©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of RCK1 Currents with a cAMP Analog via Enhanced Protein Synthesis and Direct Channel Phosphorylation (*)

Gal Levin (1)(§), Tal Keren (1)(§), Tuvia Peretz (1), Dodo Chikvashvili (1), William B. Thornhill (2), Ilana Lotan (1)(¶)

From the (1)Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat Aviv 69978, Israel and the (2)Department of Physiology and Biophysics, Mount Sinai School of Medicine, Mount Sinai Hospital, New York, New York 10029-6574

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have recently shown that the rat brain Kv1.1 (RCK1) voltage-gated K channel is partially phosphorylated in its basal state in Xenopus oocytes and can be further phosphorylated upon treatment for a short time with a cAMP analog (Ivanina, T., Perts, T., Thornhill, W. B., Levin, G., Dascal, N., and Lotan, I.(1994) Biochemistry 33, 8786-8792). In this study, we show, by two-electrode voltage clamp analysis, that whereas treatments for a short time with various cAMP analogs do not affect the channel function, prolonged treatment with 8-bromoadenosine 3`,5`-cyclic monophosphorothioate ((S)-8-Br-cAMPS), a membrane-permeant cAMP analog, enhances the current amplitude. It also enhances the current amplitude through a mutant channel that cannot be phosphorylated by protein kinase A activation. The enhancement is inhibited in the presence of (R)-8-Br-cAMPS, a membrane-permeant protein kinase A inhibitor. Concomitant SDS-polyacrylamide gel electrophoresis analysis reveals that this treatment not only brings about phosphorylation of the wild-type channel, but also increases the amounts of both wild-type and mutant channel proteins; the latter effect can be inhibited by cycloheximide, a protein synthesis inhibitor. In the presence of cycloheximide, the (Sp)-8-Br-cAMPS treatment enhances only the wild-type current amplitudes and induces accumulation of wild-type channels in the plasma membrane of the oocyte. In summary, prolonged treatment with (S)-8-Br-cAMPS regulates RCK1 function via two pathways, a pathway leading to enhanced channel synthesis and a pathway involving channel phosphorylation that directs channels to the plasma membrane.


INTRODUCTION

Protein phosphorylation by protein kinase A (PKA)()is an important cellular mechanism for the modulation of K channel function(1) . For example, the anomalous rectifier channel in Aplysia neurons (2) and the Ca-activated K channel in Helix neurons (3) are activated by PKA-dependent phosphorylation. The ``S'' K channel in Aplysia sensory neurons (4) is inhibited, and the delayed rectifier K channel in Aplysia bag cell neurons (5) and the K channel in hippocampal neurons (6) are modulated by cAMP analogs via activation of PKA. In cardiac cells, the delayed rectifier K channel function is up-regulated by PKA-mediated phosphorylation(7, 8, 9, 10) . In artificial lipid bilayers, PKA activation affects the probability of opening and the Ca/voltage sensitivity of reconstituted rat brain Ca-activated K channels(11) . Correlation of PKA-mediated modulation of intrinsic channel characteristics with the direct phosphorylation of a K channel has been demonstrated for three distinct voltage-gated K channels belonging to the Shaker subfamily (e.g. see Ref. 12). Thus, by integrating electrophysiological and molecular biology techniques in a Xenopus oocyte expression system, the inactivation gating of Shaker B and Shaker D channels (13) and the open time that a single Kv1.2 channel spends in different conductance states (14) were shown to be regulated by PKA-induced phosphorylation of the channels. Actual phosphorylation by PKA of K channel proteins has been demonstrated biochemically for a mixture of Shaker channels (predominantly Kv1.2) that were purified from bovine brain (15) and whose probability of opening, upon reconstitution in lipid bilayers, can be increased by cAMP-dependent phosphorylation (16) as well as for the Kv1.3 channel that resides in membranes of T cell lines (17) and is inactivated by PKA activity(18) .

We recently showed that the Shaker RCK1 K channel (rat brain Kv1.1) (19, 20) is expressed mainly in the form of two protein products that migrate on SDS-polyacrylamide gels as distinct bands of 54 and 57 kDa and also as a minor constituent of high molecular mass proteins(21) . We also showed that the RCK1 channel is partially phosphorylated in its basal state in Xenopus oocytes and can be further phosphorylated in vivo by PKA activation, and we identified the site of phosphorylation(21) . In the present study, we characterize the functional consequences of the PKA-induced phosphorylation of RCK1 and show that prolonged treatment with a cAMP analog enhances the RCK1 current via direct phosphorylation of the channel, probably by inducing accumulation of channels in the plasma membrane. We also show that this treatment affects the level of synthesis of the channel protein.


EXPERIMENTAL PROCEDURES

Materials

Chemicals were from Sigma (Rishon Le Zion, Israel) unless stated otherwise. Vanadate (sodium orthovanadate) and okadaic acid were from Alomone Labs (Jerusalem, Israel). 8-Bromoadenosine 3`,5`-cyclic monophosphorothioate ((S)-8-Br-cAMPS; sodium salt) was from Biolog (Bremen, Federal Republic of Germany). The [S]methionine/cysteine mixture and [-P]ATP were from Amersham Corp. Calcineurin (protein phosphatase 2B) and protein phosphatase 2A were from Upstate Biotechnology, Inc. Antiserum was generated against a 23-amino acid peptide that corresponds to the N terminus of RCK1 (SGENADEASAAPGHPQDGSYPRQ) as described(21) .

Construction of cRNAs of Wild-type and Mutant RCK1 Channels

RCK1 cDNA was subcloned into a vector to yield a SupEx-RCK1 construct that confers high levels of expression as described(21) . cRNA was synthesized by NotI linearization and T7 RNA polymerase.

Substitution mutants of RCK1 (S446A, R443C,S446A, and R47A,K53Q) were generated by the oligonucleotide-directed in vitro mutagenesis method using the Amersham mutagenesis kit. Oligonucleotides encoding the desired mutations were annealed to the single-stranded template of SupEx-RCK1 and served as primers for the synthesis of the mutant channel sequences. The oligonucleotides were as follows (underlined nucleotides encode the mutated residue): 5`- CCGCCGCAGCGCCTCTACTAT-3` (S446A) and 5`-CTCCGGGCTGGGCTTC-GAGACGCAGCTCCAGACTCTGGC-3` (R47A,K53Q). R443C,S446A was generated as described(21) . The nucleotide sequences of the mutants were confirmed by DNA sequencing.

Oocytes, Drug Treatments, and Electrophysiological Recording

Xenopus laevis frogs were maintained and dissected, and oocytes were prepared as described(22) . For electrophysiological studies, oocytes were injected with 0.2-0.4 ng of cRNA. For biochemical studies, the cRNA concentrations used were up to 100-fold higher. Injected oocytes were incubated at 22 °C for 1-2 days in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl, 5 mM Hepes (pH 7.5)) supplemented with 1.8 mM CaCl, 2.5 mM sodium pyruvate, 100 µg/ml streptomycin, and 100 units/ml penicillin (NDE solution) (22) and then assayed either electrophysiologically or biochemically. (S)-8-Br-cAMPS was added to NDE solution during incubation, starting several hours after the injection; 10 µg/liter CHX was added to NDE solution 1 h before (S)-8-Br-cAMPS was added and during incubation. Electrophysiological recordings were performed as described(23, 24) using a Dagan 8500 two-electrode voltage clamp amplifier with a series resistance compensation circuit and low resistance agar cushion electrodes (<0.2 megaohms) (25) and the pCLAMP software (Axon Instruments, Inc., Foster City, CA) for data acquisition and analysis. Oocytes were placed in a 1-ml bath constantly perfused with ND96 solution supplemented with 1 mM CaCl, and their current amplitudes were determined by stepping up the membrane potential from a holding potential of -80 to 0 mV for 300 ms. Current-voltage relationships were obtained by 300-ms depolarization steps from -100 mV (50-ms prepulse). Net current was estimated by subtraction of scaled leak current elicited by a voltage step to -90 mV.

Evaluation of the Homogenization Procedure

Six to eight oocytes were homogenized in 100 µl of medium composed of 20 mM Tris (pH 7.4), 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 50 µg/ml phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 1 µM pepstatin, and 1 mM 1,10-phenanthroline. Debris was removed by centrifugation at 1000 g for 10 min at 4 °C. After the addition of Triton X-100 to a final concentration of 4%, followed by microcentrifugation for 15 min at 4 °C, antiserum was added to the supernatant. Having realized during the course of the study that one of the products of the RCK1 channel (the 57-kDa species) represents the phosphorylated form of the channel (see ``Results''), we examined whether the homogenization process itself favors dephosphorylation by enzyme(s) in the oocyte extract and thereby introduces experimental artifacts by altering the relative proportion of the phosphorylated species. Indeed, inclusion of the protein phosphatase inhibitors okadaic acid (50 nM), vanadate (0.5 mM), and KF (50 mM) (or only 50 nM okadaic acid) in the homogenization solution increased the relative proportion of the 57-kDa species (shown in Fig. 4B). It was concluded that during homogenization in Triton X-100, spontaneous dephosphorylation of the channel occurred. Homogenization of the oocytes in boiling 4% SDS solution (as described below), which should destroy any enzymatic activity, yielded a relative fraction of the phosphorylated species similar to that obtained upon inclusion of phosphatase inhibitors (data not shown). Thus, in the following experiments, homogenization of whole oocytes was carried out in boiling SDS, and separation of plasma membranes from the rest of the cells and homogenization of each fraction (see below) were done in Triton X-100 solution supplemented with the phosphatase inhibitors. Metabolic Labeling with [S]Methionine/Cysteine, Homogenization, and Immunoprecipitation from Whole Oocytes-Oocytes were injected with RCK1 cRNA and incubated at 22 °C for 4 h in NDE solution and then in NDE solution containing 0.5 mCi/ml [S]Met/Cys until homogenization. Six to eight whole oocytes were homogenized as described (26) in 100 µl of solubilization buffer (4% SDS, 10 mM EDTA, 50 mM Tris (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 1 mM 1,10-phenanthroline) and heated to 100 °C for 2 min. Following the addition of 100 µl of HO and 800 µl of immunoprecipitation buffer (190 mM NaCl, 6 mM EDTA, 50 mM Tris (pH 7.5), 2.5% Triton X-100), homogenates were centrifuged for 10 min at 1000 g and 4 °C to remove the insoluble yolk and pigment particles. The supernatant was incubated for 16 h with antiserum. The antibody-antigen complex was incubated for 3 h at 4 °C with protein A-Sepharose and then pelleted by centrifugation for 1 min at 8000 g. Immunoprecipitates were washed four times with immunowash buffer (150 mM NaCl, 6 mM EDTA, 50 mM Tris (pH 7.5), 0.1% Triton X-100, 0.02% SDS); the final wash contained no Triton X-100. Samples were boiled in SDS gel loading buffer and electrophoresed on SDS-8% polyacrylamide gel together with standard molecular mass markers (45-205 kDa).


Figure 4: Digitized PhosphorImager scan of SDS-PAGE analysis of wild-type 57- and 54-kDa protein species labeled by [S]Met/Cys. A, the 57-kDa species is the phosphorylated form of the 54-kDa species. Immunopurified protein products from whole oocytes, not injected (control) or injected with the wild-type cRNA (WT), were not (lanes2, 3, 6, and 8) or were (lanes1, 4, 5, and 7) subjected to phosphorylation by PKA-CS in the absence (lanes3, 6, and 8) or presence (lanes1, 2, 4, 5, and 7) of different concentrations of ATP. After phosphorylation by PKA-CS in the presence of ATP, the immunopurified protein was subjected to dephosphorylation by protein phosphatase 2B (PP2B) in the presence of Ca-calmodulin (lane4). The left and rightpanels are two separate experiments. B, during immunopurification, the 57-kDa protein in the oocyte homogenate undergoes spontaneous dephosphorylation that can be prevented by okadaic acid (OA). Whole oocytes were homogenized in Triton X-100 (see ``Experimental Procedures'') in the presence (lane2) or absence (lane1) of 50 nM okadaic acid. Thinarrows indicate molecular mass markers in kilodaltons.



Plasma Membrane Preparation

Plasma membranes were separated mechanically as described(21) . Defolliculated oocytes were incubated for 10 min in ice-cold hypotonic solution (5 mM NaCl, 5 mM Hepes, 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 1 mM 1,10-phenanthroline) supplemented with phosphatase inhibitors as specified above. Plasma membranes together with the vitelline membranes (extracellular collagen-like matrix), which appeared as a transparent sheet, were removed manually by the use of watchmaker's forceps. The remainder of the cell, consisting of cytoplasm and intracellular organelles (here collectively termed cytosol), was usually left as an almost intact sphere. The two fractions were collected separately by a Pasteur pipette and the membrane fraction pelleted by centrifugation for 10 min in a microcentrifuge. 10-15 cytosolic fractions or 20-30 plasma membranes were homogenized in 300 µl of medium composed of 20 mM Tris (pH 7.4), 5 mM EDTA, 5 mM EGTA, 100 mM NaCl, 50 µg/ml phenylmethylsulfonyl fluoride, 1 mM iodoacetamide, 1 µM pepstatin, and 1 mM 1,10-phenanthroline supplemented with phosphatase inhibitors. Debris was removed by centrifugation at 1000 g for 10 min at 4 °C. After the addition of Triton X-100 to a final concentration of 4%, followed by centrifugation for 15 min at 4 °C, the antiserum was added to the supernatant. The following steps were done as described for whole oocytes.

In Vitro Phosphorylation

This was done essentially as described(21) . The immunoprecipitate was washed twice with phosphorylation buffer (25 mM Hepes, 5 mM MgCl, 5 mM EGTA, 0.2% Triton X-100 (pH 7.4)). Phosphorylation was performed by incubation of the pellet with 1 µg of the catalytic subunit of PKA (PKA-CS; reconstituted in 5 mM dithiothreitol) and either 10 µCi of [-P]ATP (3000 Ci/mmol) or unlabeled ATP at the indicated concentrations in phosphorylation buffer for 5 min at 30 °C. The reaction was stopped by washing twice with 1 ml of radioimmunoassay buffer (50 mM sodium phosphate buffer (pH 7.4), 50 mM KF, 75 mM NaCl, 2.5 mM EDTA, 0.01% NaN, 25 mM Tris (pH 7.4)).

Dephosphorylation

Dephosphorylation was performed essentially as described(21) . The immunoprecipitate was washed with a solution containing 40 mM Hepes/Tris (pH 7.6), 40 mM NaCl, 0.1 mM EDTA, 0.5 mM MgCl, 0.5 mM MnCl, 0.5 mM CaCl, 0.1% Triton X-100, and 1 mg/ml bovine serum albumin. To the pellet were added protein phosphatase 2B (2.4 µg) and calmodulin (1 µM) or protein phosphatase 2A (0.1 unit), dissolved in a total volume of 50 µl of the wash solution, and the mixture was incubated at 36 °C for 10 min. Dephosphorylation was stopped by dilution with the wash solution at 0 °C. The immunoprecipitate was washed twice in this solution.

Quantification of Labeling Intensities and Generation of a Digitized PhosphorImager Scan

Gels were dried and placed in a PhosphorImager cassette (Molecular Dynamics, Inc.) for 1 day (for whole oocytes or cytosol) or 2-4 days (for plasma membranes); and using the software ImageQuant, a digitized scan was derived in the grey scale viewing mode, and its display was optimized by adjusting the range of the grey scale according to the intensity of the labeled products (for plasma membrane fractions, the range was narrower than for cytosolic fractions). The scans were converted to .tif files, processed by Adobe photoshop software, labeled with Corel Draw software, and sent to an Iris Smartjet 4012 printer as .prn files. Relative intensities of protein bands were estimated quantitatively by the software; comparison of intensities between cytosolic and plasma membrane fractions was performed on a single scan of both fractions.

Statistical Analysis

Data are presented as means ± S.E.; paired t test was used to calculate the statistical significance of differences between two populations.


RESULTS

Functional Consequences of Short and of Prolonged (S)-8-Br-cAMPS Treatment

We have previously shown that incubation of Xenopus oocytes, which were injected with the cRNA encoding the rat brain RCK1 channel, for 0.5 h in the presence of 0.5 mM (S)-8-Br-cAMPS, a membrane-permeant analog of cAMP, results in phosphorylation of the expressed RCK1 channel protein, and we identified the amino acid stretch Arg-Arg-Ser-Ser-Ser in the C terminus as the target for phosphorylation(21) . In the present study, we pinpointed the site of phosphorylation to Ser-446 (see below). To determine the electrophysiological consequences of the (S)-8-Br-cAMPS effect, we examined simultaneously both the wild-type and S446A mutant channels. Using the two-electrode voltage clamp technique, we have examined the whole cell currents through these channels and found no differences in their kinetics or voltage dependence (Fig. 1, A, B, and D). Both were of the delayed rectifier type(20) . Also, the two currents had similar amplitudes when tested in oocytes injected with comparable amounts of either wild-type or S446A cRNA (the levels of expression of both proteins, as analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), were also similar (data not shown)).


Figure 1: Two-electrode voltage clamp analysis of the voltage dependence of activation of K currents expressed in Xenopus oocytes from RCK1 wild-type and S446A cRNAs. Oocytes injected with either wild-type (WT) or S446A cRNA were placed in NDE solution in the absence (A and B) or presence (C) of (S)-8-Br-cAMPS (Sp) and incubated for 20 h until their currents were assayed. A-C, families of currents were evoked by voltage steps from -100 mV to depolarized voltages (V). Shown are representative traces of currents evoked by voltage steps to the indicated values. Currents were corrected for leak at all voltages (see ``Experimental Procedures''). D, shown are steady-state activation curves of currents evoked as described for A-C. Conductances (G) at all voltages were derived from the equation G = I/(V - V), where I is current via channels and V is the reversal potential for K in oocytes bathed in ND96 solution (2 mM K) taken as 100 mV. Conductances, normalized to the maximal conductance (G), are plotted as a function of voltage. For each treatment, five oocytes were assayed, and means ± S.E. are presented.



Neither incubation with 0.5 mM (S)-8-Br-cAMPS nor injection of cAMP or (S)-8-Br-cAMPS into the oocyte (up to 50 pmol) had any detectable effect on either the wild-type or S446A channel amplitude for up to 30 min from the start of the treatment. However, longer incubations (lasting from 6 to 20 h) in the presence of 0.5 mM (S)-8-Br-cAMPS were followed by a statistically significant increase in both wild-type and S446A current amplitudes by 75 and 60%, on the average, respectively (Fig. 2). The effect was dose-dependent in oocytes of the same donor (Fig. 2, lowerinset), but varied among different donors, ranging between -10 and +280% (with one extreme case of +600%). The prolonged treatment with (S)-8-Br-cAMPS did not affect the steady-state activation curve (Fig. 1, C and D).


Figure 2: Two-electrode voltage clamp analysis of the effect of (S)-8-Br-cAMPS on amplitudes of wild-type and S446A currents, elicited by voltage steps from -80 to 0 mV, in the absence (left) and presence (right) of CHX. Groups of 10-20 oocytes injected with either wild-type (WT) or S446A cRNA were placed in NDE solution in the presence (treated) or absence (control) of 0.5 mM (S)-8-Br-cAMPS (Sp) and incubated for 6-20 h until their current amplitudes were assayed and averaged for each group. The ratio between mean amplitudes of treated and control groups was calculated. Each column is the mean ratio of several such experiments (the number of experiments is indicated above the columns). Errorbars represent S.E. When 10 µg/ml CHX was used, it was introduced into NDE solution 1 h prior to and during incubation with (S)-8-Br-cAMPS.**, p < 0.005; *, p < 0.05. Upper inset, effect of increasing CHX concentrations on wild-type currents (I). Oocytes were incubated with CHX 1 h prior to cRNA injection and during the following 20 h until their current amplitudes were assayed. Lower inset, dose dependence of the (S)-8-Br-cAMPS effect on wild-type currents. Oocytes were incubated for 20 h with or without the indicated (S)-8-Br-cAMPS concentrations until their current amplitudes were assayed.



To determine whether the effect occurred through activation of PKA, we coincubated oocytes with 0.5 mM (S)-8-Br-cAMPS and 4 mM (R)-8-Br-cAMPS, a membrane-permeant PKA inhibitor that competes with (S)-8-Br-cAMPS for binding to the PKA regulatory subunit (with an affinity of an order of magnitude lower than that of (S)-8-Br-cAMPS (27)), and showed that the (S)-8-Br-cAMPS effect was significantly reduced (p < 0.001) (Fig. 3). Incubation with (R)-8-Br-cAMPS alone caused a small increase of the currents, possibly because of its partial agonist properties at high concentrations(28) .


Figure 3: Antagonism of the (S)-8-Br-cAMPS effect on wild-type current amplitudes by the PKA inhibitor (R)-8-Br-cAMPS. Each column represents the (S)-8-Br-cAMPS (Sp) effect (under conditions as described for Fig. 1) in the absence or presence of the indicated concentrations of (R)-8-Br-cAMPS (Rp), which was introduced 4 h prior to and during the 20-h (S)-8-Br-cAMPS incubation.**, p < 0.0005; *, p < 0.03. Numbers indicate oocytes assayed; errorbars represent S.E. WT, wild-type.



Fig. 2also shows that in the presence of 10 µg/ml CHX, an inhibitor of protein synthesis, treatment with 0.5 mM (S)-8-Br-cAMPS for 10-16 h increased the wild-type current by 224%, while the S446A current was not affected. CHX was applied 1 h prior to and during (S)-8-Br-cAMPS application. Note that even 5 µg/ml CHX was sufficient to almost completely inhibit the expression of wild-type currents through newly synthesized channels (Fig. 2, upperinset). Thus, under conditions of synthesis arrest (see below), prolonged treatment with (S)-8-Br-cAMPS appears to modulate only the wild-type and not the S446A channel.

Physiological Significance of the RCK1 Protein Doublet

We have previously demonstrated that immunopurification of RCK1 proteins from extracts of S-labeled Xenopus oocytes injected with RCK1 cRNA yields two main labeled products (doublet) with molecular masses of 57 and 54 kDa as verified by SDS-PAGE analysis (Ref. 21; see also Fig. 4A, lane6). Both products were shown to be N-glycosylated to the same extent(21) , and we could not detect any O-glycosylation (data not shown). Since only the 57-kDa product was shown to be phosphorylated, we attempted to determine whether the apparent doublet could be explained in terms of phosphorylation. If one of the species is the phosphorylated derivative of the other, it should be possible to detect, upon phosphorylation in vitro, an increase of this species at the expense of the other and vice versa upon dephosphorylation. Indeed, phosphorylation of the immunopurified RCK1 product that was labeled by [S]Met/Cys by PKA-CS in the presence of ATP shifted part of the 54-kDa species to the 57-kDa species; the shift was dependent on ATP concentration (Fig. 4A, lanes1 and 5). Subsequent dephosphorylation by protein phosphatase 2B (Fig. 4A, lane4) resulted in a shift to 54 kDa of the whole amount of the 57-kDa species, both the fraction existing basally in the oocyte and the in vitro PKA-phosphorylated fraction. These data indicate that the species that migrates as 57-kDa protein (we refer to it as the 57-kDa species) represents the channel that is basally phosphorylated in the oocyte or is phosphorylated by PKA activity. Phosphorylation of several other proteins has been shown to cause a similar shift in their electrophoretic mobilities (e.g. see Ref. 29).

Further experiments have indicated that during immunopurification, the channel protein undergoes spontaneous partial dephosphorylation in the oocyte homogenate, resulting in under-representation of the 57-kDa species. The extent of dephosphorylation varied among experiments with oocytes from different donors and could be prevented by inclusion of 50 nM okadaic acid (a phosphatase inhibitor) in the oocyte homogenate (Fig. 4B; in this experiment, the spontaneous dephosphorylation was 100%). Additional studies were carried out to evaluate the spontaneous dephosphorylation and to eliminate it so as to obtain adequate representation of the protein doublet in the experiments described below (see ``Experimental Procedures'').

Protein Products of RCK1 Mutants

Our next objective was to determine whether the 57-kDa species was phosphorylated at the site of PKA-induced phosphorylation. In a previous study, we demonstrated that the double mutant R443C,S446A cannot be phosphorylated by PKA in vitro or in vivo(21) , and in the present study, we further showed that the mutant S446A also cannot be phosphorylated by the catalytic subunit of PKA in the presence of [-P]ATP (Fig. 5A) (the faint labeling of oocytes injected with S446A mRNA and of uninjected oocytes (control) probably represents endogenous K channels(21) ). We compared the protein products of R443C,S446A and S446A with those of the wild type and of an additional mutant, R47A,K53Q, at sites irrelevant to PKA-induced phosphorylation. Fig. 5B shows that neither of the phosphorylation site mutants was expressed as a 57-kDa product (lanes2 and 3) and that this species was expressed only by the wild type and the irrelevant mutant (lanes4 and 1, respectively). We concluded that the 57-kDa species represents a phosphorylated form of the RCK1 channel at Ser-446. This finding explains our previous observation that only the 57-kDa species was P-labeled by the basal, the in vivo cAMP-induced, and the in vitro PKA-catalyzed phosphorylation of the RCK1 channel (Ref. 21; see also Fig. 5A, lane2).


Figure 5: RCK1 mutants that cannot be phosphorylated by PKA activation are not expressed as the 57-kDa species. A, S446A is not a substrate for PKA phosphorylation. Shown is a digitized PhosphorImager scan of SDS-PAGE analysis of immunopurified protein products from whole oocytes either not injected (lane3) or injected with wild-type (lane2) or S446A (lane1) cRNA that was phosphorylated by PKA-CS in the presence of [-P]ATP (after prior dephosphorylation by protein phosphatase 2B in the presence of Ca-calmodulin; see Ref. 21 and ``Experimental Procedures''). B, protein products of S446A and R443C,S446A mutants do not include the 57-kDa species. Shown is a digitized PhosphorImager scan of SDS-PAGE analysis of [S]Met/Cys-labeled protein products immunopurified from whole oocytes injected with wild-type cRNA (WT; lane4) or with other mutant cRNAs (lanes 1-3) homogenized in the presence of 4% SDS and boiled at 100 °C to eliminate spontaneous dephosphorylation (see ``Experimental Procedures''). Thinarrows indicate molecular mass markers in kilodaltons.



Unexpectedly, the phosphorylation site mutants were not always expressed as 54-kDa species. The major product of R443C,S446A migrated always as an 56-kDa protein (Fig. 5B), while S446A was expressed differently in oocytes from different donors: as a main product of either 56 kDa (Fig. 5B) or 54 kDa (see Fig. 7A) and as a doublet of 54 and 56 kDa (data not shown). Examination of the 56-kDa species indicated that in contrast to the wild-type 57-kDa species, its relative proportion could not be increased or decreased by in vitro PKA-induced phosphorylation or protein phosphatase 2B-induced dephosphorylation, respectively, and it was not sensitive to the presence of okadaic acid or other wide-range phosphatase inhibitors (sodium orthovanadate and KF) in the homogenization solution (data not shown).


Figure 7: Effect of prolonged treatment with (S)-8-Br-cAMPS on protein phosphorylation and level of protein synthesis in the plasma membrane and the rest of the cell (cytosol). A, digitized PhosphorImager scan of SDS-PAGE analysis of [S]Met/Cys-labeled wild-type (WT; lanes3 and 4) and S446A (lanes1 and 2) protein products immunopurified from 30 plasma membranes (rightpanel) or 10 cytosols (leftpanel) of oocytes incubated for 20 h with 0.5 mM (S)-8-Br-cAMPS (Sp). Homogenization was done in the presence of protein phosphatase inhibitors (orthovanadate, KF, and okadaic acid) to eliminate spontaneous dephosphorylation (see ``Experimental Procedures''). Shown are two scans of cytosol and plasma membrane (PM) fractions (only one-third of the equivalent volume of membrane fractions was loaded on the lanes of cytosolic fractions) after 1 and 4 days of exposure, respectively. Thinarrows indicate molecular mass markers in kilodaltons. B, histogram derived from the labeling intensities of the protein products in scans of several experiments similar to those shown in A. Shown is the averaged effect of prolonged (6-20 h) treatment with (S)-8-Br-cAMPS on the extent of increase in phosphorylation of the wild-type channel in the plasma membrane and in the cytosol, calculated as the -fold increase in the ratio WT(57/54) (see ``Results'') with and without (S)-8-Br-cAMPS treatment (yaxis). Note that in this experiment, lower molecular mass products (besides the 54- and 57-kDa species) were also apparent; these were not taken into account as usually they were negligible and their biosynthetic significance is not known. Errorbars represent S.E., and the number of experiments is indicated.**, p < 0.025.



Biochemical Analysis of Prolonged Treatment with (S)-8-Br-cAMPS

In this part of the study, we took advantage of the fact that the 57-kDa band represents the phosphorylated RCK1 protein at Ser-446, so that RCK1 phosphorylation was examined by [S]Met/Cys labeling. The extent of phosphorylation was evaluated by the ratio of 57- and 54-kDa protein levels (quantitated by the ratio of intensities of labeling of 57- and 54-kDa bands; WT(57/54)). Fig. 6A shows that incubation with 0.5 mM (S)-8-Br-cAMPS for 16 h increased the extent of wild-type channel phosphorylation (compare lanes1 and 2). In five similar experiments, prolonged (6-20 h) treatment with (S)-8-Br-cAMPS increased the extent of wild-type channel phosphorylation 1.7-fold on the average (Fig. 6B, upperpanel; in one experiment not included in the average, the increase was 8.7-fold). Strikingly, Fig. 6A shows that incubation for 16 h with (S)-8-Br-cAMPS had an additional consequence that affected, however, both the wild-type (lanes1 and 2) and S446A (lanes5 and 6) channels: it increased the total protein levels. In several similar experiments, the amounts of wild-type and S446A proteins showed average increases of 2.7- and 1.9-fold, respectively (Fig. 6B, lowerpanel). Fig. 6A also shows the effect of 16-h (S)-8-Br-cAMPS treatment on the wild-type (lanes3 and 4) and S446A (lanes7 and 8) proteins in the presence of a 10 µg/ml concentration of the protein synthesis inhibitor CHX. In this experiment, which was performed twice, (S)-8-Br-cAMPS treatment failed to increase the amount of protein; however, in both experiments, it still increased the extent of phosphorylation of the wild-type channel 2.5- and 2.1-fold (the box in Fig. 6A contains an enhanced PhosphorImager scan of lanes3 and 4 to provide better resolution of the RCK1 products in the presence of CHX).


Figure 6: Effect of prolonged treatment with (S)-8-Br-cAMPS on phosphorylation and level of protein expression in the presence and absence of CHX. A, digitized PhosphorImager scan of SDS-PAGE analysis of [S]Met/Cys-labeled wild-type (WT; lanes 1-4) and S446A (lanes 5-8) protein products immunopurified from whole oocytes at the end of a 20-h incubation without (lanes1, 3, 5, and 7) or with (lanes2, 4, 6, and 8) 0.5 mM (S)-8-Br-cAMPS (Sp). 1 h prior to and during incubation with (S)-8-Br-cAMPS, 10 µg/ml CHX was introduced (lanes3 and 4 and lanes7 and 8, respectively). The box contains an enhanced scan of lanes3 and 4 (achieved by adjustment of brightness and contrast of the scan) enabling better resolution of the protein products. Homogenization was performed in the presence of 4% SDS with boiling at 100 °C to eliminate spontaneous dephosphorylation (see ``Experimental Procedures''). Thinarrows indicate molecular mass markers in kilodaltons. B, histograms derived from the labeling intensities of the protein products in scans of experiments similar to those shown in A. Upper panel, averaged effect of prolonged (6-20 h) treatment with (S)-8-Br-cAMPS on the extent of increase in phosphorylation of the wild type, calculated as the -fold increase in the ratio WT(57/54) (see ``Results'') with and without (S)-8-Br-cAMPS treatment (yaxis). Lower panel, averaged effect of prolonged (6-20 h) treatment with (S)-8-Br-cAMPS on the level of expression (synthesis) of wild-type and S446A proteins in the presence and absence of CHX, calculated as the -fold increase in total intensity of labeling of the products (yaxis). CHXcolumns represent means of two experiments, the results of which are given in the ``Results.'' Errorbars represent S.E., and the number of experiments is indicated. *, p < 0.05;**, p < 0.025.



The above analysis was performed on proteins that were immunopurified from homogenates of whole oocytes. The large size of the oocyte also allows manual separation of its plasma membrane from the rest of the cell (``cytosol''), with no apparent contamination from intracellular membranes(21) . Analysis of proteins immunopurified from plasma membrane showed that (S)-8-Br-cAMPS treatment increased the extent of phosphorylation 2.2-fold on the average, whereas in homogenates of cytosol of the same cells, the average increase in extent of phosphorylation was significantly (p < 0.01) smaller, only 1.2-fold (Fig. 7B); a typical experiment is shown in Fig. 7A. Treatment with (S)-8-Br-cAMPS increased the amounts of total protein in plasma membranes of oocytes expressing wild-type and S446A channels 3.5 ± 1.5-fold (n = 5; p < 0.05) and 2.1 ± 0.9-fold (n = 4), respectively.

Application for 12-20 h of H-89, a specific blocker of the catalytic site of PKA(30) , decreased the amount of total synthesized RCK1 channels to 0.70 ± 0.11-fold (n = 5; p < 0.01) of the control value (see a typical experiment in Fig. 8) and also of S446A channels to about the same extent (data not shown). However, application of the competitive PKA inhibitor (R)-8-Br-cAMPS (up to 2 mM) did not decrease the amount of synthesized RCK1 (data not shown). Basal phosphorylation apparently was not altered significantly by these two PKA inhibitors (see Fig. 8for H-89 and data not shown for (R)-8-Br-cAMPS).


Figure 8: Effect of prolonged treatment with H-89 on wild-type channels. A, digitized PhosphorImager scan of SDS-PAGE analysis of [S]Met/Cys-labeled wild-type protein products (WT) immunopurified from whole oocytes treated for 20 h with 50 µM H-89 (lane1) or with 0.5 mM (S)-8-Br-cAMPS (Sp; lane2) or not treated (lane3). Oocytes were homogenized in the presence of 4% SDS with boiling at 100 °C to avoid spontaneous dephosphorylation. Thinarrows indicate molecular mass markers in kilodaltons. B, histogram derived from the labeling intensities of the protein products in the scan shown in A. Shown is a comparison of effects of prolonged H-89 and (S)-8-Br-cAMPS treatments on the level of expression of wild-type proteins, calculated as the -fold change in total intensity of labeling of the products from treated oocytes, as compared with products from untreated oocytes.



Comparison of the Relative Plasma Membrane Protein Fractions (F) of Wild-type and S446A Channels

Division of the amount of channel protein in the plasma membrane (summation of 54- and 57-kDa intensities) by that in the cytosol of the same oocytes yields F. A comparison of F in oocytes expressing wild-type channels with that in oocytes expressing S446A channels showed that F in the wild type was larger than that in S446A. Thus, for a given amount of channel protein, more protein is concentrated in the plasma membrane of oocytes expressing wild-type channels than in the plasma membrane of oocytes expressing S446A channels. This is true for both untreated (basal) and (S)-8-Br-cAMPS-treated oocytes (for results from a typical experiment, see Fig. 9A). Normalized F (F(wild-type)/F(S446A)) values indicate that in the basal state, the RCK1 protein is 2.6-fold more concentrated in the plasma membrane of oocytes expressing wild-type channels than in the plasma membrane of oocytes expressing S446A channels, and in (S)-8-Br-cAMPS-treated cells, the ratio increases significantly (p < 0.05) to 5.2-fold (Fig. 9B).


Figure 9: Histograms showing the relative accumulation of wild-type channels in the plasma membrane as compared with S446A channels in the absence (basal state) and presence of prolonged (6-20 h) treatment with (S)-8-Br-cAMPS. A, F, calculated as the ratio of total protein intensities (57 + 54 kDa) between the plasma membrane and the cytosol (yaxis; see ``Results'') of wild-type and S446A proteins, derived from an experiment similar to that shown in Fig. 7. The intensities of plasma membrane and cytosolic fractions were estimated from the same scan (not as shown in Fig. 7; see ``Experimental Procedure''). B, average F of the wild type normalized to F of S446A (see ``Results''). Errorbars represent S.E., and the number of experiments is indicated above the columns.**, p < 0.005; *, p < 0.025.




DISCUSSION

Having previously shown that the RCK1 channel can be phosphorylated by PKA in vivo(21) , we set out in this study to find the physiological correlate of the phosphorylation. This was done by concomitant electrophysiological and biochemical analyses of the effects of (S)-8-Br-cAMPS, a membrane-permeant cAMP analog. An additional aim was to determine the physiological meaning of the 57-kDa species, as it appeared to be the only RCK1 protein product being P-labeled upon phosphorylation. We found that the 57-kDa species represents the phosphorylated form of the RCK1 channel at Ser-446, the target for basal and PKA-induced phosphorylation, whereas the 54-kDa product is the unphosphorylated species. This enabled us to follow (S)-8-Br-cAMPS-induced phosphorylation by S labeling. Thus, by comparing the relative intensities of S-labeled 57- and 54-kDa species, we could monitor the phosphorylated protein fraction and simultaneously follow the biosynthesis of the channel proteins.

Prolonged Treatment with (S)-8-Br-cAMPS Enhances the RCK1 Current Amplitude and the Synthesis of the Channel Protein

In the electrophysiological study of PKA-induced phosphorylation, we used the S446A mutant channel, which cannot be phosphorylated by PKA, as a tool to discriminate between effects due to direct phosphorylation of the channels and indirect effects such as phosphorylation of auxiliary proteins. A comparison of wild-type and S446A currents with respect to amplitudes, kinetics, and voltage dependence revealed that the point mutation did not affect the intrinsic channel properties. In addition, we could not detect any electrophysiological effects on whole cell currents of either the wild type or S446A following short treatment (0.5 h) with (S)-8-Br-cAMPS, even though we have shown that such treatment causes phosphorylation of RCK1 channels in vivo(21) . Prolonged treatment with (S)-8-Br-cAMPS (for at least 6 h), however, resulted in enhancement of the wild-type current amplitudes, which could be inhibited by a PKA inhibitor ((R)-8-Br-cAMPS) that competes with cAMP for binding to the regulatory subunit of PKA(27) . Since the S446A currents were similarly enhanced by prolonged (S)-8-Br-cAMPS treatment, we concluded that the enhancement probably involves PKA-induced phosphorylation of cellular protein(s) other than the channel protein itself, which would indirectly affect the channel.

Concomitant biochemical analysis revealed that prolonged (S)-8-Br-cAMPS treatment caused an increase in the protein level of both wild-type and S446A channels; the increased level of protein in the plasma membrane fraction (2-3-fold) could account for the increased current amplitudes (1.7-fold). The observation that prolonged treatment with H-89, a PKA inhibitor at the catalytic site, caused the opposite effect, namely, a decrease in the basal protein levels, may indicate that regulation of protein levels in the native oocyte is already under some control by PKA activity. In this context, we can try to explain the failure of 2 mM (R)-8-Br-cAMPS, another PKA inhibitor, to cause the same effect by arguing that it is due to its being a competitive inhibitor with a binding affinity for the PKA regulatory subunit that is 100 times lower than that of cAMP (27). The (S)-8-Br-cAMPS-induced increase in the protein level was blocked in the presence of CHX, which inhibited protein synthesis, thereby excluding the involvement of post-translational regulation. Also, the involvement of transcription regulation is excluded as the channel is expressed from cRNA. Hence, it seems that regulation of the RCK1 protein level is a result of modification of protein(s), most probably PKA-sensitive, involved either in the stabilization of mRNA species in the oocyte or in the translation machinery, leading to enhanced protein synthesis.

There are several reported examples of the possible involvement of protein kinase activity in enhanced mRNA stability. Treatment with a cAMP analog resulted in a 10-fold increase in the half-life of mRNA for phosphorylpyruvate carboxykinase in a rat hepatoma cell line(31) . Activation of protein kinase C by phorbol esters increased mRNA levels of corticotropin-releasing hormone in a cell line(32) . Also, the half-life of -adrenergic receptor mRNA in rat C6 glioma cells has been increased by treatments with agents that increase cAMP levels(33) . In contrast, chronic inhibition of PKA activity in Chinese hamster ovary cells expressing mKv1.1, the mouse homolog of RCK1, resulted in increased mRNA levels(34) . The possible regulation of protein synthesis by PKA activity has not been widely studied. Studies that might be relevant to our own report that phosphorylation in vitro by the catalytic subunit of PKA activates purified eucaryotic elongation factor-2 kinase, which regulates the activity of eucaryotic elongation factor-2 by phosphorylation and hence affects the translocation step in the elongation phase of protein synthesis. Also, cAMP, possibly acting through PKA, blocks transmission of signals from Ras to Raf-1 and thereby prevents activation of the mitogen-activated protein kinase cascade (for review, see Ref. 35), which, in turn, is involved in the control of translation initiation (for review, see Ref. 36). Taken together, these findings suggest that if cAMP analogs affect RCK1 protein synthesis in oocytes, it should be either at the elongation phase or, by interacting with the mitogen-activated protein kinase cascade, at the initiation phase of protein synthesis.

Phosphorylation of the RCK1 Channel Protein Enhances Its Current Amplitude and Increases the Amount of Channels in the Plasma Membrane

At this point, we were still concerned with the physiological role of the direct phosphorylation of the channel by PKA. As expected, biochemical analysis showed that the prolonged (S)-8-Br-cAMPS treatment, in addition to its enhancing effect on synthesis, also caused phosphorylation of the wild-type but not of the S446A channel. Namely, the treatment resulted in an increase in the fraction of the 57-kDa species relative to the basal (unstimulated) state. This direct phosphorylation of the channel by (S)-8-Br-cAMPS was still prominent in the presence of the protein synthesis inhibitor CHX, even though enhancement of synthesis did not occur, indicating that phosphorylation and enhanced synthesis are two separate effects. Having isolated the direct phosphorylation effect by the addition of CHX, we could test its physiological consequences. Amplitudes of the wild-type but not of the S446A currents were found to be enhanced by 2.2-fold, indicating that direct channel phosphorylation plays a role in the enhancement of RCK1 channel function.

A possible mechanism underlying the enhanced RCK1 channel function by direct channel phosphorylation could be inferred from the observation that in the basal state and significantly more so in the (S)-8-Br-cAMPS-stimulated state, the plasma membrane protein fraction of the wild-type channel was considerably larger than that of the S446A channel, which is not phosphorylated in either of the states. It thus seems that phosphorylation induces accumulation of channels in the plasma membrane. A similar phenomenon was described for the Torpedo acetylcholine receptor, stably integrated in mouse fibroplasts (37, 38, 39) presumably through phosphorylation of the -subunit, leading to longer lifetimes and increased efficiency of subunit assembly. In our case, however, it seems more plausible that increased expression of RCK1 channels in the plasma membrane following (S)-8-Br-cAMPS treatment is the result of enhanced mobilization of the phosphorylated channel to the plasma membrane. Our observation that the relative fraction of the phosphorylated 57-kDa species in the plasma membrane increased significantly more than in the rest of the cell hints at the possibility that somehow, possibly through a cytoskeleton-associated mechanism, cAMP-dependent phosphorylation of the RCK1 protein enhances its transport to the plasma membrane of the oocyte. More important, a recent study on axons of Aplysia bag cells (40) has shown that elevation of cAMP concentration leads to a 2-3-fold enhancement of the average rate of organelle transport along microtubule tracks.

Further Considerations

Two questions remain to be resolved. (i) In the basal state, the plasma membrane protein fraction of the wild-type channel is larger than that of S446A. Why are the wild-type currents not larger than those of S446A? (ii) It is surprising that enhancement of RCK1 by (S)-8-Br-cAMPS treatment is similar in the presence and absence of CHX. In the latter case, both effects of (S)-8-Br-cAMPS should be manifested, and apparently, the wild-type current should be more enhanced. Moreover, one would expect that under this condition the wild-type current amplitude would be enhanced above that of the mutant by direct phosphorylation; however, this is not the case. These questions can be resolved if one assumes that while the direct phosphorylation of the RCK1 channel causes accumulation of channels in the plasma membrane, it also has an overlapping negative effect that counterbalances the positive one. The negative effect is inhibited under conditions of protein synthesis arrest. Thus, under normal physiological conditions, the enhancement by (S)-8-Br-cAMPS of the wild-type current is predominantly governed by the increased synthesis and is therefore not significantly larger than that of the S446A current. Under conditions of protein synthesis arrest, however, the (S)-8-Br-cAMPS effect is due only to the positive effect of direct channel phosphorylation, and therefore, the wild-type, but not the mutant, current amplitude is enhanced.

The negative effect could arise from interaction of an endogenous factor (with a half-life of the order of several hours) with the phosphorylated form of the channel. This could possibly lead to alteration of intrinsic channel characteristics (see Refs. 11 and 13) that have better chances of being resolved by the single channel analysis that we will carry out in the future. Another possibility is that this factor recruits a certain fraction of the phosphorylated channel to ``a nonfunctional plasma membrane pool'' (e.g. see Ref. 41). It has been noticed (reviewed in Ref. 34) that changes in cellular PKA activity can variably affect the expression of closely related ion channels in different cell types. Thus, we suggest that the postulated endogenous factor might not be unique to the oocyte. Variable levels of expression of this factor in different cell types may regulate the balance between different and overlapping PKA effects on the same K channel.

There are two additional implications of this study. The first derives from the fact that the extent of basal phosphorylation was not significantly affected by either of the two PKA inhibitors used. This may indicate that despite the occurrence of basal phosphorylation at Ser-446, which is the site of PKA phosphorylation, the channel is not basally phosphorylated by PKA. This observation is in accord with results concerning K channels (mainly Kv1.2), closely related to RCK1, that were copurified with an unidentified kinase that phosphorylates the channels, probably at sites phosphorylated by exogenously applied PKA(15) . The second implication is that the oocyte homogenate contains a phosphatase that can be inhibited by 50 nM okadaic acid and that can dephosphorylate the RCK1 channel protein. Modulation by phosphatases of K channels has been reported for the voltage-gated Shaker B and Shaker D channels (13) and for the Ca-activated channel(11) . Despite our demonstration that protein phosphatase 2B can dephosphorylate the channel in vitro, it seems unlikely that this is the identity of the endogenous phosphatase since the IC of okadaic acid for protein phosphatase 2B is 5 µM(42) . However, the IC values of okadaic acid for protein phosphatase 2A and protein phosphatase 1 are in the nanomolar range(43) , indicating that either one of them could account for the endogenous phosphatase activity. Further studies should address the possible modulation of the RCK1 channel by dephosphorylation reactions.


FOOTNOTES

*
This work was supported by grants from the Israel Academy of Sciences and the United States-Israel Binational Science Foundation (to I. L.) and by National Institutes of Health Grant NS-29633 (to W. B. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Equal contributors.

To whom correspondence should be addressed. Tel.: 972-36409863; Fax: 972-36409113.

The abbreviations used are: PKA, protein kinase A (cAMP-dependent protein kinase); PKA-CS, catalytic subunit of protein kinase A; (S)-8-Br-cAMPS, 8-bromoadenosine 3`,5`-cyclic monophosphorothioate; CHX, cycloheximide; PAGE, polyacrylamide gel electrophoresis; F, plasma membrane protein fraction; [S]Met/Cys, [S]methionine/cysteine.


ACKNOWLEDGEMENTS

We thank Dr. Nathan Dascal and Dr. S. Blumberg for helpful discussions and for reading the manuscript.


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