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J Biol Chem, Vol. 275, Issue 6, 3749-3754, February 11, 2000

Conditional and Unconditional Inhibition of Calcium-activated Potassium Channels by Reversible Protein Phosphorylation^*

Sarah K. Hall^§¶ and David L. Armstrong

From the  Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709 and ^§ Physiology Unit, School of Biosciences, Cardiff University, Cardiff CF10 3US, Wales, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Large conductance, calcium-activated potassium channels (BK_Ca or maxi-K) are important determinants of membrane excitability in many cell types. We used patch clamp techniques to study the biochemical regulation of native BK_Ca channel proteins by endogenous Ser/Thr-directed protein kinases and phosphatases in cell-free membrane patches from rat pituitary tumor cells (GH₄C₁). When protein kinase activity was blocked by removing ATP, endogenous protein phosphatases slowly increased BK_Ca channel activity approximately 3-fold. Dephosphorylated channels could be activated fully by physiological increases in cytoplasmic calcium or membrane depolarization. In contrast, endogenous protein kinases inhibited BK_Ca channel activity at two functionally distinct sites. A closely associated, cAMP-dependent protein kinase rapidly reduced channel activity in a conditional manner that could be overcome completely by increasing cytoplasmic free calcium 3-fold or 20 mV further depolarization. Phosphorylation at a pharmacologically distinct site inhibited channel activity unconditionally by reducing availability to approximately half that of maximum at all physiological calcium and voltages. Conditional versus unconditional inhibition of BK_Ca channel activity through different protein kinases provides cells with a powerful computational mechanism for regulating membrane excitability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Large conductance, calcium-activated potassium channels (BK_Ca or maxi-K channels) are uniquely powerful determinants of electrical activity in the nervous, endocrine, and vascular systems, since they respond directly to both membrane depolarization and calcium accumulation (1). The sensitivity of BK_Ca channels to such physiological stimuli can be modified by enzyme pathways mediating reversible phosphorylation of the ion channel proteins or other closely associated regulatory proteins (2). Such reversible protein phosphorylation is the primary mechanism for regulating ion channel activity on the physiological time scale of seconds and minutes (2-4). Modification of the behavior of BK_Ca channels has profound effects on the frequency and duration of action potentials in excitable cells, thereby controlling the physiological function of these cells.

Electrophysiological measurements indicate that the activity of BK_Ca channels in native cells may be up- or down-regulated by reversible protein phosphorylation. For example, protein kinase-induced phosphorylation enhances calcium-activated potassium currents in smooth muscle (5, 6), whereas in photoreceptors (7), hippocampal neurones (8-11), and neurendocrine cells (12), the currents are inhibited by kinase activity. BK_Ca channels exist as multimeric protein complexes composed of two integral membrane subunits, the pore-forming  subunit and the regulatory  subunit (1). All  subunits are apparently coded by the same gene, the slo gene, but alternative RNA splicing during development produces functionally distinct channel proteins in different cell types (13-15). Hence, opposite modulatory effects of protein phosphorylation are likely to reflect differential modulation of BK_Ca channel subtypes derived from alternately spliced mRNA transcripts or could arise as a result of variations in the  and  subunit composition of the channel complexes (1, 16) or more indirect signaling cascades. We have used the patch-clamp technique to investigate the modulation of BK_Ca channel behavior by reversible protein phosphorylation at the molecular level by recording channel opening behavior in cell-free patches of native membrane. Thus, we report studies of BK_Ca channel modulation under controlled conditions in the absence of alternative splicing or diffusible regulatory proteins, where the open probability (P_o) of the channels was determined under a variety of experimental conditions to promote or inhibit protein phosphorylation.

In mammalian endocrine and nervous systems, most examples of BK_Ca channel regulation by hormones and neurotransmitters involve channel inhibition by protein phosphorylation (1, 9-11). Similarly, the BK_Ca channels in rat pituitary tumor cells (GH₄C₁ cells) are also inhibited by phosphorylation. In these cells, maximal stimulation of either protein kinase A (PKA) or C (PKC) completely suppresses BK_Ca channel activity in the physiological voltage range (17, 18); conversely, hormones that inhibit secretion from GH₄C₁ cells have been shown to stimulate BK_Ca channels through protein dephosphorylation (12, 19). Thus, the BK_Ca channels in GH₄C₁ cells are regulated in the same way as many other physiologically relevant examples of BK_Ca channels in the brain. For this reason, we have chosen to study the effects of reversible protein phosphorylation on the calcium and voltage dependence of single native BK_Ca channels from GH₄C₁ cells. Previous studies at the single-channel level have focused on BK_Ca channels that are stimulated by protein phosphorylation (5, 20-23). In this paper, we report evidence of BK_Ca channel modulation using a cell-free system that qualitatively and quantitatively reproduces the effects of hormones on intact cells.

This is the first study of native BK_Ca channels in native membranes that systematically varies all three of the molecular mechanisms known to regulate these channels: voltage, intracellular free calcium concentration, and protein phosphorylation. BK_Ca channel modulation has not previously been examined at sufficiently high open probabilities to address the molecular mechanism of modulation across the complete range of channel activity. To measure the effects of reversible protein phosphorylation on BK_Ca channel activity reproducibly over the entire physiological range, 0.01  P_o  0.9, we have found it essential to use 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)¹ and its dibromo analogue to buffer calcium on the cytoplasmic side of the membrane patches. EGTA, which saturates at submicromolar concentrations of calcium (24-26), is an ineffective buffer of physiological calcium transients. In contrast, BAPTA and dibromo-BAPTA bind calcium rapidly and independently of pH and physiological magnesium concentrations with an affinity that allows effective buffering of free calcium at concentrations up to and above 10 µM (27). With this experimental precaution, our measurements have revealed a novel property of BK_Ca channel regulation by protein phosphorylation. Our findings have important implications for the control of BK_Ca channel proteins and demonstrate that reversible protein phosphorylation cascades play an integral role in controlling cellular excitability through their actions on BK_Ca channels.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- BK_Ca channel activity was studied in GH₄C₁ cells, an immortal cell line derived from rat anterior pituitary tumor cells (28). Cells were maintained at 37 °C in sterile Ham's F-10 culture medium supplemented with fetal bovine serum (2.5%, vol), equine serum (12.5%, vol), and antibiotics (penicillin and streptomycin). Cells were plated onto plain or collagen-coated glass coverslips 1-6 days before patch clamp recording, and the culture medium was replaced every 3-4 days. Cells from culture passage 2-28 were used in these studies.

Electrophysiological Recording-- The activity of single BK_Ca channels was investigated using conventional patch clamp recording techniques (29). Patch pipettes were fabricated from borosilicate glass capillary tubing (7052; Garner Glass Co, Claremont, CA), the pipette shank was coated with Sylgard (Dow Corning, Midland, MI), and the tips were fire-polished just before use. These pipettes had resistances in the range of 4-8 megaohms when filled with electrolyte (see below for composition). Single-channel activity was recorded at room temperature from cell-attached patches of membrane on intact cells and from cell-free, inside-out patches using an Axopatch 1C amplifier and TL-1 interface (Axon Instruments, Burlingame, CA). Data were recorded and analyzed using pClamp software, version 6.0.1 (Axon Instruments). Voltage-dependent channel activity was measured over the range 40 to 100 mV, with the applied voltage stepped at random. BK_Ca channels were identified by their conductance, ~120 picosiemens in a physiological [K⁺] gradient. The stochastic behavior of ion channels in these patches was interpreted using standard analytical procedures. Channel open probability (P_o) was determined from continuous records of channel activity over 10, 20, or 30 s at a given potential; P_o may show some variability when measured over shorter periods (30). Excised membrane patches commonly contained more than one active BK_Ca channel. The number of active channels could be counted with confidence (N  3) when all the channels were stimulated to open simultaneously, giving high open probabilities (P_o near 1) during a 10-s period. In these patches, P_o was calculated according to the relationship,

(Eq. 1)

where i = 1 to N, N is the maximum number of channels open simultaneously at the most depolarized membrane potentials (positive to +60 mV; N  3), t_t is the total time of the recording and t_i is the cumulative time during which exactly i channels are open. When channel behavior in multichannel patches was not monitored across the complete voltage range, activity is expressed as N*P_o. Data are expressed as the mean value ± S.D. from multiple independent measurements (n  3). Statistical analyses were made by Student's t test (2-tailed) for paired or unpaired data as appropriate; the null hypothesis was rejected when p < 0.05. Boltzmann curves were generated by best fit to complete data sets using the Origin 4.0 software package (Microcal Software Inc., Northampton, MA); P_o(min) was constrained at 0, and where appropriate, P_o(max) was constrained at 0.87 (mean value from experimental data).

Solutions and Drugs-- For cell-attached recording, intact cells were bathed in high [K⁺] solution with the following composition: 140 mM KCl, 10 mM HEPES (pH 7.2 with NaOH), 1 mM MgCl₂, 1 mM CaCl₂, 30 mM glucose, such that E_K = 0 mV. Pipette-filling solution had the following composition: 135 mM NaCl, 5 mM KCl, 10 mM HEPES (pH 7.4 with NaOH), 1 mM MgCl₂, 1 mM CaCl₂, 0.001 mM tetrodotoxin. After seal formation (>30 gigaohms), cell-free patches were excised into a similar high [K⁺] solution containing the calcium buffers BAPTA or dibromo-BAPTA (1 mM). Free calcium concentration ([Ca²⁺]) was adjusted in the ranges 10⁸ to 5 × 10⁷ M (with BAPTA as the calcium buffer) and 10⁶ to 10⁵ M (with dibromo-BAPTA as the calcium buffer) by the addition of appropriate amounts of a stock solution of CaCl₂ (1 M), according to the relationship,

(Eq. 2)

Use of 1 mM BAPTA or dibromo-BAPTA allowed accurate buffering of [Ca²⁺] at the intracellular face of the membrane and avoided the artifacts associated with the slow Mg²⁺- and pH-dependent buffering by EGTA (27).

Okadaic acid was used to inhibit the activity of specific protein phosphatases (31). Consistent effects of okadaic acid were obtained by careful treatment of the inhibitor. Stock okadaic acid solutions were prepared in sterile, dry dimethyl sulfoxide (Me₂SO) and stored in airtight containers at 20 °C for up to 2 months. Stock solutions were diluted in bath solution to give a final Me₂SO concentration of 0.1%; the activity of okadaic acid was preserved by limiting the thawing and refreezing of stock solutions and avoiding sonication of experimental solutions.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

When GH cells are voltage-clamped through perforated patches in a physiological salt solution, BK_Ca channels dominate the steady-state membrane conductance at depolarized voltages (12, 32, 33). In contrast, individual channels in cell-attached patches on resting cells often showed surprisingly low activity: P_o < 0.1 even at +40 mV. However, when membrane patches were excised into an ATP-free bathing solution (Fig. 1), the mean open probability (P_o) increased slowly but significantly over 10-15 min, reaching a new steady-state level that was 258 ± 167% greater than the activity measured immediately following patch excision (n = 11; p < 0.01). In many patches, an increase in the number of BK_Ca channels open simultaneously (N) was also observed. The rate of increase in channel activity was not affected by varying calcium concentration the range 0.1 to 1.0 µM or by varying the membrane potential of the patch from 0 to +40 mV (not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   BKCa channel activity runs up in ATP-free solution. A, continuous records of unitary BKCa currents recorded at 0 mV in the same excised, inside-out patch of membrane from a GH4C1 cell immediately after excision into ATP-free solution with 1 µM Ca and 22 min later. B, time course of channel activity (N*Po calculated from continuous 20-s records) following patch excision into ATP-free solution (filled circles). Run up was prevented in cells that had been treated with 1 nM okadaic acid for 10 min before patch excision (open circles).

Dephosphorylated BK_Ca Channels Respond to Voltage and Calcium in the Physiological Ranges-- Once BK_Ca channel activity had stabilized in MgATP-free solution, it remained stable for the duration of the recording (up to 90 min, Fig. 2A). Nevertheless, the mean open probability of the channels in the patch could be increased independently by further depolarization (V) or by increasing the free calcium concentration ([Ca²⁺]_i) at the intracellular face of the membrane, up to a maximum P_o = 0.86 ± 0.11 (n = 10 complete P_oV curves at 5 different [Ca²⁺] in 8 patches) (Fig. 2B). These effects were reproducible and fully reversible and showed no sign of inactivation during prolonged stimulation. At calcium concentrations between 0.1 and 10.0 µM, an e-fold increase in P_o over the linear range from 0.1 to 0.8 was produced by increasing the voltage 14.6 ± 6.6 mV (n = 8 independent curves, in 6 patches). On average, increasing free calcium 10-fold at the cytoplasmic surface reduced the voltage required to produce half-maximal activity (V_1/2) by 55mV without changing the intrinsic voltage dependence of activity (Fig. 2C). Thus, when protein kinases are inhibited, native BK_Ca channels are extremely sensitive to changes in voltage and calcium in the physiological ranges. The dissociation constant (K_D) for Ca²⁺ binding was 1.8 µM (calculated from the linear fit to the data in Fig. 2C (34)). Hence, although depolarization greater than any action potential (~+55 mV) would be required for half-maximal channel activity at the resting calcium level in the cell (~0.1 µM), half-maximal channel activity would be sustained below the resting membrane potential of the cell (~55 mV) as long as free Ca²⁺ levels underneath the plasma membrane exceeded 10.0 µM.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Dephosphorylated BKCa channels respond to physiological calcium and voltage. A, time course of BKCa channel activity (N*Po calculated from continuous 20-s records at discrete intervals throughout the experiment) during changes in the membrane voltage (Vm) and cytoplasmic calcium ([Ca2+]) in the prolonged absence of exogenous ATP. Following run-up, activation by physiological [Ca2+] and voltage was fully reversible. B, voltage dependence of BKCa channel activity measured in the same patch in 0.1 µM (filled circles) and 1 µM (filled squares) free calcium following development of stable channel activity in the absence of exogenous ATP. The activity of dephosphorylated channels increases steeply with depolarization (e-fold/14.6 mV). C, plot of the voltage required for half-maximal channel activity (V1/2) as a function of [Ca2+] on the former cytoplasmic side of the membrane in the prolonged absence of exogenous ATP. Increasing [Ca2+] 10-fold shifts the voltage dependence of channel activity by 55 mV in the depolarizing direction (data fit with a straight line (r = 0.85), which was then used to determine Kd and  (34)).

Protein Dephosphorylation Increases BK_Ca Channel Activity-- Previous studies show that protein dephosphorylation stimulates BK_Ca channel activity in GH₄C₁ cells (12, 19); this behavior is similar to that of BK channels in other neuronal cell types (3, 9-11). The increase in BK_Ca channels activity we observed following patch excision into ATP-free solutions is consistent with this published data. Two independent pharmacological manipulations of the patches provide additional evidence that endogenous protein phosphatases are responsible for the increase in BK_Ca channel activity. First, inhibition of serine/threonine-directed protein phosphatases with the structurally unrelated microbial toxins 1 µM microcystin (n = 3) or 1 nM okadaic acid (n = 5) to selectively inhibit the PP2A family of phosphatases (35) completely prevented the increase in channel activity when they were added before patch excision (Fig. 1B). In contrast, if the inhibitors were not added until after channel activity had increased in ATP-free solution, they had no effect on channel activity (not shown), which rules out direct effects of the toxins on channel gating. Second, subsequent addition of MgATP at the intracellular face of the membrane rapidly reduced activity back to the low level recorded immediately following patch excision (Fig. 3A; n = 6). This effect of MgATP was not readily reversible, but neither free Mg²⁺ ions alone (as 1 mM MgCl₂; n = 4) nor 1 mM MgADP (n = 4) nor 0.1 mM GTP (n = 2) could mimic this effect of MgATP. These findings demonstrate a specific requirement for MgATP, which indicates a role for protein phosphorylation in the suppression of BK_Ca channel opening. In support of this conclusion, the effect of MgATP was prevented when PKA was inhibited by preaddition of 10 µM Rp-cAMPS with 1 µM wiptide (PKI_5-22 (Peninsula Labs)).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   MgATP reverses run-up and causes conditional inhibition of BKCa channel activity. A, time course of BKCa channel activity (N*Po calculated from continuous 20-s records) following patch excision into 1 µM Ca before and after the addition of 1 mM MgATP. B, continuous records of unitary currents recorded at 0 mV in the same excised, inside-out patch of membrane: 1 min after excision into ATP-free solution, 20 min later, and then 27 min after the addition of 1 mM MgATP to the bathing solution at the former cytoplasmic face of the membrane. C, voltage dependence of BKCa channel activity at constant calcium (1 µM Ca) in the same patch before (filled circles) and after (filled squares) the addition of 1 mM MgATP at the intracellular face of the membrane. Note that the intrinsic voltage dependence of channel activity, indicated by the slope of the relationship, does not change.

Protein Phosphorylation Reduces the Sensitivity of BK_Ca Channels to Calcium-- The activity of BK_Ca channels in dephosphorylated membrane patches was reduced dramatically by subsequent protein phosphorylation (Fig. 3). The addition of 1 mM MgATP (but not ADP or GTP; see above) to the solution at the intracellular face of the excised patch caused a shift of 19 ± 10 mV (n = 5 patches; p < 0.05) toward more depolarizing potentials in the voltage dependence of BK_Ca channel activity without changing the slope of the relationship (Fig. 3C). We interpret the reduction in activity as a decrease in channel sensitivity to calcium because the same effect was observed at two calcium concentrations (1 and 5 µM) and because the intrinsic voltage-dependence of channel activity remained unchanged. The decrease in P_o was achieved predominantly by a decrease in the frequency of channel opening with no significant change in the mean open time. Nevertheless, the maximum attainable channel activity was not suppressed by MgATP in excised patches; channels could be stimulated maximally by raising calcium or by depolarizing further (Fig. 3). In three patches, P_o(max) was 0.76 ± 0.07 following run up in the absence of ATP and 0.71 ± 0.10 following the addition of MgATP (no significant difference; p > 0.1).

The inhibitory effect of MgATP on channel activity was not potentiated by subsequent addition of low concentrations (10 nM) of okadaic acid or microcystin (not shown), indicating that the basal activity of PKA was substantially higher than that of the phosphatase. This conclusion is consistent with the low activity of BK_Ca channels in cell-attached patches observed before excision and is supported by the observation that incubating cells in 1 nM okadaic acid and 1 mM cpt-cAMP for 10 min before patch excision also had no significant effect on channel activity in cell-attached patches (n = 4). However, the open probability of the channels after patch excision from cells treated with okadaic acid could not be increased beyond P_o = 0.47 ± 0.19 (n = 10 complete P_oV curves over a range of [Ca²⁺] in 6 patches) at any calcium concentration (50 µM) or voltage (+100mV) we examined (Fig. 4). In all but one of the patches treated with okadaic acid before excision, maximal opening probability was <0.5. In contrast, in every single one of the dephosphorylated patches and those patches that were rephosphorylated after excision, maximal channel open probability could be driven to P_o > 0.7. Hence, we interpret this suppressed channel activity as an effect on all of the channels rather than on a subset of the channel population. Our data indicate that in intact cells there is an additional kinase regulating the activity of BK_Ca channels to produce a significantly different physiological effect on cell excitability. PKA-mediated phosphorylation reduces the sensitivity of BK_Ca channels to increases in voltage and calcium, but phosphorylation by the second kinase reduces the maximum attainable channel activity by half. In other words, the inhibition by PKA is conditional, but the inhibition by the second kinase is unconditional.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Protein phosphorylation reduces BKCa channel availability unconditionally. A, voltage dependence of BKCa channel activity at 1 µM [Ca2+] (filled squares) and 10 µM [Ca2+] (filled circles) on the same patch from a cell that was incubated with 1 nM okadaic acid and 0.1 mM cpt-cAMP before excision into bath solution containing 1 mM MgATP. B, continuous records of unitary currents in a single patch at the same voltage (+40 mV) but two different [Ca2+] at the cytoplasmic face of the channels.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The data reported here establish clearly that native calcium-activated potassium channels with the largest conductance (BK_Ca or maxi-K) are inhibited by endogenous protein kinases and stimulated by endogenous protein phosphatases that remain closely associated with the channels in cell-free patches from an immortalized endocrine rat pituitary cell line (GH₄C₁). Our results are entirely consistent with the physiological stimulation of excitability and secretion in the anterior pituitary by hypothalamic neuropeptides, which increase protein kinase activity through G_s- and G_q-coupled receptors (36, 37). Such kinase-mediated inhibition demonstrates that BK_Ca channels in GH₄C₁ cells behave in the same way as the calcium-activated potassium currents in hippocampal neurons of the mammalian central nervous system (3, 8-11). In contrast, most previous single-channel studies of BK_Ca channel modulation have been conducted on channels that are stimulated by protein kinases and inhibited by protein phosphatases, using either native channels in nonneuronal tissues (5, 21, 22) or recombinant channels that are expressed heterologously in Xenopus oocytes, where the channels are much less sensitive to calcium and phosphorylation (38-40). Nevertheless, single-channel studies in neurons have also shown stimulation of BK_Ca channel activity by protein kinases (20, 23, 40). Similar diversity in the response to protein kinases and phosphatases has been reported for BK_Ca channels in cells from the vascular system (42-44). As yet there is only one slo gene encoding the pore-forming subunit of the BK_Ca channel in any single species; hence, such diametrically opposed differences in modulation by phosphorylation must reflect alternative splicing of the gene or interaction with additional regulatory subunits or more indirect signaling cascades through which the protein kinases produce their effects.

We have used a number of criteria to identify the specific biochemical mechanism of the observed "run-up" of BK_Ca channel activity in excised membrane patches, although we have not characterized as clearly all of the enzymes or their substrates that are responsible for modulating the availability of BK_Ca channels for opening. This is the first study to investigate the effect of protein phosphorylation on BK_Ca channel activity at maximal open probabilities. As a result, we have discovered that pharmacologically distinct protein kinases produce functionally distinct effects on channel behavior. One kinase, probably PKA, reduces stimulation of BK_Ca channel activity by calcium and voltage without reducing the availability of the channels. For an enzyme, this would correspond to a reduction in K_m without a change in V_max. A second, unidentified kinase reduces the availability of the channels at all calcium concentrations and voltages, corresponding to a decrease in V_max. The exact mechanisms by which the functional consequences of protein phosphorylation are achieved remain unclear. Both  and  subunits of the channel complex contain potential sites for phosphorylation by protein kinase A and protein kinase C (16). The C-terminal "tail" of the  subunit has also been demonstrated to confer the calcium-sensing properties of BK_Ca channels (38); thus, the addition or removal of charged phosphate groups could alter the calcium sensitivity of channel activity by influencing the "Ca²⁺ sensor" in the protein. Alternatively, co-expression of the  subunit has been shown to enhance the calcium sensitivity of BK_Ca channels (13, 39, 45) so reversible phosphorylation could modulate channel behavior by promoting or inhibiting interactions between the  and  subunits in the heteromultimeric channel complex.

Fig. 5 illustrates one hypothesis that integrates the results of this study with previously published results from intact GH₄C₁ cells (12,18) and from BK_Ca channels isolated from rat brain and reconstituted into lipid bilayers (41, 46). When protein phosphorylation is prevented, the channels respond robustly to physiological increases in voltage and calcium. Phosphorylation at one of the modulatory sites inhibits channel activity in a conditional manner that can be overcome by increasing cytoplasmic calcium 3-fold or depolarizing the membrane by an additional 20 mV. Phosphorylation of this "conditional" site requires PKA activity and is protected by low concentrations of okadaic acid, which leads us to postulate that it is regulated by a type 2A-like protein phosphatase. In support of this proposal, it has been shown in bilayers that the inhibitory effect of PKA on brain BK_Ca channels is reversed selectively by PP2A but not PP1 (41).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Diagramatic summary of the experimental results. Boltzmann curves generated from the experimental data (all values given in text). Solid lines represent the Po/V relationships when ATP was omitted; broken lines represent the relationships with ATP present. A, proteins dephosphorylated. The activity of dephosphorylated BKCa channel proteins is sensitive to [Ca2+] and voltage in the physiological ranges (method: patches excised in the absence of exogenous nucleotides). B, phosphorylation at PKA/PP2A site only. Rephosphorylation of the excised patch reduces the [Ca2+] sensitivity of channel activity conditionally, without altering Po(max) (Method: ATP added following stable run up of channel activity). C, phosphorylation at both PKA/PP2A and PKC/PP1 sites. Maximally phosphorylated channel proteins have reduced [Ca2+] sensitivity, and Po(max) is reduced unconditionally to ~50% (Method: intact cells bathed in okadaic acid and patches excised into MgATP and cAMP).

Our results also demonstrate a second pharmacologically and functionally distinct phosphorylation site that regulates channel availability. Phosphorylation at this site modulates BK_Ca channel behavior in an unconditional manner by reducing the maximum attainable open probability to P_o <0.5, even with unphysiologically high calcium concentrations or depolarized membrane potentials. PKC is a powerful inhibitor of BK_Ca channel activity in intact GH₄C₁ cells (18) and in neurons (9). In our experiments, however, unconditional inhibition is lost when the membrane patch is excised from the cell, which implies either that this kinase is not functionally coupled to the channels in cell-free patches or that basal protein phosphatase activity is much greater than the activity of the kinase at this site. Preliminary experiments in which cell-free patches were treated with MgATP and a higher concentration (100 nM) of okadaic acid indicate that channel availability is suppressed under these conditions,² which implies a direct role for another phosphatase less sensitive to okadaic acid than PP2A. Protein phosphatase 1 (PP1) is much less sensitive to okadaic acid than PP2A (31), and PP1 selectively reverses the effects of PKC on BK_Ca channels in other systems (23, 46).

In summary, we have identified two functionally distinct effects of endogenous protein kinases and phosphatases on the activity of calcium- and voltage-activated potassium channels. Phosphorylation at a putative PKA/PP2A site reduces the calcium sensitivity of BK_Ca channels without altering the voltage dependence of activation or suppressing maximum channel availability. In contrast, phosphorylation at a putative PKC/PP1 site reduces the availability of the channels at all calcium concentrations and voltages in the physiological range. Such conditional and unconditional regulation by two pharmacologically distinct signaling pathways provides the nervous and neuroendocrine systems with a powerful computational process at the cellular level. Activation of G_s-coupled receptors would inhibit BK_Ca channel activity and, hence, stimulate cell excitability in a conditional manner, an effect that could be reversed when the intracellular calcium concentration increased or the membrane depolarized further. In contrast, activation of G_q-coupled receptors would inhibit BK_Ca channel activity and stimulate excitability unconditionally until the effects of PKC were reversed by protein phosphatase activity.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Cardiff School of Biosciences, P. O. Box 911, Cardiff University, Museum Ave., Cardiff CF10 3US, Wales, UK. Tel.: 44 1222 875164; Fax: 44 1222 874094; E-mail: hallsk@cardiff.ac.uk.

² S. K. Hall, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: BAPTA, 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; PKA and PKC, protein kinases A and C, respectively; PP1, protein phosphatase 1; PP2A, protein phosphatase 2A.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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