Correspondence to: Steve A.N. Goldstein, Section of Developmental Biology and Biophysics, 295 Congress Avenue, New Haven, CT 06536. Fax:(203) 737-2290 E-mail:steve.goldstein{at}yale.edu.
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Abstract |
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Potassium-selective leak channels control neuromuscular function through effects on membrane excitability. Nonetheless, their existence as independent molecular entities was established only recently with the cloning of KCNKØ from Drosophila melanogaster. Here, the operating mechanism of these 2 P domain leak channels is delineated. Single KCNKØ channels switch between two long-lived states (one open and one closed) in a tenaciously regulated fashion. Activation can increase the open probability to 1, and inhibition can reduce it to
0.05. Gating is dictated by a 700-residue carboxy-terminal tail that controls the closed state dwell time but does not form a channel gate; its deletion (to produce a 300-residue subunit with two P domains and four transmembrane segments) yields unregulated leak channels that enter, but do not maintain, the closed state. The tail integrates simultaneous input from multiple regulatory pathways acting via protein kinases C, A, and G.
Key Words: background conductance, 2 P domain, protein kinases C, A, and G, open rectifier , ORK1
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INTRODUCTION |
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Potassium currents that develop without delay in response to voltage steps and pass current across the physiological voltage-range are called leak (or background) conductances when recorded in native cells (-aminobutyric acid, and serve to establish the resting membrane potential and modify the duration, frequency, and amplitude of action potentials (
Cloning and expression of KCNKØ (previously ORK1) of Drosophila nerves and muscles revealed a leak-type channel that functions like an open, potassium-selective portal in an electric field (
Although single KCNKØ channel currents develop without delay in response to voltage steps (as expected for an open, nonvoltage-gated channel), constant field current formulations (
Here, we show that the opening and closing of KCNKØ is strictly regulated. Single KCNKØ channels are seen to open in long-lived bursts lasting many minutes and to enter an equally long-lasting closed conformation (Clong)1 in a voltage-independent fashion (300-residue amino-terminal segment that is pore-forming, and an
700 carboxy-terminal tail that mediates dwell time in Clong. The tail is found to be essential only for regulation: its deletion yields fully functional channels that enter Clong but cannot remain closed. Finally, regulated gating is seen to employ multiple second messenger pathways that utilize distinct carboxy-terminal KCNKØ residues and act separately or concurrently. Despite their functional and structural independence, pathways using PKC, PKA, and PKG all serve to control dwell time in the closed state Clong.
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MATERIALS AND METHODS |
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Molecular Biology
The cloning and sequence of KCNKØ (previously ORK1) has been described (
Electrophysiology
Xenopus laevis oocytes were isolated and injected with 46 nl containing 0.22 ng cRNA. Whole-cell currents were measured 13 d after injection by two-electrode voltage clamp (Warner Instruments Corp.). Data were filtered at 1 kHz and sampled at 4 kHz. The patch-clamp technique was used to record single channels in on-cell patches 24 d after cRNA injection using an EPC-9 amplifier (HEKA Elektronik) and stored on videocassettes. For analysis, records were sampled at 20 kHz or 940 Hz using ACQUIRE software (Bruxton Corporation, Inc.) and digitally filtered at 3 kHz or 100 Hz, respectively. Kinetic analyses were performed on patches judged to contain only one channel on the basis of the single current level. Closed and open durations were determined using a half-amplitude threshold-detected technique (
Unless otherwise noted, the bath solution for two-electrode voltage clamp experiments contained (in mM): 20 KCl, 78 NaCl, 1 MgCl2, 0.3 CaCl2, 5 HEPES, pH 7.5, with NaOH. For patch-clamp experiments, both pipet and bath solutions contained (in mM): 140 KCl, 2 MgCl2, 5 EGTA, 5 HEPES, pH 7.4, with KOH. All kinase modulators were purchased from Calbiochem-Novabiochem">Calbiochem-Novabiochem. All experiments were conducted at room temperature.
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RESULTS |
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Modulators of Protein Kinase C Regulate Activity of KCNKØ Channels
KCNKØ subunits contain 42 canonical consensus sequences for phosphorylation in the 700-residue carboxy-terminal tail region, which is predicted to be intracellular; as 11 sites are for PKC, the PKC activator PMA was evaluated. When KCNKØ was expressed in Xenopus laevis oocytes, exposure to 50 nM PMA had a dramatic effect on channel activity, increasing mean whole-cell currents up to 11-fold (Fig 1A and Fig B). Similar results were produced by steady exposure to 5 or 100 nM PMA for 10 or 20 min (not shown, n = 810). Conversely, the inactive PMA analogue 4-phorbol-12,13-didecanoate had no effect (500 nM, n = 10; not shown). When PMA-treated cells were bathed in a nonspecific protein kinase inhibitor (2 µM staurosporine), the current augmentation was reversed (Fig 1A and Fig B). Similarly, bisindolylmaleimide I (4 µM), a specific PKC inhibitor, reversed the effect of PMA treatment (Fig 1 C). After staurosporine or bisindolylmaleimide I, reexposure to PMA again increased KCNKØ channel currents (Fig 1 C).
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Upregulation did not change the attributes of macroscopic KCNKØ currents; they continued to develop instantaneously in response to changes in membrane voltage and were noninactivating (Fig 1 B). Activated KCNKØ channels were also unchanged in their selectivity for potassium over sodium; a 10-fold increase in bath potassium concentration (achieved by isotonic substitution for sodium) altered whole-cell reversal potentials by 52 ± 1 mV in PMA or control solution (n = 4).
Single KCNKØ Channels Occupy Long-lived Open Burst and Closed States
To assess the mechanism by which PKC activators and inhibitors altered macroscopic KCNKØ currents, single channels were studied. Channel behavior was first evaluated in untreated cells. Fig 2 A shows a single KCNKØ channel in an on-cell patch held at 60 mV for 17 min. The record reveals transitions of the channel between long-lived open burst and closed conformations that last for many minutes. Expanding a portion of the record during an open burst (Fig 2 A, inset) reveals the presence of short intraburst closures that are quantified below. The mean duration of open bursts (Oburst) and long-lived closures (Clong) were 50 and
70 s, respectively (Table 1). KCNKØ channels in untreated cells were open approximately one third of the time (open probability, Po, Table 1).
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Residence of Single KCNKØ Channels in the Long-lived Closed State Is Regulated
Single KCNKØ channels behaved quite differently when treated with 50 nM PMA (Fig 2 B). The PKC activator virtually eliminated residence in the long-lived closed state. The open probability for single KCNKØ channels during exposure to PMA was 0.993 ± 0.003 (Table 1). Conversely, application of staurosporine depressed the open probability to <0.05 by increasing the duration of long closures and decreasing the frequency and duration of open channel bursts (Table 1).
To further explore the influence of PMA on channel state, recordings of single KCNKØ channels were first analyzed at a bandwidth of 100 Hz, and then reanalyzed at 3 kHz to assess brief closed times. Closed time distributions in the absence of the PMA were best fit by four time constants (Fig 3 B); these represented three brief closed states present in open bursts and the long-lived closed states (Clong) that separated open bursts. Channels in untreated cells showed mean dwell times for closures within bursts of 0.14, 4, and 100 ms while the time constant for the long-lived interburst closed state was 71.4 s (Table 1). Dwell times for openings within bursts were well-fit by a single time constant of 2 ms (Fig 3 A) and multichannel patches showed no evidence for additional open states. The mean open burst duration for KCNKØ channels in untreated cells was 50 s (Table 1). After PMA treatment, closed times were also well-fit by four exponential components (Fig 3 D). PMA did not change the mean duration of the three intraburst closed states or alter the mean open time (Table 1). However, PMA reduced the frequency of long-lived closures 8-fold and their duration 30-fold (Table 1).
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To assess the effect of staurosporine, patches were first treated with PMA to confirm that only a single KCNKØ channel was present. 14 min after PMA was replaced by staurosporine, profound current suppression was observed. Four single KCNKØ channels, studied for a total of 29 min, demonstrated just five open bursts (that were approximately fivefold shorter than open bursts under control conditions) and nine long closures that lasted no less than 45 s with four longer than 150 s (the time when the experiment was concluded by discarding the patch or reexposure to PMA). This offered a rough estimate for open probability of <0.05 (Table 1).
No change in the single channel current amplitude was observed in >100 min of the recording of single KCNKØ channels in control, PMA-exposed, or staurosporine-treated cells (Fig 2). This argued that changes in macroscopic currents were due to altered gating of KCNKØ channels. Consistent with this conclusion was the uniform stepwise decrease in the current observed when four KCNKØ channels in one patch were first fully activated by PMA and subsequently driven into the long-lived closed state by exposure to staurosporine (Fig 3 E).
The Carboxy-terminal Portion of KCNKØ Is Required to Regulate Long Closures
The pore-forming portion of KCNKØ subunits has two P domains and four predicted transmembrane segments extending from amino acid 1 to 264; the residues that follow are hydrophilic in nature and predicted to be cytoplasmic (Fig 4 A). To assess the role of the carboxy-terminal region in regulation, subunits were produced that contained only residues 1298 (KCNK
299-1001). The truncated subunits lacked most of the carboxy terminus and 10/11 consensus sites for PKC-mediated phosphorylation. The channels formed with KCNK
299-1001 subunits were fully functional except that they showed no regulation by PKC modulators: the subunits were unaffected by exposure to either PMA or staurosporine (Fig 4B and Fig C). Thus, oocytes expressing wild-type KCNKØ channels showed an
11-fold increase in macroscopic currents when treated with PMA for 20 min, whereas KCNK
299-1001 channels showed no change (Fig 4 D).
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The Carboxy Terminus Does Not Affect Ion Selectivity or Unitary Current Amplitude
Although KCNK299-1001 channels were unresponsive to PMA and staurosporine, their other functional attributes were well-preserved. Like the wild type, KCNK
299-1001 channels showed macroscopic currents that developed instantaneously with changes in transmembrane voltage and were noninactivating (Fig 4 B). Channels formed with truncated subunits showed the same selectivity for potassium over sodium as wild-type channels (a 10-fold change in bath potassium produced the same shift in reversal potential of 52 ± 2 mV, n = 4). Further, wild-type and KCNK
299-1001 channels displayed the same relative permeability for monovalent cations (K+ > Rb+ > Cs+ >> Na+ and Li+) based on whole-cell reversal potential measurements (Table 2). This indicated that carboxy-terminal residues were not essential for normal selective ion permeation through KCNKØ channels. Moreover, single wild-type and KCNK
299-1001 channels exhibited the same unitary conductance: 64 ± 2 pS for truncated channels, and 63 ± 1 pS for wild type (Fig 5).
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The Carboxy Terminus Is Not a Channel Gate
Single channel recordings supported the idea that deletion of the carboxy terminus did not change the states visited by KCNKØ channels, but rather it appeared primarily to alter the stability of the long-lived closed conformation, Clong. Thus, single wild-type and KCNK299-1001 channels were almost indistinguishable within bursts from -120 to 60 mV (Fig 5A and Fig B). Like the wild type, mutant channels revealed one open and three brief closed states within open bursts and one long-lived closed state (Fig 6, Table 1). Furthermore, time constants for the four closed states were similar for KCNK
299-1001 and wild-type channels activated by PMA; although, mutant channels visited their longest closed state more frequently, producing a shorter mean burst duration (Table 1). These findings suggested that residues 2991,001 were critical to sustaining the long-lived closed state, whereas residues 1298 were sufficient to form the ion conduction pathway and closing gates.
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Multiple Regulatory Pathways Act Independently via the KCNKØ Carboxy Terminus
In addition to 11 classical consensus sites for PKC-mediated phosphorylation, the carboxy terminus of KCNKØ has all of the following: 1 site for tyrosine kinase (TK); 2 for protein kinase B; 14 for casein kinase II; 8 for protein kinase A (PKA) or G; and 5 for PKG. Baseline whole-cell KCNKØ currents changed <20% when exposed to agents that inhibit TK (100 µM tyrphostin A25, n = 8; 100 µM genistein, n = 8) or PKB (100 nM wortmannin, n = 5). These agents were also without effect when applied after PMA activation (n = 56).
In contrast, activation of PKA by a mixture of 3-isobutyl-1-methylxanthine (IBMX, 1 mM) and forskolin (20 µM) or cytoplasmic microinjection of 8-Br-cAMP (to an internal concentration of 450 µM) produced an approximately fourfold increase in KCNKØ current (not shown, n = 6). Similarly, the PKG activator 8-Br-GMP (microinjected to
450 µM) also produced an approximately fourfold increase in the KCNKØ current (not shown, n = 6). Upregulation by these agents was not due to surreptitious activation of PKC or stimulation of a PKC-dependent pathway. Although the PKC inhibitor bisindolylmaleimide I blocked activation by PMA (Fig 7A and Fig B), it did not modify stimulation by IBMX and forskolin (Fig 7 A) or 8-Br-cGMP (Fig 7 B). Reciprocally, it was seen that PKC activation was not via a PKA-dependent mechanism, whereas the PKA inhibitor H89 (5 µM) suppressed the response to IBMX and forskolin, it did not ablate PMA-induced activation (Fig 7 C). As for PKC, PKA and PKG did not alter the function of truncated KCNK
299-1001 channels that lacked the carboxy terminus (not shown).
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That channel activation by PKA or PKG proceeds despite concurrent inhibition of PKC (Fig 7A and Fig B) and that activation of PKC proceeds despite PKA inhibition (Fig 7 C) indicates that the regulatory pathways function independently. That PKC activation further increases currents previously upregulated by PKG and PKA (Fig 7 D) indicates that the pathways can act concurrently. Activation of PKA after PKG-induced upregulation yielded no significant additional increase in current (Fig 7 D); this suggested PKA and PKG alter channel function by a similar mechanism. Like activation mediated by PKC, upregulation by PKA increased the open probability of KCNKØ channels by decreasing the frequency and duration of visits to the long-lived closed state, Clong (Fig 7, EG, and Table 1).
KCNKØ Residues that Mediate PKC and PKA Regulation Are Distinct
To identify residues involved in PMA-induced upregulation, each serine or threonine in the 11 consensus sites for PKC-mediated phosphorylation was mutated individually to a nonpolar residue; 10 point mutants behaved like wild type when exposed to PMA (Fig 8 A). Conversely, the subunit mutated to leucine at position 270 (KCNKØ-S270L) formed channels that activated abnormally. Thus, wild-type KCNKØ currents rose in response to PMA in two phases: first, readily, and then more slowly (Fig 8 B). In contrast, KCNKØ-S270L channels showed rapid development of peak currents like wild-type channels (4.4 ± 0.2 and 4.6 ± 0.3-fold increase at 5 min, n = 812, respectively) but, thereafter, returned slowly toward baseline without evidence for a slow phase of current activation.
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Six additional truncation mutants were also studied (Fig 8 C). Deletion of 40 or 80 residues from the carboxy terminus yielded channels that responded to 50 nM PMA like wild type (961-1001 and
921-1001). Conversely, larger deletions produced channels that showed minimal (
872-1001,
736-1001, and
619-1001) or no response to PMA (
407-1001 and
299-1001). Studies of these mutants suggested a role for residues between 872 and 921 in the rapid phase of current activation as
872-1001 channels responded only slowly to PMA (Fig 8 B). Further changes were not seen with mutation of both local PKC consensus sites (S880L,S914V), motifs like those that bind synapsins (PPPPP889,890,894,895,896AAAAA), proteins with SH3 domains (P794A), or with WW domains (PP848,849AA; not shown).
Combining mutations that removed fast activation (872-1001) and the slow second phase response (S270L) was sufficient to eliminate upregulation by PMA at both the macroscopic and microscopic levels (Fig 8 D). Single KCNKØ-S270L
872-1001 channels under control conditions revealed a low open probability (0.04 ± 0.01, 64 min, n = 4) similar to wild-type channels in the presence of staurosporine (Table 1); this was due to an increased frequency of long-lived, interburst closures (Clong), without a significant change in their duration, for the mutant channels compared to wild type (Table 1). Conversely, the combined mutations did not alter the frequency or duration of brief closures, mean open time, or single-channel conductance (Fig 8 E). Whereas KCNKØ-S270L
872-1001 channels were unresponsive to activators of PKC, they retained their sensitivity to PKA modulators. Thus, PMA did not alter activity of the mutant, whereas IBMX and forskolin activated both mutant and wild-type KCNKØ channels similarly (Fig 8 F). This indicated that PKC and PKA regulators acted at distinct sites in the KCNKØ carboxy terminus to alter open probability despite their common effector mechanism: control over dwell time in the long-lived closed state.
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DISCUSSION |
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KCNKØ Leak Channel Gating: Tightly Regulated Opening and Closing
Strictly regulated, potassium-selective leak conductances appear fundamental to excitability, synaptic transmission and neural plasticity (
Two Functional Domains: Pore-forming and Regulatory
KCNKØ subunits were found to have two functional segments: one with a pore and gates and one with channel regulatory apparatus. KCNKØ residues 1298 contain two P domains and four predicted transmembrane segments. On its own, this segment can form an ion conduction pathway and attain open and closed conformations like those observed with complete KCNKØ channels (Fig 4 and Fig 6). Thus, KCNK299-1001 channels show single channel conductance (Fig 5), ion selectivity (Table 2), and gating kinetics like PMA-activated wild-type channels (Table 1). The large carboxy terminus is essential for regulation of channel function. Channels without residues 2991,001 cannot maintain the long-lived closed state (Table 1) and are unaffected by activators or inhibitors of PKC (Fig 4), PKA, or PKG (not shown). Moreover, upregulation of wild-type KCNKØ channels does not alter unitary conductance (Fig 2), intraburst gating kinetics (Table 1), or ion selectivity (Table 2). These findings support the idea that the carboxy terminus does not contribute physically to channel gates or act as a blocking particle (
PKC, PKA, and PKG: Independent Collaborators in Regulation of KCNKØ Closed State Dwell Time
Regulation of KCNKØ involves at least two regulatory pathways (PKC and PKA/G) that can act independently or concurrently (Fig 7) through distinct carboxy-terminal residues (Fig 8). Despite their functional and structural independence, both pathways control the frequency and dwell time of KCNKØ channels in a single conformation, the long closed state (Table 1). Indeed, the response to PMA or IBMX and forskolin is similar at the single-channel level at steady-state (Table 1). Conversely, macroscopic current development is greater with PMA than IBMX and forskolin (Fig 8 F) and fails to saturate (Fig 7, AD). This suggests PMA might also act by other mechanisms to increase the current. As PMA treatment does not alter membrane capacitance significantly (not shown), we speculate that quiescent KCNKØ channels already in the membrane may emerge from an even more deeply closed state. This mechanism has been seen with upregulation of other channels by PKC (
Potassium channels are well recognized targets for protein kinases and phosphatases (
The KCNK Superfamily: Regulated Leak Channels
The 2 P domain potassium channel superfamily has grown rapidly since isolation of TOK1, the nonvoltage-dependent outward rectifier of Saccharomyces cerevisiae with a predicted 2P/8TM topology (
Another attribute shared by KCNK leak channels is regulated activity. Opening and closing of KCNKØ channels was shown here to depend quite strictly on activation (or inhibition) of PKC, PKA, or PKG. Regulation of other KCNK channels is notable if somewhat less aggressive. Activity of KCNK2 channels was moderately depressed by activators of PKC and PKA (
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Footnotes |
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N. Zilberberg and N. Ilan contributed equally to this work.
1 Abbreviations used in this paper: Clong, long-lasting closed conformation; IBMX, 3-isobutyl-1-methylxanthine; Oburst, open burst; Po, open probability; TK, tyrosine kinase.
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Acknowledgements |
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We are grateful to F. Sigworth and N. Goldstein for thoughtful advice during the course of these studies.
This work was supported by grants from the National Institutes of Health (to S.A.N. Goldstein), the Human Frontier Science Program (to N. Zilberberg), and the Bi-national Agricultural Research and Development Fund (to N. Ilan).
Submitted: 2 June 2000
Revised: 11 September 2000
Accepted: 2 October 2000
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