Department of Physiology and Program in Neuroscience, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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The regulation by protein kinase C (PKC) of recombinant voltage-gated potassium (K) channels in frog oocytes was studied. Phorbol 12-myristate 13-acetate (PMA; 500 nM), an activator of PKC, caused persistent and large (up to 90%) inhibition of mouse, rat, and fly Shaker K currents. K current inhibition by PMA was blocked by inhibitors of PKC, and inhibition was not observed in control experiments with PMA analogs that do not activate PKC. However, site-directed substitution of potential PKC phosphorylation sites in the Kv1.1 protein did not prevent current inhibition by PMA. Kv1.1 current inhibition was also not accompanied by changes in macroscopic activation kinetics or in the conductance-voltage relationship. In Western blots, Kv1.1 membrane protein was not significantly reduced by PKC activation. The injection of oocytes with botulinum toxin C3 exoenzyme blocked the PMA inhibition of Kv1.1 currents. These data are consistent with the hypothesis that PKC-mediated inhibition of Kv1.1 channel function occurs by a novel mechanism that requires a C3 exoenzyme substrate but does not alter channel activation gating or promote internalization of the channel protein.
phorbol ester; Shaker; voltage clamp; Xenopus oocyte
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INTRODUCTION |
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VOLTAGE-GATED K CHANNELS are a diverse family of proteins that function in the regulation of action potentials, cardiac pacemaking, signal integration, and neurotransmitter release in excitable cells. In nonexcitable cells, K channels help modulate hormone secretion, cell volume regulation, cell proliferation, and lymphocyte differentiation. The highly localized expression of the different molecular species of K channels (10) suggests that K channels may be specialized for different cellular functions. However, little comparative information is available regarding the second messenger regulation of the many different but related molecular forms of voltage-gated K channels. An understanding of how the different K channels are regulated by neurochemicals is critical to understanding the diverse and important functions of these channels.
The phosphorylation of K channels by protein kinase C (PKC) may regulate channel function. In several cell types, native K channel currents are affected by agents that directly activate PKC or by receptor systems that activate PKC through a second messenger cascade. For example, PKC activation inhibits a K current in cultured endothelial cells (38) and K currents in brain stem respiratory neurons (5) but enhances a K current in ventricular cells (36). In the Xenopus oocyte expression system, several types of recombinant, voltage-gated K channels have been shown to be modulated by PKC (18, 19, 21, 23, 29), and in some cases there is evidence that the K channel protein is the substrate for PKC phosphorylation (2, 3, 4, 8). However, the mechanisms and sites of kinase regulation of K channel activity are not known for many types of cells and K channels.
Xenopus oocytes are a useful system for studying the regulation of ion channel activity. The oocytes express a variety of native components of second messenger cascades including the G proteins Go and Gq (25), phospholipase C (14), and PKC (26). Thus the molecular mechanisms of receptor-channel coupling can be studied by using the coexpression of foreign cell surface receptor and channel proteins linked by the native oocyte second messenger pathways.
In the present study, we tested for possible physiological differences in the modulation by PKC of recombinant voltage-gated K channels. We found that PKC activation had different consequences for K currents encoded by genes from the Shaker (Kv1), Shab (Kv2), Shaw (Kv3), and Shal (Kv4) subfamilies. PKC-mediated downregulation of Kv1.1 currents occurs by a novel mechanism that is inhibited by the C3 exoenzyme of botulinum toxin without an effect on channel gating or a significant reduction in K channel protein present at the membrane surface.
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MATERIALS AND METHODS |
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Expression of K channels.
Plasmids containing the cDNAs encoding voltage-gated K channels were
linearized, and capped RNAs were synthesized in vitro with Ambion
(Austin, TX) RNA polymerase kits. Similarly, plasmids containing turkey
P2Y or human
P2U receptor cDNAs (gifts from T. K. Harden, Univ. of North Carolina, Chapel Hill, NC) were linearized and transcribed. RNA was purified by use of the RNAid kit (Bio 101, Vista, CA) and stored at 80°C in diethyl pyrocarbonate
(DEPC)-treated water. Transcription reaction products were checked for
size by agarose gel electrophoresis, and RNA concentrations were
determined by spectrophotometry (Beckman DU-7000) by using the equation
1 A260 unit = 40 µg/ml RNA, where
A260 is
absorbance at 260 nm. K channel cDNAs were generous gifts
from M. Covarrubias (mKv4.1), R. Joho (rKv2.1), O. Pongs (rKv1.1 and
rKv3.4), L. Salkoff (rKv4.2), and B. Tempel (mKv1.1). We also used
ShakerH4 from
Drosophila melanogaster and a deletion mutant
Sh
(residues 6-46 deleted) that removes fast,
N-type inactivation (13).
Mutagenesis. Mutations were introduced into the mKv1.1 K channel cDNA in a Bluescript KS(+) vector (Stratagene) by oligonucleotide-directed mutagenesis. Miniprep DNA was prepared from isolated colonies with a DNA isolation kit (Promega). Mutant DNA was identified by dideoxy sequencing with Sequenase-modified DNA polymerase (United States Biochemical) and electrophoresis on 6% polyacrylamide urea sequencing gels. For each site studied, two mutant colonies were sequenced and prepared and tested separately.
Electrophysiology.
Macroscopic K currents from oocytes were recorded with a two-electrode
voltage clamp equipped with an OC-725B amplifier (Warner Instrument,
Hamden, CT). Voltage-measuring and current-passing electrodes were
filled with 3 M KCl and had resistances between 0.3 and 1.1 M.
Currents were sampled at 5 kHz and filtered at 2 kHz with an eight-pole
Bessel filter (Frequency Devices, Haverhill, MA). All recordings were
done at room temperature (~20°C). Oocytes were clamped at
70 or
80 mV (as noted in figure legends) and pulsed every 1-5 s to various test potentials to activate K channels.
Biotinylation and recovery of surface proteins. Kv1.1-expressing oocytes (54-60/treatment) were washed twice with 2 ml of frog Ringer solution and then incubated in a labeling solution of 1 mg/ml EZ-Link NHS-SS-biotin [sulfosuccinimidyl 2-(biotinamido) ethyl-1,3-dithiopropionate; Pierce, Rockford, IL] dissolved in frog Ringer solution, pH 7.4. We used modifications to the methods of Stern-Bach et al. (28). Cells were labeled for 2 h at 21°C with gentle mixing on a laboratory shaker and then washed twice for 10 min each in frog Ringer solution with gentle mixing. Biotin-labeled oocytes were then washed once with 1 ml ice-cold homogenization buffer (20 mM Tris · HCl, 200 mM NaCl, pH 7.4) containing a protease inhibitor cocktail: 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml aprotinin, 1 µM pepstatin (all from Boehringer Mannheim), and 2 µg/ml antipain and 1 µg/ml leupeptin (both from Sigma). Oocytes were then homogenized by several passages through a pipette tip in 200 µl ice-cold homogenization buffer with protease inhibitors. Membrane-bound proteins were solubilized by 1.5% Triton X-100 (Sigma). Samples were incubated for 1 h on ice and centrifuged for 30 min (16,000 g at 8°C). The supernatant was carefully removed to a fresh tube. A 5-µl aliquot was removed and represented the total protein (T) fraction. The remaining supernatant was incubated for 2 h with a 20-µl bed volume of UltraLink immobilized monomeric avidin beads (Pierce; 50% slurry equilibrated in homogenization buffer with 1.5% Triton X-100) with gentle mixing on a laboratory rotator at 8°C. The resulting biotin-avidin complex was washed five times by suspension in 1 ml homogenization buffer (with the protease inhibitor cocktail and 1.5% Triton X-100). The samples were centrifuged in a PicoFuge (Stratagene) (1 min, 325 g), and the supernatant was carefully removed. Membrane proteins were released from the biotin-avidin complex by adding 25-50 µl loading buffer with reducing agent [0.13 M Tris · HCl (pH 6.8), 10% glycerol, 2% SDS, 150 mM dithiothreitol, 0.01% bromphenol blue] and gently mixed for 15 min at 21°C. Beads were removed by centrifugation for 2 min at 10,000 g. The resulting supernatant was collected as the membrane-bound protein (M) fraction. In some experiments, protein concentration was determined by the bicinchoninic acid protein assay (Pierce), according to the manufacturer's instructions.
SDS-PAGE and Western blots. The oocyte lysates were denatured in Laemmli sample buffer (161-0737; Bio-Rad) for 5 min at 100°C and electrophoresed on an SDS-7.5 or 10% polyacrylamide gel. To obtain a sufficient signal on the Western blot, we generally loaded the gel with 1-2 µl of the T fraction (represents total Kv1.1 protein in 0.25-0.5 oocytes) and the full volume of the M fraction (represents membrane Kv1.1 protein in 50 oocytes). After electrophoresis, separated proteins were transferred to an Immobilon-P polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA) by electroblotting. Rainbow colored protein molecular weight markers (Amersham Life Sciences) were used.
The PVDF membrane was rinsed in distilled water and blocked with 4% Blotto [4% nonfat dried milk in Tris-buffered saline, pH 7.6, with 0.1% Tween 20 (TBS-T)] for 1 h at room temperature or overnight at 4°C. The blots were incubated with primary antibodies in sealable pouches. We used 1 µg/ml anti-rat Kv1.1 monoclonal antibody (Upstate Biotech or gift from J. Trimmer), diluted in 4% Blotto, for 1 h at room temperature on a shaking platform. The anti-Kv1.1 antibody was raised against residues 458-476, part of the putative intracellular COOH terminus of rat brain Kv1.1. After primary antibody incubation, the blot was washed five times in TBS-T for 5 min each. The blot was then incubated with the secondary antibody for 1 h on a shaking platform in a Pyrex dish. We used a 1:80,000 dilution of goat anti-rat IgG conjugated with horseradish peroxidase (Sigma) and diluted in 4% Blotto. Finally, the blot was washed three times in TBS-T for 10 min each. Immunolabeled proteins were detected by the ECL Plus enhanced chemiluminescence detection system (Amersham Life Science). X-ray films were scanned and analyzed with a model GS-700 scanning densitometer and Molecular Analyst software (Bio-Rad). A crude synaptosomal membrane fraction was prepared from freshly dissected adult mouse brains as described by Trimmer (33), and an aliquot (6 µg protein/lane) was included as a positive control for Kv1.1 expression. Extracts from uninjected oocytes do not react with the Kv1.1 antibody (data not shown). We probed the M and T fractions with an anti-actin primary antibody (Sigma) that specifically recognized an ~46-kDa band present in the mouse brain extract and in purified actin, run as a positive control for the antibody. By Western blotting, native oocyte actin was detected only in the T fractions, not in the M fractions prepared from either uninjected oocytes or Kv1.1-expressing oocytes (data not shown). We probed for actin because it is a ubiquitous cytoskeletal protein that is known to associate with some plasma membrane proteins, but actin itself is not an integral membrane protein. Thus the lack of anti-actin reactivity in the M fraction is consistent with a lack of contamination of the M fraction with proteins not embedded in the plasma membrane.Drugs.
Phorbol 12-myristate 13-acetate (PMA), phorbol 12-monomyristate (PMM),
4-phorbol, and staurosporine were from Sigma Chemical. Phorbol
esters were prepared as stock solutions in DMSO and then diluted into
standard bath solution before recording; the final concentration of
DMSO was
0.1%. Bisindolylmaleimide I and ATP (Na+ salt) were from Boehringer
Mannheim. For pharmacological experiments, staurosporine (prepared in
DMSO) and the C3 exoenzyme of botulinum toxin (prepared in DEPC-water)
were injected into oocytes 2 days after injecting channel RNA. On the
basis of an estimate of 500 nl for oocyte volume, staurosporine was
injected to achieve a final (estimated) cytoplasmic concentration of
0.9 µM. C3 exoenzyme was injected at 50-100 pg/oocyte. Control
experiments for staurosporine included 0.1% DMSO-injected oocytes.
Control experiments for C3 exoenzyme included injection of an equal
concentration of C3 exoenzyme that had been heated to >110°C for
2 h and allowed to cool to room temperature before injection. Oocytes
injected with C3 or "heat-inactivated" C3 were then incubated at
different temperatures before electrophysiology, as described in
RESULTS.
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RESULTS |
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PMA application inhibits Shaker K currents.
Bath application of the PKC activator PMA (500 nM) caused large
inhibition of Shaker subfamily K
channel currents expressed in frog oocytes (Fig.
1A).
PMA applied to oocytes expressing mouse Kv1.1
(n = 8), rat Kv1.1
(n = 7),
Drosophila Sh and
ShakerH4 (n = 4), and mouse Kv1.3
(n = 3) caused nearly complete
inhibition in some cases, with an average of 73% current inhibition.
The time courses and magnitudes of the inhibition were the same
regardless of the Shaker channel
species. Voltage-gated K channels from the Shab (Fig.
1B) and
Shal (Fig.
1C) subfamilies were much less
affected by PMA application than were K channels from the
Shaker subfamily (Fig.
1D). At the membrane potential
indicated in the legend for Fig. 1, levels of current
inhibition were compared, either at the peak current (for inactivating
currents) or at the end of a 60- to 80-ms pulse if currents lacked
N-type inactivation. Because of the selectively large inhibitory effect
of PMA on Shaker subfamily K currents,
we further studied the inhibition of mKv1.1 K channels.
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Pharmacological specificity.
Phorbol esters such as PMA are membrane-permeant drugs that bind with
high affinity to the diacylglycerol site of PKC. To test whether the
inhibition of mKv1.1 K channel currents by PMA was due to the
activation of native oocyte PKC, we used inactive phorbols and
inhibitors of PKC activation. Two structural analogs of PMA that do not
activate PKC in in vitro phosphorylation assays (for review see Ref.
22), 4-phorbol and PMM, were ineffective inhibitors of K channel
current (Fig. 2, B,
C, and
F). The analog 4
-phorbol always
caused a small and reversible inhibition of current (Fig.
2B), unlike the persistent
inhibition produced by PMA (Fig.
2A). PMM caused a small but
insignificant (unpaired t-test;
P < 0.05) inhibition of mKv1.1 K
current 15 min after a 1-min application of 500 nM drug (Fig. 2,
C and
F).
Receptor-dependent coupling of PKC to K currents.
The inhibition of Shaker subfamily K
channel currents by PMA persisted with up to 2 h of washing. This might
be explained by either the persistent activation of PKC by PMA that
accumulates and cannot be washed from the oocyte membrane or the
presence of a stable phosphorylation event. To distinguish between
these ideas, we studied K channel modulation by receptor-stimulated PKC
activation. We coinjected oocytes with RNA encoding the mKv1.1 K
channel and a P2Y purinergic
receptor, previously shown to activate phospholipase C in stably
transfected astrocytoma cells (11). In coinjected oocytes (Fig.
3A), the P2Y agonist
ATP (100 µM) mimicked the effect of PMA inhibition of mKv1.1 K
current (n = 6). ATP had no persistent
effect on mKv1.1 K channels in oocytes injected only with channel RNA
(Fig. 3B; n = 4 cells).
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Current gating after inhibition by PKC.
We plotted conductance-voltage
(G-V)
relationships and scaled current traces during the inhibition of K
current to study the impact of PKC activation on current gating. We
observed overlap of the control and inhibited-K-current
G-V
profiles. The large inhibition of the K current by PMA (Fig.
4A) or
ATP application to cells coexpressing K channels and
P2Y receptors (Fig.
4B) was not accompanied by a
significant change in the voltage range of activation or the slope of
the activation curve. Furthermore, mKv1.1 K channel currents were
inhibited by PKC activation without a significant effect on current
kinetics (Fig. 4C). Scaling the inhibited current to match the steady-state control current showed that
inhibition by PMA did not alter macroscopic activation kinetics (Fig.
4D). This demonstrates that the
large inhibition measured cannot be simply explained by an effect on K
channel activation gating. This is in contrast to other reports that
PKC phosphorylation may change the voltage dependence of ion channel
activation gating, possibly through direct phosphorylation of the
channel protein (1, 24). With activating pulses up to 10 s
long, PMA inhibition also did not alter C-type inactivation rates (data
not shown). In ShakerH4 channels with
intact N-type inactivation (Fig.
1A) and in fast-inactivating
channels of the Shal subfamily (Fig. 1C; rKv4.2), PMA inhibition also did
not change the rate of inactivation (see also Ref. 21).
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PKC activation does not inhibit all K channel subfamilies. PKC activation does not inhibit to the same degree all recombinant voltage-gated K channels expressed in oocytes. As reported previously for a human Shaw subfamily K channel (8), we confirmed that PMA application largely removed the N-type inactivation of rKv3.4 (in 5 of 5 cells; data not shown). In coinjected oocytes, we found that functional coupling to purinergic receptor activation largely removed the N-type inactivation of rKv3.4 (in 3 of 3 cells; data not shown). Because this PKC-mediated pathway has been well studied (2, 8), it provided an interesting comparison for the time course of the PKC-mediated event we studied. We observed that the inhibition of inactivation of rKv3.4 by either PMA or purinergic receptor activation required 5-10 min to reach maximal effect and was a relatively stable event, similar to the PKC-dependent inhibition of Shaker subfamily K channels. Thus the time courses and levels of stability of the PKC phosphorylation events for the different groups of oocytes studied may be similar even though PKC activation may have different consequences for the function of different K channel proteins. In cells expressing multiple types of K channels, PKC activation could be an important and complex process for regulating cellular excitability and signaling.
Site-directed mutagenesis of PKC phosphorylation sites. We identified only three potential PKC phosphorylation sites on mKv1.1 channels, based on the consensus sequence identified by Woodgett et al. (37): Ser/Thr-X-Lys/Arg, where X is generally an uncharged residue. Residues 318 and 322 in mKv1.1 channels are present in the S4-S5 linker, and residue 442 is in the S6 region, all thought to be intracellular in current working models of the channel. The PKC phosphorylation sequence at Thr-318 is conserved in most voltage-gated K channels (except Kv1.5 and Kv1.6), whereas the phosphorylation sequence at Ser-322 is conserved throughout the Shaker subfamily and in Drosophila Shab and Shaw. The phosphorylation sequence at Ser-442 in mKv1.1 is shared only by Kv1.1 channels of the Shaker subfamily.
Using mutagenesis experiments, we tested the hypothesis that PKC inhibition of mKv1.1 K channel current is due to phosphorylation of one of these three consensus PKC sites, present in four-fold symmetry in the homotetrameric K channel. We replaced, one at a time, the native serine or threonine residue at position 318, 322, or 442 in mKv1.1 with alanine or valine. These conservative substitutions remove the PKC phosphorylation sequence. The mutant channels were sequenced, expressed, and tested for inhibition by PMA. Channel mRNAs for T318A and S442A mutants gave rise to K currents with no functional differences in macroscopic gating or current stability recorded. In addition, both mutants demonstrated K current inhibition after PMA application, with effects not significantly different (unpaired t-test) from those found in control experiments. K currents from the T318A mutant were inhibited by 83.3 ± 6.8% (n = 6), and currents from the S442A mutant were inhibited by 69.5 ± 6.3% (n = 5). In addition, channels expressing both the T318A and S442A mutations were also inhibited by PKC activation (76.8 ± 10%; n = 5). For comparison, wild-type mKv1.1 currents were inhibited by 73.4 ± 5.2% (n = 8). The only difference in these mutant channels was a current amplitude smaller than that for wild-type channels, after injections of the same quantity of RNA (generally 2- to 3-fold-smaller currents). The mutant S322A did not express functional K channel currents, even though all three of the mutant RNAs directed membrane protein expression that could be identified on a Western blot with a Kv1.1-specific primary antibody (not shown). We conclude that at least two of the three consensus PKC phosphorylation sites are not required for the PKC-mediated inhibition of mKv1.1 K current. We cannot rule out the possibility that additional, unidentified phosphorylation sites exist in the native, folded protein.Assay for channel internalization. As shown in Fig. 4, the inhibition of mKv1.1 K channel current was not accompanied by changes in the channels' G-V relationship or kinetics of activation, suggesting that the channels that contribute the remaining current are not functionally different from the channels that account for the inhibited current. Instead, the inhibition of K current may reflect a reduction in the number of functional K channels by, for example, downregulating the K channels from the membrane surface. Indeed, several pumps, transporters and ion channels (7, 9, 34), and G protein-coupled receptors (12, 35) are thought to be up- or downregulated by insertion or internalization from the plasma membrane, and these processes may also require a phosphorylation event.
The possible movement of biotin-labeled Kv1.1 K channels from the membrane surface to a nonmembrane (cytosolic) compartment was determined by avidin recovery of the biotinylated surface proteins and subsequent immunoblotting for Kv1.1 protein. Groups of oocytes were placed in single wells of a 24-well plate and exposed for 2 min to one of the following, dissolved in extracellular recording solution: 0.01% DMSO, 500 nM 4
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Botulinum toxin sensitivity. The specific components of the regulatory pathway of the PKC-mediated inhibition of Kv1.1 K channels are not known. Mechanisms ascribed to other channel regulatory pathways, such as phosphorylation of the channel protein and either modulation of the voltage sensor or translocation from the membrane to the cytosol, seem unlikely to explain this large inhibition of K current by PKC activation. In a search for alternative mechanisms, we discovered that the C3 exoenzyme of botulinum toxin blocked the inhibition of Kv1.1 by PKC activation. The known targets of C3 toxin are low-molecular mass (20-25 kDa) G proteins, specifically RhoA and RhoC, which are selectively modified by ADP-ribosyltransferase activity, preventing their interaction with effector proteins (6, 27, 31).
Two days after injection with Kv1.1 channel RNA, we injected oocytes with C3 exoenzyme and then incubated the oocytes at different temperatures for 1-2 h. Figure 6 shows the effect of PMA application on C3-injected oocytes. C3 inhibited PKC-mediated K channel inhibition in a temperature-sensitive fashion. The temperature dependence is, perhaps, not surprising if C3 exoenzyme mediates ADP-ribosylation of a substrate required for K current inhibition. In control experiments, the C3 exoenzyme was heated to >110°C for 2 h, allowed to cool, and then injected into oocytes. This heat-inactivated preparation did not inhibit PKC-mediated inhibition of K channel current (Fig. 6, A and B). Alone, none of these treatments altered the K channel current kinetics, gating, or amplitude, compared with injection of heat-inactivated C3 toxin or no injections.
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DISCUSSION |
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A membrane-permeant activator of PKC inhibits cloned Shaker subfamily K channel currents expressed in Xenopus oocytes. The inhibition of K current is specific for a phorbol ester known to activate PKC, and the effects of PMA application are prevented by two inhibitors of PKC. Agonist stimulation of coexpressed purinergic receptors also inhibits the K channel currents, mimicking the effect of PKC activation. Together, these data strongly suggest that direct activation of PKC through membrane-permeant activators, or indirect activation through cell-surface receptors coupled to phospholipase C, inhibits K channel currents of the Shaker subfamily.
Cell surface receptors that couple to PLC may also inhibit K channel currents in native cells. PKC is a ubiquitous enzyme, present in high concentrations in brain and other tissues (16), and many neurochemicals activate phosphatidylinositol 4,5-bisphosphate hydrolysis, including ATP, histamine, thrombin, and bradykinin. The regulation of neuronal and glial K channels by transmitter- or hormone-activated PKC may be an important physiological mechanism for the modulation of membrane excitability and K homeostasis. Likewise, there may be important roles for PKC regulation of nonneuronal K currents by endogenous hormones and messenger molecules (see, e.g., Ref. 29).
The molecular mechanisms of PKC regulation of K channels are not completely understood. In some cases, phosphorylation of channel proteins themselves may be the structural basis for PKC-mediated events. Consensus protein phosphorylation sites are prevalent in the deduced amino acid sequences of cloned ion channel proteins. Commonly located in regions thought to be intracellular or near the cytoplasmic face are sequences targeted by PKC, cAMP-dependent protein kinase, and other posttranslational modification sites. Busch et al. (3) showed that a specific Ser-to-Ala mutation in a cloned voltage-dependent K channel subunit (ISK) from rat kidney prevented K current inhibition by PKC activators. Also, Cai and Douglass (4) reported biochemical evidence that PKC phosphorylates a voltage-gated K channel (hKv1.3) in T lymphocytes. And Covarrubias et al. (8) suggested that inactivation of a Shaw subfamily K channel (Kv3.4) is regulated by direct phosphorylation of the channel protein. In the present study, however, site-directed substitution of the consensus PKC phosphorylation sites on mKv1.1 channels did not prevent the inhibition of K current by PMA. A similar result was reported for the inhibition by PKC activation of rKv4.2 K currents (21). Although we cannot rule out the possibility of phosphorylation at novel or dormant sites in the folded channel protein, at least two consensus PKC sites in Shaker subfamily K channels are not likely to be directly phosphorylated by PKC, on the basis of mutagenesis data. This suggests that the substrate for phosphorylation may be a protein that associates with or indirectly regulates the Kv1.1 channel protein.
We found that the C3 exoenzyme of botulinum toxin blocks the inhibitory effect of PKC activation on Shaker subfamily K currents. The C3 exoenzyme of botulinum toxin ADP-ribosylates small-molecular weight G proteins of the ras superfamily. A major target of C3 exoenzyme is thought to be RhoC (27), which has a known role in the regulation of actin microfilament assembly. For example, C3 exoenzyme causes morphological changes in mammalian cells due to disassembly of actin microfilaments (6). Thus one hypothesis is that small GTP-binding proteins, downstream from PKC activation, mediate the inhibition of Shaker subfamily K channel currents through interactions with actin microfilaments.
In most cases, protein kinases are thought to be "modulators" or modifiers of ion channel gating. Perozo and Bezanilla (24) found that PKC activation shifts the voltage dependence of activation gating of a squid giant-axon delayed rectifier K channel. They suggested that phosphorylation of the channel protein modulates the voltage-sensing capabilities of the channel through an electrostatic interaction of added phosphate groups with the voltage sensor. Also for squid axon, Augustine and Bezanilla (1) found that MgATP modulates K conductance through an endogenous, unidentified protein kinase. Similarly, PKC activation caused a rightward shift in the voltage dependence of activation gating of a rat kidney K channel, and mutagenesis experiments were used to identify the serine substrate for the phosphorylation event (3). In addition, phosphorylation of Kv2.1 was reported to cause a depolarizing shift in activation gating (20). In all of these studies, the inhibitory effects of PKC may be explained by a phosphorylation-mediated positive shift in the voltage dependence of K channel gating. The addition of phosphate groups to the K channel protein is proposed to increase the negative surface charge on the cytoplasmic side of the membrane, thus changing the local electric field, rendering the channels less sensitive to activating stimuli.
However, gating changes are not the only mechanism by which PKC activation alters channel function. In our experiments and those of others on Kv4.2 and Kv4.3 K channels (21), there was no evidence for a PKC-induced change in K channel gating. Moran et al. (18), however, noted a small change in the voltage dependence of activation and inactivation gating of ShakerH4 channels. Together with our results that the consensus PKC phosphorylation sites are not required for the PKC-induced inhibition of mKv1.1 K channels, our data suggest that PKC inhibits K channel function. This could occur by reducing the number of functional channels so that after PKC inhibition of current, the remaining channels are normal, or by reducing the single-channel conductance of each channel.
One mechanism for a change in the number of functional channels is a
selective endocytotic mechanism, as has been reported for
hormone-activated endocytosis of K channels in starfish oocyte plasma
membranes (17). Such a process has also been implicated in the receptor
and second messenger regulation of the plasma membrane stability of
adrenergic receptors (12, 35), Na+
channels (9), and
Na+-K+-ATPase
(34). Interestingly, the time course of agonist-induced -adrenergic
receptor or activity-induced Na+
channel internalization parallels the time course of the inhibition of
K channel current in our studies. However, we assayed for changes in
the density of Kv1.1 channel protein present at the cell surface before
and after PKC stimulation in oocytes. Although the K channel current is
90% inhibited by PKC activation, there is no concomitant reduction in
the amount of Kv1.1 protein detected in membrane extracts, indicating
that PKC stimulation does not cause internalization of the K channel
protein. This is consistent with the lack of observable change in the
oocyte membrane capacitance after PKC activation. We conclude
therefore, that PKC activation causes K channels present at the cell
surface to become nonfunctional or significantly reduces the average
single-channel conductance.
Interestingly, the regulation of K channel function by PKC is clearly
different for the different molecular species of K channel. The large,
nonmodulatory, PKC-mediated inhibition of K channel activity
exemplified by mKv1.1 is also seen for other
Shaker subfamily K channels. However,
K channels of the Shab,
Shaw, and
Shal subfamilies are only slightly or
not at all inhibited by PKC activation, and, in some cases,
inactivation gating is also modulated by PKC. Covarrubias et al. (8)
first noted the elimination by PKC of N-type inactivation in a human
Kv3.4 channel and have evidence for PKC-mediated phosphorylation of
four serines within the inactivation "gate" (2). Likewise, we
observed PKC-mediated increases in current amplitude and slowing of
inactivation kinetics in rKv3.4, but not in all channels demonstrating fast inactivation (e.g., not Sh or rKv4.2). The different effects of
PKC activation on different K channels are likely explained by
differences in channel structure, possibly at PKC phosphorylation sites, or functional regions that may interact with other
membrane-associated or cytoplasmic proteins.
Understanding the physiological importance of PKC regulation of K channels depends on knowledge about the molecular species of K channel subunits that account for the native K currents. In addition, the diversity of PKC and phospholipase isoenzymes suggests that there may be cellular or regional specificity to receptor-activated PKC coupling in native cells. The coupling between neurotransmitter or hormone receptors and voltage-dependent K channels could have important effects on the roles of K channels in determining the resting membrane potential, action potential duration, speed of repolarization, and regulation of K homeostasis and synaptic plasticity. Regulation of K channel availability would thereby alter neurochemical communication and other important functions served by K channels.
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ACKNOWLEDGEMENTS |
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We thank Heather Hill for help with early experiments, Viet Hoa Hoang for help with oocyte preparations and Western blots, Gary Yellen for generously donating the data acquisition software, and T. Kendall Harden for cDNAs for purinergic receptors. We are grateful to Manuel Covarrubias, Rolf Joho, Olaf Pongs, Larry Salkoff, and Bruce Tempel for K channel cDNAs. We thank James Trimmer for a gift of anti-Kv1.1 and discussions on protein extracts and Scott O'Grady for helpful discussions. We thank Wildcat Farms for beef liver for feeding frogs.
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FOOTNOTES |
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This study was supported by grants from The Minnesota Medical Foundation and the National Institutes of Health.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. M. Boland, Dept. of Physiology, Univ. of Minnesota, 6-255 Millard Hall, Minneapolis, MN 55455 (E-mail: bolan007{at}tc.umn.edu).
Received 4 November 1998; accepted in final form 7 April 1999.
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