Protein Kinase C (PKC) Activity Regulates Functional Effects of Kvβ1.3 Subunit on KV1.5 Channels

Background: Kvβ1.3 fast inactivation conferred onto Kv1.5 is PKC-dependent. Results: PKC inhibition shifts Kvβ1.3-induced inactivation curve without altering Kv1.5-Kvβ1.3 interaction. A Kv1.5 channelosome is characterized. Conclusion: Kv1.5 channelosome is composed of several PKC isoforms (βI, βII, and θ), Kvβ1.3 and RACK1 in HEK293 and in rat ventricular cells. Significance: This is the first evidence of a cardiac Kv1.5-Kvβ1.3-RACK1-PKC macromolecular complex. Kv1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (IKur). The regulatory Kvβ1.3 subunit converts Kv1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by Kvβ1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack Kvβ subunits) transiently cotransfected with Kv1.5+Kvβ1.3 and also rat ventricular and atrial tissue to study native α-β subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that Kv1.5 and Kvβ1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, Kvβ1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed Kvβ1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of Kv1.5 and Kvβ1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between Kv1.5, Kvβ1.3, the receptor for activated C kinase (RACK1), PKCβI, PKCβII, and PKCθ in HEK293 cells. A very similar Kv1.5 channelosome was found in rat ventricular tissue but not in atrial tissue.

The outward potassium current I Kur , 7 the main current responsible for human atrial repolarization, is generated following the activation of K v 1.5 channels. The slow and partial inactivation and the voltage dependence of these channels underlie their key role in the regulation of the atrial action potential duration (1,2). This slow inactivation is modified by the assembly of K v 1.5 subunits with ␤ subunits (K v ␤1.2, K v ␤1.3, and K v ␤2.1) present in the human myocardium (3,4). The K v ␤1.3 subunit provides a number of functions, including a fast, partial inactivation component, a greater degree of slow inactivation, a shift of the activation curve toward more negative potentials, a 7-fold decrease in the sensitivity of the channel to the block induced by antiarrhythmic drugs and local anesthetics, and a decrease in the degree of stereoselective blockage (5)(6)(7).
I Kur is highly susceptible to adrenergic regulation, which is differentially modulated by ␣and ␤-stimulation (8) via protein kinases C (PKC) and A (PKA), respectively (9 -11). This phenomenon is very important because the expression levels of ␣-and ␤-adrenergic receptors are altered in several cardiac pathologies as well as the release of catecholamines (12,13). In fact, cardiac hypertrophy is associated with an up-regulation of different PKC isoforms (14 -17). Similarly, ␦-calmodulin kinase II (␦-CaMKII) expression increases during atrial fibrillation (18). Furthermore, one of the most effective treatments for atrial fibrillation is the oral administration of ␤-blockers (19), which induce a pharmacological remodeling that is capable of reversing the electrical dysfunction typically observed during atrial fibrillation (20,21). PKC and PKA activities are also required for the Kv1.5 modulation by the auxiliary subunits K v ␤1.2 and K v ␤1.3 (9,10,22). Indeed, PKC inhibition by calphostin C reverses the K v ␤1.3-dependent electrophysiological effects (9). Calphostin C, a potent and selective inhibitor of multiple protein kinase C (PKC) isoforms, acts via interaction with the regulatory diacylglycerol (DAG) binding site (23).
PKCs comprise a cluster of at least 11 different isoforms that include three subfamilies according to their sensitivities to second messengers such as Ca 2ϩ and DAG. Classical isoforms (PKC␣, -␤I, -␤II, and -␥) are activated by Ca 2ϩ and DAG, whereas novel isoforms (PKC␦, -⑀, -, and ) are sensitive to DAG but insensitive to Ca 2ϩ . Finally, atypical isoforms (PKC and /) do not require Ca 2ϩ or DAG for their activation, whereas they are dependent on ceramide, arachidonic acid, and other lipids (24 -26). Upon activation, most PKC isoforms undergo subcellular relocation depending on the cell type (27). This specific compartmentation is further fine-tuned by interactions with specific PKC adaptor proteins named receptor for activated C kinase (RACK) (28).
PKC isoforms translocate to different subcellular sites after their activation by specific second messengers, eliciting unique cellular effects (29,30) that are dependent on the substrate to be phosphorylated (26,31). The interaction of PKCs with several substrates is believed to occur through a family of proteins that are collectively named RACKs (32). These adaptor proteins do not have any intrinsic functional effects. However, they are able to translocate bound proteins to different subcellular locations through their protein-protein interaction domains (WD40) (33). Thus, they are capable of binding several enzymes concomitantly to form an enzymatic complex that is localized close to the substrate of the enzyme. This process allows them to regulate different cellular responses (32). RACK1 is one of the adaptor proteins that has been identified as an anchoring protein for PKC. Additionally, several ion channels (K Ca 1.1, Ca v 1.2, K ir 3.1, TRPC3, and NMDA receptor) have been reported to be modulated by PKC via this scaffold protein (34 -39). The binding of RACK1 to PKC␤II enhances the enzymatic activity of the latter by 4 -6-fold (34,40).
In the present study, we analyzed the effects of PKC inhibition on the K v 1.5ϩK v ␤1.3 interaction. None of the isoformselective PKC inhibitors removed the K v ␤1.3-mediated fast inactivation. By silencing all of the PKC isoforms with siRNA or inhibiting PKCs with calphostin C, fast inactivation was abolished at membrane potentials up to ϩ60 mV. However, at membrane potentials more positive than ϩ100 mV, fast inactivation was evident, which indicated that K v ␤1.3 and K v 1.5 remained assembled after PKC inhibition and that without PKC activity, the voltage dependence of K v ␤1.3-induced inac-tivation is dramatically shifted in the positive direction. Additionally, we demonstrate that K v 1.5ϩK v ␤1.3 channels interacted with RACK1, PKC␤I, PKC␤II, and PKC, either directly or through scaffold proteins, generating an emerging K v 1.5 channelosome in HEK293 cells and ventricular tissue.

EXPERIMENTAL PROCEDURES
Expression Plasmids, Cell Culture, and Transient Transfection-Human K v 1.5 and K v ␤1.3 in pBK have been extensively characterized (4). Human K v 1.5 (Ϫ22 to 1,894 nucleotides) and K v ␤1.3 (Ϫ53 to 1,500 nucleotides) were inserted into the same pBK vector, with the K v 1.5 subunit placed 3Ј to the K v ␤1.3 subunit and preceded by an internal ribosome entry sequence, thus generating a bicistronic messenger RNA as described previously (9). The gene encoding K v ␤1.3 was subcloned between the SacII and NotI restriction sites within the polylinker of the pCMV-Tag5A vector, which generated a recombinant K v ␤1.3-Myc protein. To generate K v 2.1-HA, the HA epitope was inserted after Gly-217 of the rat K v 2.1 cDNA, placing the epitope in the extracellular S1-S2 loop (41). In some experiments, a construct with an HA tag introduced into the K v 1.5 S1-S2 loop was used (kindly provided by Prof. D. J. Snyders).
To knock down all PKC isoforms, we transfected HEK293 cells with PKC-specific small interfering RNAs (siRNAs) purchased from Santa Cruz Biotechnology, according to the manufacturer's instructions. The PKC siRNA contains five targetspecific 19 -25-nucleotide siRNAs that are designed to knock down gene expression. The most efficient transfection results were obtained with 50 nM of the siRNA duplexes transfected 72 h prior to the experiments.
Electrophysiological Recordings and Data Acquisition-The intracellular pipette filling solution contained the following (in mM): 80 potassium aspartate, 42 KCl, 3 phosphocreatine, 10 KH 2 PO 4 , 3 MgATP, 5 HEPES-K, and 5 EGTA-K (adjusted to pH 7.25 with KOH). The bath solution contained the following (in mM): 140 NaCl, 4 KCl, 1.8 CaCl 2 , 1 MgCl 2 , 10 HEPES-Na, and 10 glucose (adjusted to pH 7.40 with NaOH). Currents were recorded using the whole-cell configuration of the patch clamp technique with a patch clamp amplifier (Axopatch-200B patch clamp amplifier; Molecular Devices) and stored on a personal computer (TD Systems) with a DigiData 1440A analog-to-digital converter (Molecular Devices). PClamp version 10 software was used for both data acquisition and analysis (Molecular Devices). Currents were recorded at room temperature (21-23°C) at a stimulation frequency of 0.1 Hz and were sampled at 4 kHz after antialias filtering at 2 kHz. The average pipette resistance ranged between 1 and 3 megaohms (n ϭ 70). Gigaohm seal formation was achieved by suction (2-5 gigaohms, n ϭ 70). After seal formation, the cells were lifted from the bath, and the membrane patch was ruptured with a brief additional suction. The capacitive transients elicited by symmetrical 10-mV steps from Ϫ80 mV were recorded at 50 kHz and filtered at 10 kHz for subsequent calculations of the capacitive surface area, access resistance, and input impedance. Thereafter, the capacitance and series resistance compensation were optimized, and usually 80% compensation of the effective access resistance was obtained. MicroCal Origin 7.05 (Origin-Lab Co) and the Clampfit utility of pClamp 9 were used to perform least squares fitting and for data presentation. Deactivation and inactivation were fitted to a biexponential process with an equation of the form y ϭ A 1 exp(Ϫt/ 1 ) ϩ A 2 exp(Ϫt/ 2 ) ϩ C, where 1 and 2 are the system time constants, A 1 and A 2 are the amplitudes of each component of the exponential, and C is the baseline value. The voltage dependence of the activation curves was fitted with a Boltzmann equation: y ϭ 1/(1 ϩ exp(Ϫ(V Ϫ V h )/s)), where s represents the slope factor, V represents the membrane potential, and V h represents the voltage at which 50% of the channels are open.
Protein Extracts, Immunoprecipitation, and Western Blot-For total protein extraction from HEK293 cells, the cells were washed twice in chilled phosphate-buffered saline (PBS) and centrifuged at 3,000 ϫ g for 10 min. The pellet was then lysed in ice-cold lysis solution (20 mM HEPES, pH 7.4, 1 mM EDTA, 255 mM sucrose supplemented with Complete protease inhibitor mixture tablets (Roche Diagnostics)), and homogenized by repeated passage (10 times) through a 25-gauge (0.45 ϫ 16 mm) needle. Homogenates were further centrifuged at 10,000 ϫ g for 5 min to remove nuclei and organelles. Samples were separated into aliquots and stored at Ϫ80°C. For immunoprecipitation assays, we isolated membrane protein from the total protein extract by an additional centrifugation at ϳ150,000 ϫ g for 90 min. The pellet was resuspended in 30 mM HEPES (pH 7.4), and the protein content was determined using the Bradford Bio-Rad protein assay (Bio-Rad). Ventricular (principal coronary arteries excluded) and atrial tissues from male Wistar rats were kindly provided by Drs. A. Cogolludo and F. Pérez-Vizcaíno (Universidad Complutense de Madrid, Spain). After dissection, cardiac tissue was frozen in liquid nitrogen and homogenized in a glass potter (300 l and 3 ml of the lysis buffer described above were used for atria and ventricles, respectively). The homogenate was centrifuged at 6000 ϫ g for 10 min at 4°C. The supernatant was collected, separated into aliquots, and stored at Ϫ80°C until its posterior analysis.
For the coimmunoprecipitation experiments, the homogenates were resuspended in 150 l of immunoprecipitation buffer (1% Nonidet P-40, 10% glycerol, 10 mM HEPES, and 150 mM NaCl supplemented with Complete protease inhibitor mixture tablets (pH ϭ 7.8) (Roche Diagnostics)) and homogenized by orbital shaking at 4°C for 1 h. 300 g of crude membrane protein was used for HEK293 cells, 500 g was used for rat atria, and 1500 g was used for the ventricular tissue. Proteins were then incubated with 20 l of immunoprecipitation buffer-prewashed Sepharose protein A/G beads (Santa Cruz Biotechnology) for 2 h at 4°C, and contaminant-bound Sepharose beads were separated by centrifugation for 30 s at 5000 ϫ g at 4°C.
The supernatant was incubated with 4 ng of polyclonal anti-K v 1.5 (Alomone Labs) or monoclonal anti-RACK1 antibody (Santa Cruz Biotechnology) for each microgram of protein, overnight at 4°C with orbital shaking. Approximately 20 -30 l of PBS-washed Sepharose protein A/G beads was then added to the mixture followed by incubation for 2 h. Sepharose beads bound to antibody-protein complexes were precipitated by centrifugation (30 s at 5000 ϫ g at 4°C), and antibody-bound beads were then washed twice with immunoprecipitation buffer and centrifuged for 30 s at 5000 ϫ g at room temperature. In the case of cardiac tissue samples, coimmunoprecipitation was performed using Pierce Direct IP kit (Thermo Scientific) following the manufacturer's instructions.
Total protein extracts and immunoprecipitated protein samples were resuspended in 1ϫ SDS (2% ␤-mercaptoethanol) and boiled at 100°C for 5 min. The samples were then centrifuged for 3 min at 5,000 ϫ g at room temperature, and 25-50 l of protein extract was separated by SDS-PAGE (7, 10, or 15% acrylamide/bisacrylamide) gels. The proteins, transferred to PVDF membranes, were probed with anti-K v 1.5, anti-Myc, anti-PKC, anti-K v ␤1, and anti-RACK1 antibodies. Secondary antibodies were developed by ECL-Plus Western blotting reagent (Amersham Biosciences).
Immunostaining and Confocal Microscopy-For immunostaining, HEK293 cells were grown on gelatin-coated coverslips in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Twenty-four hours after transfection, the cells were washed three times with PBS. For antibody-induced patching experiments, after 30 min of incubation with blocking solution (10% goat serum, 5% nonfat dry milk, PBS), the cells were incubated with the S1-S2 K v 1.5 external epitope antibody (diluted 1:1000) or anti-HA (diluted 1:250) in HEPES-based culture medium for 1 h at room temperature (43). Next, the cells were fixed with 4% paraformaldehyde in PBS for 10 min and blocked overnight (PBS ϩ 5% w/v dry milk). The cells were washed and permeabilized three times with PBS-CHAPS and then incubated with anti-Myc antibody (1:500; PBS-CHAPS with 10% goat serum). Next, the cells were washed three times with PBS-CHAPS and incubated with Alexa Fluor 488 antirabbit (1:500) and Alexa Fluor 594 anti-mouse (1:500) antibodies from Molecular Probes. The samples were mounted with Aqua Poly/Mount (Polysciences, Inc). Stained cells were visualized using LSM510 ZEISS (Carl Zeiss) or Leica TCS SP5 confocal microscopes, and images were analyzed with Zeiss, Leica, and ImageJ software (44).
Statistical Analysis-Data are presented as the mean Ϯ S.E. One-way analysis of variance was used to compare more than two groups. Statistical significance was set at p Ͻ 0.05. The curve-fitting procedure used a nonlinear least squares (Gauss-Newton) algorithm; the results are displayed in a linear and semilogarithmic format together with the difference plot. Goodness of fit was determined using the 2 -square criterion and by inspection of systematic nonrandom trends in the difference plot.

Modulation of K v ␤1.3-induced Fast Inactivation Is Not
Driven by a single PKC Isoform-Although PKC inhibition prevents K v ␤1.3-induced fast inactivation of K v 1.5 channels (9 -11), the identity of the specific isoform responsible for this phenomenon remains unknown. To that end, we first determined which PKC isoforms are expressed in HEK293 cells. Supplemental Fig. S1 shows that HEK293 cells express all PKC isoforms (classical: ␣, ␤, and ␥; novel: ␦, ⑀, and ; and atypical: and /) with the exception of the novel PKC isoform.
Next, we performed electrophysiological experiments using different PKC inhibitors. Fig. 1 shows representative current traces obtained under control conditions and after PKC inhibition with different inhibitors. The characteristics of these PKC inhibitors are shown in Table 1. With the exception of calphostin C, PKC inhibitors (Gö6976, Gö6983, hispidin, and PKC-PI) failed to abolish K v ␤1.3-induced fast inactivation. Only after PKC inhibition with calphostin C did the current-voltage relationship (IV) exhibit the linearity observed when the K v 1.5 current is recorded in the absence of K v ␤1.3. Different mechanisms of action of PKC inhibitors may explain these results. Table 2 shows the values obtained for the degrees of inactiva-FIGURE 1. Modulation of K v ␤1.3-induced fast inactivation is not due to a single PKC isoform. HEK293 cells were transiently transfected with K v 1.5ϩK v ␤1.3. Representative current traces elicited by 250-ms depolarizing pulses from a holding potential of Ϫ80 mV to voltages between Ϫ80 and ϩ60 mV in 10-mV steps and the IV relationships in which the current magnitude measured at the end of 250-ms pulses was plotted versus the membrane potential are shown. Tail currents were obtained upon return to Ϫ40 mV. The specific inhibitor used is indicated above each trace family. tion, V h , and the inactivation and deactivation kinetics () of the currents generated after treating the cells with various PKC inhibitors. Remarkably, all of the tested PKC inhibitors decreased the degree of fast inactivation and slowed the fast time constant of the inactivation process (Table 2).
siRNA-induced Down-regulation of PKC Mimics Effects of Calphostin C-The results presented above could be explained by either (a) an effect that requires inhibition of different PKC isoforms or (b) a direct effect of calphostin C that is independent of its PKC inhibitory properties. To differentiate between these two possibilities, we performed a series of experiments in which all PKC isoforms were silenced using siRNA (PKC siRNA). The greatest PKC silencing was observed 72 h after transient transfection of siRNA duplexes at a concentration of 50 nM (Fig. 2A). In the present study, PKC siRNA led to an ϳ75% decrease in expression of the various PKC isoforms (Fig.   2B, supplemental Table S1). Fig. 2C shows non-inactivating electrophysiological recordings of the K v 1.5ϩK v ␤1.3 channels after PKC siRNA treatment. Voltage-dependent potassium currents were evoked by applying depolarizing pulses from a holding potential of Ϫ80 mV to different voltages between Ϫ80 to ϩ60 mV in 10-mV steps. Fig. 2D shows the IV relationships measured at the end of the 250-ms pulses under these experimental conditions. Under control conditions, the IV relationship reached a plateau at around ϩ15 mV. Binding of the inactivating particle of the K v ␤1.3 subunit is a highly voltagedependent process; at more positive test potentials, this binding is more likely to occur. PKC siRNA eliminated fast inactivation, and thus, the IV relationships obtained under these experimental conditions resembled those obtained after activation of K v 1.5 channels in the absence of K v ␤1.3 subunits (7). Hence, this treatment mimicked the effects of calphostin C on the current generated after activation of K v 1.5ϩK v ␤1.3 channels, which ruled out a possible direct effect of calphostin C. K v 1.5 and K v ␤1.3 Still Colocalize in Cell Membrane during PKC Inhibition-If the inhibition of PKC affects the assembly of these subunits, a physical dissociation of both proteins in the cell membrane should be detected. To further test the above hypothesis, we used two different experimental approaches. First, we performed a series of experiments in which the K v 1.5 and K v ␤1.3-Myc subunits were stained using antibody-induced patching. This method of immunostaining is based on the for-mation of antibody-induced membrane patches after labeling an external epitope of the K v 1.5 channel located in the S1-S2 loop (using a custom-made polyclonal antibody that recognizes this epitope). Fig. 3A shows the colocalization of K v 1.5 and K v ␤1.3-Myc (n ϭ 28). This pattern is specific because, as expected, K v 2.1 and K v ␤1.3-Myc (n ϭ 8), which do not coassemble (45,46), did not colocalize (Fig. 3B). Interestingly, we did not detect differences in the degree of colocalization between control and calphostin C-treated cells (n ϭ 29) (Fig. 3D).
Given the widespread cellular distribution of K v ␤1.3, we next analyzed via immunoprecipitation whether K v 1.5 and K v ␤1.3 remained associated with K v 1.5 following calphostin C-mediated PKC inhibition. Fig. 4A shows that K v 1.5 protein was immunoprecipitated in the presence but not in the absence of anti-K v 1.5 antibody. As shown in Fig. 4B, we did not observe any K v ␤1.3-Myc signal in untransfected cells or in cells transfected with K v 1.5 alone. However, the K v ␤1.3 subunit remained associated with K v 1.5 under control conditions and after calphostin C treatment, which suggests that these proteins do not dissociate following treatment with calphostin C.
Positive Potentials Up to ϩ100 mV Reveal Presence of K v ␤1.3-Assembly of the K v ␤1.3 subunit causes fast inactivation of the K v 1.5 outward current (45,(47)(48)(49). Therefore, it was surprising to observe that even when these subunits remained assembled, K v ␤1.3-induced fast inactivation was abolished either by inhibition of PKC with calphostin C or by silencing PKC. K v 1.5ϩK v ␤1.3 current is activated at Ϫ30 mV and inactivates at potentials positive to 0 mV, exhibiting a degree of inactivation of 70 Ϯ 2% (n ϭ 23) when measured at ϩ60 mV (Fig. 1). After PKC inhibition with calphostin C-or siRNA-mediated PKC silencing, K v 1.5ϩK v ␤1.3 currents showed a degree of slow inactivation that ranged between 20 and 30%, whereas pulse steps to potentials positive to ϩ60 mV revealed the presence of the typical K v ␤1.3-induced fast inactivation (Fig. 5A). Fig. 5A also shows the mean values of the inactivation degrees obtained at ϩ100 mV recorded in at least four experiments. These results further suggest that K v 1.5 and K v ␤1.3 remain assembled after PKC inhibition.
This outcome suggests that after PKC inhibition, the inactivation curve of K v 1.5-K v ␤1.3 current is shifted toward more positive membrane potentials. To test this hypothesis, a series of experiments was performed in which a double-pulse protocol was applied (Fig. 5B). These results show that the inactivation curve in calphostin C-treated cells was shifted toward more positive potentials (V h ϭ Ϫ26.5 Ϯ 0.6 versus Ϫ8.5 Ϯ 1.9 mV in the presence and in the absence of calphostin C, respectively; n ϭ 10; see Fig. 5C and Table 3). Moreover, the degree of inactivation in calphostin C-treated cells decreased from 47.3 Ϯ 5.7% (n ϭ 5) to 34.6 Ϯ 2.2% (n ϭ 7) (p Ͻ 0.05).
Effects of PIP 2 and OAG on K v ␤1.3-induced Fast Inactivation-It has been proposed that PIP 2 associates with the N terminus of the K v ␤1.3 subunit, and when the ␤ subunit dissociates from PIP 2 , it assumes a hairpin structure that can enter the central cavity of an open K v 1.5 channel, triggering N-type inactivation. Therefore, it has been suggested that K v ␤1.3-induced fast inactivation is mediated by equilibrium binding of the N terminus of K v ␤1.3, which switches between binding to phosphoinositides (PIPs) and the inner pore region of K v 1.5 channels (50). Moreover, stimulation of ␣1-receptors activates PLC␥, which is capable of cleaving PIP 2 into IP 3 and DAG, thus activating classical and novel PKCs. Thus, the effects of PIP 2 and OAG (a nondegradable DAG analog) were analyzed by adding them to the internal solution (Fig. 6). Cells dialyzed with PIP 2 exhibited a lower degree of fast inactivation just after patch rupture in comparison with control cells (55 Ϯ 4% versus 68 Ϯ 6%, n ϭ 4, p Ͻ 0.05) (Fig. 6A). Also, after 8 min of dialysis with PIP 2 , the degree of N-type inactivation increased significantly (from 55 Ϯ 4% to 70 Ϯ 4%, n ϭ 4, p Ͻ 0.05) (Fig. 6A). These effects could be due to a decrease of the PIP 2 concentration due to its cleavage into IP 3 and DAG. In fact, the degradation of PIP 2 would increase the ability of the N terminus of K v ␤1.3 to inactivate K v 1.5 (50). In contrast, cells dialyzed with OAG exhibited a degree of N-type inactivation similar to control cells, both after patch rupture and after 8 min of dialysis with OAG (65 Ϯ 3% to 67 Ϯ 2%, n ϭ 5, p Ͼ 0.05) (Fig. 6B). These results are in agreement with the involvement of classical and novel PKC isoforms in the effects of calphostin C and PKC siRNA on the fast K v ␤1.3-induced inactivation. To test whether the PIP 2 effects at different times (t ϭ 0 or 8 min) were due to the cleavage of PIP 2 into DAG and IP 3 , a series of experiments in which cells previously incubated with a PLC inhibitor (U73122, 10 M) and dialyzed with PIP 2 was performed (Fig. 6C). Under these experimental conditions, the degree of fast inactivation was reduced (from 69 Ϯ 2% to 62 Ϯ 2%, n ϭ 9, p Ͻ 0.01), the time constant of the fast inactivation was increased, and the contribution of the fast component of inactivation was decreased.
The K v 1.5 Macromolecular Complex Contains K v 1.5, K v ␤1.3, RACK1, PKC␤I, PKC␤II, and PKC-Several ion channels have been reported to be modulated by PKC via RACK1 (34). We hypothesized that K v 1.5, K v ␤1.3, and PKC form a functional complex in which PKC activity is an essential requirement for the induction of fast and incomplete channel inactivation by K v ␤1.3. To test this hypothesis, immunocytochemistry experiments were performed (Fig. 7). Fig. 7A shows confocal images of cells transfected with K v 1.5-HA and K v ␤1.3, in which we stained for K v 1.5 and RACK1. Fig. 7B shows confocal images of cells transfected with K v 1.5 and K v ␤1.3-Myc, in which we stained for K v ␤1.3 and RACK1. As shown, under both experimental conditions, colocalization was consistent between K v 1.5 and RACK1, as well as between K v ␤1.3 and RACK1. Given the widespread immunolocalization patterns, we confirmed subunit association using immunoprecipitation experiments. We immunoprecipitated K v 1.5 channels and blotted for PKC␤II to confirm the presence of this enzyme in the protein complexes. As shown in Fig. 8A, PKC␤II signal was absent in PKC siRNA-transfected cells. Furthermore, blots against RACK1 confirmed the interaction of K v 1.5 with this adaptor protein (Fig. 8B). Moreover, reverse coimmunoprecipitation (immunoprecipitation of RACK1) yielded similar results, revealing the presence not only of PKC␤II (data not shown) but also of the K v 1.5 and K v ␤1.3 subunits (Fig. 8C). Collectively, these results demonstrate the presence of a functional complex or channelosome that includes K v 1.5, K v ␤1.3, RACK1, and PKC␤II. This interaction was demonstrated in controls and was found to be absent in PKC-silenced cells (Fig. 8A). On the other hand, because calphostin C inhibits PKC by binding to the DAG binding site, the reduced level of PKC␤II in calphostin C-treated cells is not surprising, as inhibited PKC␤II may not be able to bind RACK1 in the channelosome.
To determine which PKC isoforms are present in this macromolecular complex, a series of coimmunoprecipitation experiments were performed in which we immunoprecipitated K v 1.5 channels and blotted for the PKC isoforms present in HEK293 cells (Fig. 9). We observed that only PKC␤I, PKC␤II, and PKC were present in the K v 1.5 channelosome.
The K v 1.5 Channelosome Is Present in Rat Ventricular Myocytes but Not in Atrial Myocytes-Finally, we performed experiments in cardiac tissue (Fig. 10). Coimmunoprecipitation experiments were performed in which we immunoprecipitated K v 1.5 channels and blotted for PKC␤I, PKC␤II, PKC, RACK1, and K v ␤1.x. Samples from ventricular homogenates presented a K v 1.5 channelosome composed of K v 1.5, K v ␤1, RACK1, PKC␤I, and PKC␤II (Fig. 10A). Importantly, atrial tissue did not display this K v 1.5 protein complex (Fig. 10B) because none of the proteins studied forming the K v 1.5 channelosome in ventricle coimmunoprecipitated with K v 1.5 in atrial tissue.

DISCUSSION
In the present study, we have analyzed the mechanisms by which calphostin C-mediated PKC inhibition abolishes K v ␤1.3induced fast inactivation of the K v 1.5 channel. We have demonstrated that the inhibition of at least classical and novel PKC isoforms is required to abolish K v ␤1.3-induced fast inactivation (9,10). Furthermore, this effect was not due to K v 1.5ϩK v ␤1.3 dissociation but due to a positive shift of the inactivation curve driven by PKC inhibition because both subunits remained assembled as shown by immunocytochemistry, immunoprecipitation, and electrophysiological experiments. We have also shown that at least K v ␤1.3, RACK1, PKC␤I, PKC␤II, and PKC are associated with K v 1.5 in HEK293 cells, forming a channelosome. Finally, for the first time, we provide evidence pointing to the existence of a native ventricular cardiac K v 1.5 channelosome, whose composition is similar to that found in HEK293 cells (with the exception of PKC, absent in this tissue according to our Western blot analyses, data not shown).
The stimulation of ␣1-receptors leads to activation of PLC␥. This enzyme cleaves PIP 2 , generating IP 3 and DAG, which activate most PKC isoforms either alone or with Ca 2ϩ , with the exception of atypical PKCs. The N terminus of the K v ␤1.3 subunit associates with membrane-bound PIP 2 , and when it dissociates from PIP 2 , it assumes a hairpin structure that enters the central cavity of an open K v 1.5 channel, inducing fast inactivation. Thus, K v ␤1.3-induced fast inactivation is mediated by a competitive binding between PIPs and the inner pore region of K v 1.5 channels for the N terminus of K v ␤1.3 (50). The present results obtained for PIP 2 and OAG are in agreement with previous studies and indicate a fine-tuned regulation of K v ␤1.3induced fast inactivation by PKC and PIP 2 . Moreover, the effects of PIP 2 in cells in which the PLC was inhibited produced a marked decrease of the K v ␤1.3-induced fast inactivation. In the absence of PIP 2 into the internal solution, the effects observed were qualitatively similar (data not shown), suggesting a role of PLC on the fast inactivation induced by this ␤ subunit.
Ϫ8.5 Ϯ 1.9* 9.8Ϯ.9* Most PKC isoforms redistribute into different subcellular compartments upon activation, depending on the PKC isoform and the cell type (36). A surprising finding of the present study was that all of the PKC inhibitors tested (Gö6976, Gö6983, hispidin, and PKC-PI), with the exception of calphostin C, failed to abolish K v ␤1.3-induced fast inactivation. This result is likely due to the different mechanisms of action of the PKC inhibitors used. Indeed, calphostin C inhibits PKC by binding to its C1 domain (DAG and phorbol ester binding site) and then irreversibly inactivates the enzyme. Gö6983 and Gö6976 are competitive inhibitors of ATP binding to the catalytic domain of PKC, whereas hispidin affects both PKC␤I and PKC␤II translocation (23,(51)(52)(53)(54). Our results suggest that inhibition of classical and novel, but not atypical, PKC isoforms counteracts K v ␤1.3-induced fast inactivation and shifts the V h of the activation curve to values closer to those of K v 1.5 observed in the absence of K v ␤1.3, as has been described previously (9). The potential dissociation of ␣-␤ caused by PKC inhibition was also analyzed in the present study. Colocalization, immunoprecipitation, and electrophysiological experiments revealed that these subunits remained assembled in the plasma membrane despite PKC inhibition by calphostin C. These results are in agreement with the notion that K v 1.5 and K v ␤1.3 subunits assemble in the endoplasmic reticulum during the early stages of their biosynthesis (55), and a 2-h incubation with calphostin C is sufficient to eliminate the typical fast inactivation induced by K v ␤1.3 (9). Moreover, previous results reported by Kwak et al. (9) showed that a tandem construction of K v 1.5 and K v ␤1.3, which generates a unique protein, showed similar responses to calphostin C (a shift in V h to more positive potentials and a loss of fast inactivation). These results indicate that mechanisms other than subunit dissociation must be involved in the abolishment of K v ␤1.3-induced fast inactivation produced by calphostin C.
Although we were able to rule out a role for atypical PKCs, the involvement of a specific PKC isoform in this process has not yet been demonstrated. In addition, experiments in which the membrane potential was depolarized to ϩ100 mV indicated that despite PKC inhibition (calphostin C or PKC siRNA), K v ␤1.3 was still capable of conferring fast inactivation and thus remained associated with K v 1.5. Furthermore, the coimmunoprecipitation experiments showed that K v ␤1.3 remained associated with K v 1.5 despite PKC inhibition by calphostin C or by PKC silencing by siRNA. These data suggest that K v 1.5 and K v ␤1.3 subunits form a stable complex following their biosynthesis in the endoplasmic reticulum and that PKC inhibition and/or activation modulates the inactivating effect of K v ␤1. 3. It has been demonstrated that residues Arg-5 and Thr-6 of the K v ␤1.3 subunit are involved in PIP 2 binding and that mutations (alanine or cysteine) at these residues dramatically increase the degree of K v ␤1.3-induced fast inactivation. It has been described that calphostin C does not modify the gating of the K v 1.5 in the absence of ␤ subunits (9). Therefore, it is likely that the effects shown in the present study involve the phosphorylation of some residues present in the K v ␤1.3 subunit. One candidate could be threonine at position 6 (Thr-6) of K v ␤1.3. Thus, we may hypothesize that PKC phosphorylation of this threonine or another threonine/serine at the N terminus of the K v ␤1.3 subunit can lead to a diminished capability to bind PIP 2 , thus avoiding the abrogation of fast inactivation caused by PIP 2 . These findings provide an explanation for the effects of PKC inhibition on the K v ␤1.3-induced inactivation, assuming that phosphorylated K v ␤1.3 cannot bind PIP 2 . Nonphosphorylated K v ␤1.3 would be able to bind PIP 2 , and this binding would abolish N-type inactivation (50). However, further experiments are necessary to elucidate the residue(s) involved in this effect.
In the past decade, myriads of protein-protein interactions involved in intracellular signaling have been described (56 -59). Determination of the subcellular localization of signal transduction proteins, enzymes, substrates, and mediators has revealed the rapid, efficient transmission of signals either from the extracellular medium or from intracellular sites as an essential feature of optimal signaling (60). Despite the high degree of homology between PKC isoforms at both sequence and struc-  tural levels, especially within the catalytic domain, each PKC isoform mediates unique subcellular functions (29,30) that are dependent on the target substrate (26,31). These localization mechanisms are especially common in plasma membrane proteins because incoming stimuli must be integrated and transmitted with a high degree of efficiency. In the present study, we have demonstrated that K v 1.5 and K v ␤1.3 form a highly stable protein complex, which is tightly regulated by classical and novel, but not atypical, PKC isoforms. Furthermore, our results show that PKC␤I, PKC␤II, and RACK1 coassemble with K v 1.5 and K v ␤1.3. Therefore, the K v 1.5 functional channelosome contains at least K v 1.5, K v ␤1.3, RACK1, PKC␤I, PKC␤II, and   3). Negative (AbϪ) and positive (Abϩ) immunoprecipitations (IP) blotted against the different PKC isoforms present in HEK293 channelosome (Fig. 9). Note that a very similar channelosome has been found in ventricular (A), but not in atrial (B), tissue. WB, Western blot; S, supernatants.
PKC. Besides, a similar channelosome has been found in ventricular tissue, but not in atrial tissue, showing a parallel distribution to that of K v ␤1.3 (47), which suggests an important role of this ␤ subunit in the constitution of the macromolecular complex.
The presence of RACK1 in other ion channel complexes has been previously reported. Indeed, RACK1 binds to the carboxyl terminus of K Ca 1.1 channels (35). Moreover, the overexpression of RACK1 with K Ca 1.1 channels in Xenopus oocytes shifts the activation curve toward more positive potentials in the absence, but not in the presence, of ectopically expressed ␤ channel subunits (35). Furthermore, PKC␤II, which is recruited by RACK1, down-regulates Ca v 1.2 activity following activation (36). Besides, the functional K ir 3.1 complex contains PKA, protein phosphatase 1 (PP1), PP2A/C, and RACK1. Within this complex, RACK1 binds directly to G␤␥ (37) and PKC (34). Recently, it has been described that TRPC3 regulates IP 3 receptor (IP 3 R) function by mediating interaction between IP 3 R and the scaffolding protein RACK1, and the importance of the Orai1-STIM1-TRPC3-RACK1-IP 3 R complexes in the fine modulation of intracellular Ca 2ϩ stimulated by agonists of these receptors has been reported (38). Thus, RACK1 is emerging as an important adaptor protein that may play very important roles in the modulation of different ion channels and in the interaction between ion pores and ancillary subunits. The variety of signaling proteins linked to RACK1 (32), which include PKC␤II (34,40), PLC␥ (61), Src (62), and dynamin-1 (63), among others, could explain the diverse effects of PKA and PKC on the K v 1.5ϩK v ␤1.3 current (8 -11) and the requirement for simultaneous inhibition of numerous PKC isoforms to abolish fast inactivation.
In conclusion, we have analyzed the mechanisms by which calphostin C abolishes K v ␤1.3-induced fast inactivation. Our experiments demonstrate that this effect is not due to the dissociation of K v 1.5 and K v ␤1.3 subunits and that these subunits remain assembled following PKC inhibition. Our experiments additionally demonstrate that PLC is also involved in the regulation of the Kv␤1.3-induced fast inactivation.
In addition, we have characterized a K v 1.5 channelosome in which K v 1.5, K v ␤1.3, RACK1, PKC␤I, PKC␤II, and PKC physically and functionally interact. Importantly, we have identified a very similar macromolecular complex in rat ventricular tissue. The description and functional characterization of this channelosome open up a variety of possible mechanisms to explain the differences between I Kur recorded in ventricle and atrium in different animal species. Differential composition of this K v 1.5 complex could constitute a primary mechanism of capital importance for the modulation of cardiac excitability.