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.
Highlights
Kv3 family voltage-dependent potassium channels have fast activation and deactivation kinetics and high activation thresholds, allowing many neurons that express these channels to fire trains of action potentials at high frequencies [1,2,3,4]
We have identified two N-terminal serine residues that are consensus sites for protein kinase C (PKC) phosphorylation and that are required for enhancement of Kv3.3 currents
All other amino acids in the protein were identical to the previously reported mouse Kv3.3b gene [21]. We used this new cDNA to characterize the properties of recombinant Kv3.3 channels and to investigate its potential modulation by protein kinase C
Summary
Cloning of Kv3.3 cDNA from Mouse Brainstem—Total RNA was isolated from whole brain of two 16-day-old SvJ129 mice using the Ultraspec-II RNA kit (Biotecx Laboratories Inc., Houston, TX). The remaining 3Ј-terminal cDNA sequence was cloned using a 3Ј-RACE-PCR strategy and was identical to the previously reported mouse Kv3.3 sequence (GenBankTM accession number Q63959) [21]. For cell-attached single channel recordings, bath solution consisted of (in mM) 175 KCl, 4 MgCl2, and 10 HEPES (pH 7.3 titrated with KOH). Because treatment of Kv3.3-expressing cells with activators of PKC typically result in near total loss of inactivation during depolarizing pulses lasting 1 s, it was not possible to determine accurate time constants for inactivation under these conditions. Averaged data are expressed as means Ϯ S.E. Site-directed Mutagenesis—Point and deletion mutations in Kv3.3 channel cDNA were introduced by full-length PCR amplification of plasmid DNA with sense and antisense mutated primers using Pfu polymerase (Stratagene, La Jolla, CA). The leakage conductance gL was 0.002 S, and C, the cell capacitance, was 0.01 nanofarads
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