Protein kinase C modulates inactivation of Kv3.3 channels

Abstract

Modulation of some Kv3 family potassium channels by protein kinase C (PKC) regulates their amplitude and kinetics and adjusts firing patterns of auditory neurons in response to stimulation. Nevertheless, little is known about the modulation of Kv3.3, a channel that is widely expressed throughout the nervous system and is the dominant Kv3 family member in auditory brainstem. We have cloned the cDNA for the Kv3.3 channel from mouse brain and have expressed it in a mammalian cell line and in Xenopus oocytes to characterize its biophysical properties and modulation by PKC. Kv3.3 currents activate at positive voltages and undergo inactivation with time constants of 150-250 ms. Activators of PKC increased current amplitude and removed inactivation of Kv3.3 currents, and a specific PKC pseudosubstrate inhibitor peptide prevented the effects of the activators. Elimination of the first 78 amino acids of the N terminus of Kv3.3 produced noninactivating currents suggesting that PKC modulates N-type inactivation, potentially by phosphorylation of sites in this region. To identify potential phosphorylation sites, we investigated the response of channels in which serines in this N-terminal domain were subjected to mutagenesis. Our results suggest that serines at positions 3 and 9 are potential PKC phosphorylation sites. Computer simulations of model neurons suggest that phosphorylation of Kv3.3 by PKC may allow neurons to maintain action potential height during stimulation at high frequencies, and may therefore contribute to stimulus-induced changes in the intrinsic excitability of neurons such as those of the auditory brainstem.

FIGURE 1.

Amino acid sequence alignment of the cloned mouse Kv3. 3 cDNA with the previously reported Kv3.3 sequence (Q63959) and a mouse EST (BE949391) from GenBank™. The N terminus of the cDNA sequence investigated in this study had differences in 8 amino acids from the previously reported Kv3.3 (Q63959) cDNA sequence but was identical to an EST BE949391 from the mouse dbEST. In the same N-terminal region, the cDNA sequence differed from the rat sequence (NP_446449) by 9 amino acids and 20 amino acids with the human (NP_004968) sequence. Identical amino acids are shown in red. Positions where differences occur are shown in blue with the amino acids that differ from the consensus sequence in black.

FIGURE 2.

Expression of Kv3. 3 in mouse brainstem. Anti-Kv3.3 immunoreactive protein from CHO cells transfected with Kv3.3 cDNA has a molecular mass of ∼100 kDa, similar to the native anti-Kv3.3 immunoreactive protein from mouse brainstem. No signal was observed in nontransfected CHO cells. Western blot was stripped and re-probed with anti-Na/K-ATPase. Presence of anti-Na/K-ATPase immunoreactive protein in both the CHO cell lanes show approximately equal amounts of protein were loaded. The protein band at ∼28 kDa is probably a proteolytic fragment of Kv3.3 channel protein, and the bands at the top of the gel are because of membrane aggregates.

FIGURE 3.

Biophysical properties of Kv3. 3 channels. A, representative current traces and current-voltage relationship for Kv3.3 currents recorded from CHO cells in the whole-cell configuration. Currents were evoked by applying 800-ms voltage steps from a holding potential of -70 to + 70 mV in 10-mV increments. Conductance values were obtained by dividing current with electrochemical driving force (I_K/(V_m - E_K)). Normalized conductance-voltage plots were obtained by dividing conductance (G) with maximal conductance (G_max) and fitting with Boltzmann equation. The plots of τ of activation (B, upper panel) and τ of inactivation (B, lower panel) were determined, respectively, by fitting the rising and inactivating phases of currents obtained in A with a single term exponential equation. C, steady-state inactivation (h_{730 ms}) of Kv3.3 was determined by holding the membrane potential from a pre-pulse potential ranging from -100 to +50 mV in 10-mV increments for 730 ms to a test voltage step of +20 mV for 250 ms. Current amplitude was normalized to the maximum current, and the resulting plot was fit with the Boltzmann equation. D, τ of recovery from inactivation was determined by stepping the cells from -100 mV to 20 mV for 2000 ms to a -100-mV step of variable durations in 100- or 7.5-ms increments immediately followed by a test step to 40 mV for 350 ms. Normalized amplitudes (I) to maximum (I_max) values at 40-mV steps were plotted against the inter-pulse interval and fitted with a single exponential decay equation. E, for determining the τ of deactivation, the cells were held at -70 mV, stepped to +50 mV for 5 ms, and then stepped from -110 to 40 mV in 10-mV increments for 10 ms. The deactivating phase of the currents was fit with a single function exponential equation. F and G show single channel properties of Kv3.3 in CHO cells. F, representative cell-attached single channel current traces and their corresponding amplitude histograms at different voltages. Only three of the 10 traces obtained for each voltage are shown; however, all 10 were used for analysis. The patch was held at -80 mV and stepped to different voltages for 1500 ms. G, mean current-voltage (I-V) relationships of channels obtained by determining current amplitude at first level at different voltages (n = 7). The slope of the line fitted on this I-V plot is 15.17 ± 1.9 pS. H, normalized N(P_o) values determined for different voltages (n = 6).

FIGURE 4.

Current-voltage relationship of Kv3. 3 currents in Xenopus oocytes. A, representative current traces for Kv3.3 currents expressed in Xenopus oocytes. Currents were obtained by applying 1000-ms voltage steps from a holding potential of -80 to +70 mV in 10-mV increments. B, current-voltage relationship. Peak current amplitude at each voltage step was divided by maximum current amplitude obtained at +70 mV and was plotted against the applied voltage. Kv3.3 currents start to activate at voltages more positive of -20 mV. The plots of τ of activation (C) and inactivation (D) were obtained, respectively, by fitting the rising and inactivating phases of the currents in A with a single term exponential equation. At 40 mV, the Kv3.3 currents activate within 2.6 ± 0.26 ms and inactivate within 359.5 ± 83.8 ms.

FIGURE 5.

Bryostatin-1 and PseudoRack1 increase peak current amplitudes and remove inactivation of Kv3. 3 currents, and pretreatment with PKC-(19-31) peptide reduces their effect. A, representative traces of Kv3.3 currents in Xenopus oocytes before and 20 min after exposure to the solvent DMSO (0.01%) (upper trace), to 10 nm Bryostatin-1 (middle trace), or pretreatment with PKC-(19-31) for 10 min before application of Bryostatin1 for 20 min (lower trace). C, representative traces of Kv3.3 currents in Xenopus oocytes before and 20 min after exposure to PseudoRack1 (5 μm)(middle traces) or after addition of the buffer ND-96 alone or after exposure to PKC-(19-31) for 10 min prior to the application of PseudoRack1 for 20 min (lower trace). E, representative traces of Kv3.3 currents before and after the application of PKC-(19-31) for 10 min. B, D, and F, quantification of the responses of Kv3.3 currents to 10 nm Bryostatin-1 (B, n = 7) or 5 μm PseudoRack1 (D, n = 4) or 5 μm PKC-(19-31) (F, n = 9) and the response of Kv3.3 currents to these PKC activators in the presence of PKC pseudosubstrate PKC-(19-31) (n = 4 and 6 for Bryostatin-1 and PseudoRack1, respectively). For application of ND-96 alone (F), n = 4. Kv3.3 currents were measured by applying voltage commands from a holding potential of -80 mV to +40, +50, +60, and +70 mV for a duration of 1000 ms. Peaks currents were measured at the start (10-60 ms) of the commands, and loss of inactivation was calculated as described in the text. For statistical analysis of the effect of activators alone, the changes in current amplitudes and inactivation of oocytes treated with the activators were compared with the drug vehicle. For statistical analysis of the effect of activators in the presence of inhibitory peptide PKC-(19-31), comparisons are between the effect of the activators alone and those of the same activators in the presence of PKC-(19-31). ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001.

FIGURE 6.

Effect of PKC activator Bryostatin1 on Kv3. 3 mutants. A, representative current traces of Kv3.3 mutants, Δ78, S3A, S4A, S9A, S32A, S33A, S46A, S53A, and S60A, and the effect of Bryostatin1 on peak currents and on inactivation of current after 20 min of drug application. Δ78 and S3A produce noninactivating currents. Some mutations (e.g. S3A) produced currents in which rapid activation was followed by a very rapid (∼20 ms) but only partial decline to a steady state, as has also been described for wild type Kv3.1 currents (31). B, group data for the effects of Bryostatin1 on peak currents of wild type Kv3.3 channels and on Kv3.3 mutants Δ78, S3A, S4A, S9A, S32A, S33A, S46A, S53A, and S60A. For simplicity only currents evoked in response to voltage commands to +70 mV are quantified. C, group data for the effects of Bryostatin1 on the inactivated component of currents at +70 mV for the same mutants as in B. Data were analyzed as described in Fig. 5. For statistical analyzes, the change in peak currents and loss of inactivation of currents for the different mutant Kv3.3 channels were compared with those for the wild type Kv3.3 channels. ^**, p < 0.01; ^***, p < 0.005. Numbers of observations (N) are indicated on the bar graphs.

FIGURE 7.

Effect of PKC activator Bryostatin1 on Kv3. 3 mutants S3D, S3C, S3T, and S9D. A, representative current traces of Kv3.3 mutants S3D, S3C, S3T, and S9D before and 20 min after exposure to Bryostatin1. Like S3A, S3D and S3C also produced noninactivating currents, and application of Bryostatin1 caused a small decrease in the peak current amplitudes at the different voltages tested. The response of mutant S3T Kv3.3 channels was similar to that of wild type Kv3.3 channels. B, group data for the effects of Bryostatin1 on peak and inactivated components of current at +40, +50, +60, and +70 mV for mutations at serine 3 (S3D, S3C, and S3T). C, group data for the effects of Bryostatin1 on peak and loss of inactivation of current for the S9D mutation (n = 8 and 5 for wild type and S9D, respectively). ^***, p < 0.0005. Protocols for measurements of peak and loss of inactivation of current are as described in the text.

FIGURE 8.

Numerical simulations of the effects of control and PKC-activated Kv3. 3 currents on firing of a model fast-firing neuron. A, simulated control (de-phospho) and PKC-activated (phospho) Kv3.3 currents evoked by test depolarizations to +10, +30, +50, and +70 mV. B, response of the model neuron to trains of depolarizing current pulses (1.0 nA, 0.25 ms) applied at frequencies of 200, 300, and 400 Hz. Horizontal dashed red line is aligned at the height of the last action potential evoked in a cell with control (de-phospho) Kv3.3 current (left traces), and demonstrates that action potential height at 300 and 400 Hz is higher with simulated PKC-activated (phospho) Kv3.3 currents (right traces). C, superimposition of the first (black) and last (red) evoked action potentials in response to the same trains as in B. Under PKC-activated (phospho) conditions, the later action potentials are systematically higher than in de-phospho conditions, and repolarization between action potentials (indicated by arrows) is enhanced under conditions of PKC activation.