A inhibitors okadaic acid and calyculin A fail to prevent ROMK1 K channel rundown [42]; whereas the rundown of low-conductance K channels in CCD cells is slowed down significantly in the presence of okadaic acid in the bath solution [35]. All in all, these results show that the rundown of low-conductance K channels in CCD cells results mainly from dephosphorylation of the channel, or an associated protein, induced by membrane-bound okadaic acidsensitive PP2A and Mg2÷-dependent (possibly PP2C) phosphatases ([35], see Table 2). The rundown of ROMK1 K channels is under the control of an Mg2+-dependent but okadaic acid-insensitive phosphatase ([42], see Table 2). After complete closure, the reactivation of ROMK1 K channels by protein kinase A is variable. When added within 60 s of the rundown, PKA reactivated the channel in --- 90% of the experiments, whereas after 60 s, kinases reactivated ROMK1 channels in less than 20% of experiments [57]. These results are intriguing and may reflect a time dependent, irreversible alteration of the channel structure or alternatively a progressive conformational change in the protein leaving the phosphorylation sites inaccessible to kinases. In the basolateral membrane of CCD cells, Hirsch and Schlatter [26] have described two different K channels with unitary conductances of 85 pS (intermediate-conductance channel, i-K channel) and 28 pS (small-conductance channel, s-K channel). After the excision, the activity of both channels declined but was reversed by a solution containing cGMP and MgATP [26]. Since cGMP and MgATP are cofactors determining the activity of cGMPdependent protein kinase (PKG), the authors concluded that a PKG may be located in the membrane near the channel (Table 2). This hypothesis was strongly supported

by the effects of KT5823, a specific PKG inhibitor, which reversed the effects of MgATP and cGMP [26]. Although not tested on the rundown, the two membrane permeable phosphatase inhibitors okadaic acid and calyculin A activate CCD K channels in cell-attached patches, which indicates that endogenous protein phosphatases, in addition to endogenous PKG, may regulate the channel via a cGMP dependent process. In the human colonic T84 cells, inwardly rectifying low conductance K channels with a unitary conductance of 30 pS (KB~c), located in the basolateral membrane, are activated by carbachol [52]. In excised patches, the channel activity runs down rapidly and this can be prevented by inducing PKA-dependent phosphorylation or by adding IBMX to the bath (Table 2). After complete rundown, PKA reopens the channel in T84 cells [52] as well as in CCD cells [57]. In T84 cells, changes in the ATP or Ca 2+ concentrations, both of which affect the channel activity, do not affect the rundown [52]. The similarity between the processes involved in the regulation of the rundown of epithelial K channels and CFFR chloride channels is quite striking and raises the question as to whether there may exist a common mechanism (i.e., a phosphatase mechanism) controlling the phosphorylation state and the opening probability of these channels. 3.3. Calcium-dependent K channels

Using the whole-cell and single channel patch-clamp recording techniques, large conductance K channels (BK channels) have been identified and fully characterized. BK channels are present in many excitable and non excitable cells and their activity depends on both the intracellular calcium concentration and the voltage. When excised in the inside-out configuration, the BK channel activity runs down (Table 2) after a variable delay [7,8]. MgATP and okadaic acid prevents the rundown, which suggests that type 1 protein phosphatase (PP1) may be closely associated with BK channels. In addition, Bielefeldt and Jackson [7] have reported that GTP3,S accelerates the rundown possibly due to its interactions with phosphatases. These authors suggested that there may exist a direct coupling between phosphatase and G proteins. In rat pituitary cells, BK channels are under the control of a membrane associated phosphatase PP2A and cGMPdependent protein kinase [58]. It was suggested moreover that PKG may regulate PP2A, which in turn controls the channel activity [58]. BK channels are also tightly regulated by endogenous kinases [7,8,12,16,37,58]. Phosphatase regulation of BK channels has also been found to occur and the inhibition of an endogenous okadaic acidsensitive phosphatase activity suffices to promote channel opening in the absence of kinase stimulation [36,37]. Similar insights have been gained by studying BK channels in smooth muscle cells from canine proximal colon [9]. In these cells, calyculin A and okadaic acid potentiate the

59

F. Becq / Biochimica et Biophysica Acre 1286 (1996) 53-63

effects of exogenously applied PKA, which shows that endogenous phosphatases again tightly regulate the channel. The exact nature of the enzyme has not yet been determined, however [9].

4. Calcium channels

Calcium channels play an important role in the electrical activity of cardiac cells, and in the excitation/contraction coupling in skeletal muscle and neurotransmitter release in neurones. In whole-cell recordings, L-type calcium channel rundown (Table 3) was found to be accelerated by releasing intracellular Ca 2. with caffeine and slowed by increasing Ca 2+ buffering capacity with EGTA [6]. ATP but not AMP-PNP prevents the rundown in guinea pig heart cells [6]. In single channel experiments, Armstrong and Eckert [1] have observed that phosphorylation by protein kinase A in the presence of MgATP prevents the rundown of Ca 2+ channels. However, the effect of phosphorylation is attenuated with time [10], which led the authors to postulate that two different processes may control the rundown: (i) an irreversible loss of activity, that can be slowed down by proteolytic enzymes, and (ii) a reversible step controlled by phosphorylation. The calcium-dependent proteinase calpain has been identified as one of the endogenous enzymes involved in the irreversible rundown of cardiac Ca 2+ channels in guinea-pig ventricular myocytes [48]. Two different phosphatases play an important role in the rundown of calcium current. Okadaic acid inhibits the rundown and increases the calcium current [24,25], which suggests that PP1 or PP2A protein phosphatases may have been involved. Secondly, the calcium-dependent phosphatase calcineurin is also involved [10,19]. Wang et al. [54] showed that after being incorporated into the planar lipid bilayer, L-type calcium channels are still under the control of okadaic acid, and that rundown still occurs just as in cell-free patch-clamp experiments. These authors therefore suggested that the down-regulation of the channel may be strongly dependent

on the activity of an endogenous okadaic acid-sensitive phosphatase. Since these data were obtained in reconstituted lipid membranes, it seems likely that either the phosphatase activity may be closely associated with the channel or the channel protein may directly mediate the enzymatic activity. Regulation of the cardiac calcium current by G-proteins has also been shown to occur; the a subunit of activated stimulatory G proteins increased the channel activity and delayed the rundown in the lipid bilayer [54]. G proteins may regulate the channel activity either directly or via phosphatases. Taken as a whole, these findings suggest that calcium channels may be under the control of various membrane-associated proteins such as phosphatases, proteinases, and G proteins, which either upor down-regulate the activity and the rundown of channels.

5. Other channels

GABA a receptors are ligand-gated chloride channels that are responsible for most of the inhibitory synaptic transmission in the mammalian central nervous system. In guinea-pig hippocampal neurones, Chen et al. [11] have established that GABAa-mediated whole-cell C1- current runs down when the intracellular solution lacks MgATP (Table 3). The channel rundown was prevented with MgATP and low levels of intracellular Ca 2+ [11]. Alkaline phosphatase causes a complete and fast rundown of GABA A currents and the presence of ATPTS blocks the effects of phosphatase. On the other hand, increasing intracellular Ca 2+ accelerates the rundown of GABA A currents. On the basis of these data, it has been postulated that the regulation of the GABA A current may involve phosphorylation-activation and CaZ+-dependent dephosphorylation-inactivation of the GABA A receptor (the latter probably via activation of the phosphatase calcineurin). Typical rundown behavior was also observed in Nmethyl-D-aspartate (NMDA) channels studied under whole-cell conditions (Table 3). Channel rundown can be prevented with an ATP-regenerating solution in the pipette.

Table 3 Regulation of the rundown of Ca-' +, GABA and NMDA channels Channel type

Cell type

Inhibition of rundown

Mechanisms of rundown

L-type Ca -'+

mammalian pituitary guinea pig myocyte invertebrate neurone guinea-pig neurone hippocampal neurone

phosphorylation by PKA

dephosphorylation

nucleotides proteinase inhibitors phosphatase inhibitors proteinase and phosphatase inhibitors

dephosphorylation proteolysis proteolysis dephosphorylation Ca-"+-dependent dephosphorylation increase intracellular calcium

Ca 2 + GABA A NMDA

low level of calcium MgATP ATP-dependent process Ca-dependent process (calcium-free, BAPTA)

Endogenous enzyme (channel-associated)

Ref. [1]

phosphatase, proteinase G proteins phosphatase, proteinase

[6,19,24] [25,48] [10] [11] [49]

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F. Becq / Biochimica et Biophysica Acta 1286 (1996) 53-63

Unlike what occurs with G A B A A receptors, N M D A channel rundown is not affected by the presence of several phosphatases, including alkaline phosphatase, PP1 and calcineurin, or by ATP,/S [49]. Conflicting results have been obtained as to whether phosphorylation/ dephosphorylation processes may be involved in the rundown of N M D A channels [40,49]. At least four different phosphatases have been found to regulate N M D A channels. In cell-attached patch-clamp recordings on dissociated adult rat dentate gyrus granule cells, Lieberman and Mody have observed that okadaic acid and the calcineurin inhibitor FK506 increased the activity of N M D A channels [39]. This result is consistent with N M D A channels being regulated by endogenous Ca2+-dependent phosphatase (calcineurin). On the other hand, PP1 and PP2A [55] and tyrosine phosphatase [56] have also been thought to possibly control the activity of N M D A channels. Interpreting all these results is possible due to the knowledge that phosphatase inhibitors (used at various doses) modulate the channel activity in cell-attached patches [40,55] and in whole-cell recordings [56]. Care must therefore be taken using (so-called specific) phosphatase (or kinase) inhibitors. Future experiments on protein phosphorylation and dephosphorylation should help to identify the phosphatases involved in the regulation of N M D A channels.

Rundown induced by

I

Rundown prevented by

I

I

I

A

I__]

I

I

Oa. br-t, xant.

I

.......

I

MgATP

B

I

Van., Mg2+

MgATP

C

I

I

® 6. M e c h a n i s m s

of ionic channel rundown

What molecular mechanisms might contribute to ionic channel rundown and its modulation? Evidence from several laboratories has now confirmed that many different ionic channels show the typical spontaneous deactivation called rundown. The data cited in this review suggest that the endogenous enzymes present even in washed membranes may modulate the activity of the channels (see Tables 1-3) and be involved in either the initiation or the prevention of rundown (Fig. 3). The multitude of cellular mechanisms possibly involved in ionic channel rundown can be divided into three main groups: (i) those depending upon Mg2+-dependent nucleotide complexes, (ii) those depending on phosphorylat i o n / d e p h o s p h o r y l a t i o n and (iii) those depending on proteolysis reactions. Fig. 3 summarizes three representative models of membrane-delimited pathways involved in the control of ionic channel rundown. It emerges from the literature cited in this review that dephosphorylation is the main process responsible for the onset of rundown (Fig. 3), and that phosphorylation prevents rundown. Proteolysis, which was newly found to play an important part in rundown (Fig. 3C), seems to require particular attention in future investigations. Finally, despite the need for the presence of cyclic nucleotides as cofactors for kinase activity to be possible, there exists no proof so far that these molecules directly affect rundown (in the absence of kinase).

Oa

It__l I I C2+ Calpastatin

I

II

I

MgATP GTP ~/S PKI

Fig. 3. Cartoons showing mechanisms of ionic channel rundown. A, cystic fibrosis (CFTR) chloride channel. B, potassium channel from CCD cells. C, voltage-dependent L-type calcium channel. The left and right hand sides of the cartoons show the mechanisms which induce and prevent rundown, respectively. Channel proteins are presented with two membrane-spanning elements integrated into the membrane bilayer. A, this diagram shows that CFTR channel activity is upregulated (+) by MgATP and PKA-dependent phosphorylation. Endogenous, membraneassociated phosphatases (PPase) promote the enzymatic dephosphorylation of specific sites (P), which in turn induces channel rundown (R). Rundown was abolished either by inducing phosphorylation or after inhibiting the associated phosphatase activity with appropriate phosphatase inhibitors (Oa; okadaic acid, br-t; bromotetramisole, xant; xanthine derivatives, see text for details). In B, the endogenous phosphatase (PPase) was insensitive to okadaic acid but inhibited by orthovanadate (Van.) and critically dependent on Mg2+. Channel activity was up-regulated (+) by a membrane-associated kinase (PKA). Some K channels (see text for details) are regulated by both enzymatic processes, but the rundown (R) appears to result from the dephosphorylation of specific sites by the endogenous phosphatase. Rundown can be prevented by phosphorylation. In C, the membrane-delimited pathway involved in the regulation of ionic channels may be more complicated due to the presence, near the channel, of enzymes including phosphatase (PPase), kinase (PKA), proteinase or G proteins, as hypothesized in the case of calcium channels in excitable cells (see text for details). The channel rundown (R) can be induced by the action of two different phosphatases, an okadaic acid-sensitive pbosphatase (noted PPase) and the calcium-dependent phosphatase calcineurine (noted Calci.). In addition, the channel rundown is also associated with proteolytic processes mediated by the membraneassociated proteinase calpaine (inhibited by calpastatin). Lastly, the rundown can be prevented by activating endogenous kinases and G proteins located near the channel.

F. Becq // Biochimica et Biophysica Acta 1286 (1996) 53-63

All the data mentioned in this review raise the question as to whether channels have any enzyme activity, or whether other membrane proteins may participate in the process. To account for the experimental data obtained so far, the protein kinase or phosphatase responsible must be located in the vicinity of the channel protein within the membrane (Fig. 3). Judging from the results of most of the studies in which enzyme cofactors, enzyme inhibitors or purified enzymes have been used, it seems likely that membrane associated enzymes may be the key to rundown rather than an enzyme activity intrinsic to the channel protein. In a study on the rundown of BK channels in posterior pituitary nerve cells, Bielefeldt and Jackson [7] published an excellent functional description of the intermolecular interactions observed between enzyme and BK channels in excised patches. BK channels from rat cortical neurones are reversibly activated by MgATP and MgADP, and this effect is antagonized by PKA inhibitors, which suggests the existence of a complex functional unit consisting of closely associated phosphatase, A type kinase and adenylate kinase [38]. It has been suggested however in some reports [47] that enzyme and channel activities are not easily separable and that they may exist an intramolecular link between channel protein and modulating enzymes. Some insights have also been gained into the way in which channel activity is modulated by endogenous enzymes. Wang et al. [54] have clearly established for example that after being incorporated into an artificial bilayer, the L-type calcium channel still was under the control of an okadaic acid-sensitive phosphatase (see Fig. 3C). Lipid bilayer experiments also showed that reconstituted Ca 2÷activated K channel from rat brain is tightly regulated by endogenous protein kinase [12]. To explain the lipid bilayer data, two hypotheses can be proposed. Either the channel itself is capable of enzyme activity, or the enzyme is so intimately associated with the channel that they both diffuse together into the bilayer. Both cases involve the existence of membrane regulatory complexes tightly associated with ionic channels. Similar conclusions were reached by Esguerra et al. [16], who demonstrated that the cloned Drosophila Slo Ca2+-dependent K channel expressed in the Xenopus oocyte is functionally associated with and modulated by an endogenous PKA-like protein kinase. Moreover, Rehm et al. [47] demonstrated that a purified K channel exhibit intrinsic protein kinase activity. The idea that there may exist an enzymatically active domain within the channel protein structure has been further supported by the deduced primary sequence of some ionic channels (e.g. the CFTR chloride channel). CFTR belongs to the ABC (ATP binding cassette) family of transport protein and contains consensus sequences for ATP-binding (and hydrolysis) sites possibly showing intrinsic ATPase activity (see Ref. [21 ]). The physiological role of rundown processes is not immediately obvious. Close inspection of the data revealed

61

however that in some cells, 'in situ rundown' occurs in response to a physiological stimulation. This was found to occur in the case of the CFTR chloride channels in epithelial cells. In cell-attached recordings from pancreatic duct cells stimulated by secretine [18] or VIP [4], the activity of CFTR channels is characterized by a wave-like pattern of activity. Long periods of CPTR channel activity showed the presence of large opening probability oscillations that could not be accounted for by a stochastic distribution of the open state durations. Moreover, the occurrence of spontaneous deactivation in cell-attached recordings has been reported in some studies [4,18,23] that mimicked the rundown observed in excised patches. It has now been clearly established that CFTR channels are up-regulated by PKA and down-regulated by phosphoprotein phosphatases (see Ref. [21] and Fig. 3A). In fact, the rundown observed in excised patches probably results from an exacerbation of the inhibitory control (phosphatase activity) that occurs physiologically in the cells. In pituitary cells, White et al [58] have studied the BK channel activity which is controlled by the inhibitory peptide somatostatin, acting via a okadaic acid-sensitive membrane-associated phosphatase. Somatostatin has been found to exert its physiological action by activating membranous tyrosine phosphatase in pancreatic cells (see Ref. [4] for discussion). In pancreatic duct cells, CPTR chloride channel (regulated by endogenous phosphatase) play an important role in the secretion of bicarbonate ions, known to be inhibited by somastotatin (see Refs. [4,18]). Although the exact nature of the phosphatase involved in the control of BK and CPTR channels has not yet been elucidated, the similarity between the mode of regulation of the channel and the mode of action of somatostatin is intriguing and may mean that the phosphatase is the final effector in the physiological effect of hormone on these channels.

7. Concluding remarks In the early days, rundown of ionic channel activity in cell-free patches or that of currents in whole-cell recordings were thought of as a major hindrance to the electrophysiological investigation of channel properties, function and regulation. Solving this problem has led however to new insights and to the discovery that endogenous membrane-associated enzymes play a primary role in the control of channel activity. Finally, it brought to light the existence of integrated membrane complexes consisting of a channel protein directly under the control of enzymes such as phosphatases, kinases, or G proteins. The opening of these ionic channels simultaneously requires the inhibition of the negative control and the onset of stimulation. The resulting multi-protein domain provides the cell with a highly specific and efficient pathway for regulating cellular ionic movements. Understanding the molecular mechanisms responsible for ionic channel rundown should yield

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F. Becq / Biochimica et Biophysica Acta 1286 (1996) 53-63

some new clues about the role of channels in the physiological and pathophysiological states of cells. The idea that enzymatic processes regulate ionic rundown now appears quite evident, based on the data available on channel rundown, and the hypothesis that an intrinsic enzyme activity might operate within the channel protein itself looks very attractive. Five years ago, R.S. Eisenberg reviewed the pros and cons of a channel being an enzyme [15] and asked "What can we learn by considering channels as enzymes?". In the light of the latest findings on the enzymatic control of ionic channel rundown, a new version of this question may be proposed, as follows: What can we learn by considering a channel as a multifunctional protein serving both as a channel and an enzyme? The latter being responsible for the chemical gating of the diffusion of ions into the pore.

Acknowledgements

I am especially indebted to Prof. John. W. Hanrahan for lively and stimulating discussions, assistance and support and to my colleagues from McGill Physiology Dept. I would like to thank Emmanuelle for her continuous support and Dr. Maurice Gola for reading the manuscript and his suggestions for the improvement thereof. This work was supported by postdoctoral fellowships from the Canadian Cystic Fibrosis Foundation and the Association Fran~aise de Lutte contre la Mucoviscidose to F.B.

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