Protein Kinase C Modulates Inactivation of Kv3.3 Channels

Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut 06520, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2008; 283(32):22283-94. DOI: 10.1074/jbc.M801663200
Source: PubMed


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.

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Available from: Stuart A Forman, Dec 24, 2015
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    • "However, incorporation of a single subunit containing the N-terminal extension is sufficient to confer inactivation on the channel, although it occurs at a slower rate than in channels composed of four inactivating subunits (MacKinnon et al. 1993). We generated a non-inactivating form of Kv3.3, Kv3.3-IR (inactivation removed), by deleting the N-terminal extension and then co-expressed it with R420H in the normal, inactivating background at ratios of 1:1 and 1:4 (IR:R420H) (Fig. 1C) (Desai et al. 2008). For comparison, wild type Kv3.3 and Kv3.3-IR channels were expressed separately. "
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    ABSTRACT: Key points Abstract Mutations in Kv3.3 cause spinocerebellar ataxia type 13 (SCA13). Depending on the causative mutation, SCA13 is either a neurodevelopmental disorder that is evident in infancy or a progressive neurodegenerative disease that emerges during adulthood. Previous studies did not clarify the relationship between these distinct clinical phenotypes and the effects of SCA13 mutations on Kv3.3 function. The F448L mutation alters channel gating and causes early-onset SCA13. R420H and R423H suppress Kv3 current amplitude by a dominant negative mechanism. However, R420H results in the adult form of the disease whereas R423H produces the early-onset, neurodevelopmental form with significant clinical overlap with F448L. Since individuals with SCA13 have one wild type and one mutant allele of the Kv3.3 gene, we analysed the properties of tetrameric channels formed by mixtures of wild type and mutant subunits. We report that one R420H subunit and at least one R423H subunit can co-assemble with the wild type protein to form active channels. The functional properties of channels containing R420H and wild type subunits strongly resemble those of wild type alone. In contrast, channels containing R423H and wild type subunits show significantly altered gating, including a hyperpolarized shift in the voltage dependence of activation, slower activation, and modestly slower deactivation. Notably, these effects resemble the modified gating seen in channels containing a mixture of F448L and wild type subunits, although the F448L subunit slows deactivation more dramatically than the R423H subunit. Our results suggest that the clinical severity of R423H reflects its dual dominant negative and dominant gain of function effects. However, as shown by R420H, reducing current amplitude without altering gating does not result in infant onset disease. Therefore, our data strongly suggest that changes in Kv3.3 gating contribute significantly to an early age of onset in SCA13.
    Full-text · Article · Jan 2012 · The Journal of Physiology
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    • "We have observed variability in the rate of Kv3.3 inactivation in oocytes, perhaps due to differences in the basal level of protein kinase C (PKC) activity. Phosphorylation of Kv3.3 by PKC slows inactivation (Desai, et al., 2008). "
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    ABSTRACT: We recently identified KCNC3, encoding the Kv3.3 voltage-gated potassium channel, as the gene mutated in SCA13. One g.10684G>A (p.Arg420His) mutation caused late-onset ataxia resulting in a nonfunctional channel subunit with dominant-negative properties. A French early-onset pedigree with mild mental retardation segregated a g.10767T>C (p.Phe448Leu) mutation. This mutation changed the relative stability of the channel's open conformation. Coding exons were amplified and sequenced in 260 autosomal-dominant ataxia index cases of European descent. Functional analyses were performed using expression in Xenopus oocytes. The previously identified p.Arg420His mutation occurred in three families with late-onset ataxia. A novel mutation g.10693G>A (p.Arg423His) was identified in two families with early-onset. In one pedigree, a novel g.10522G>A (p.Arg366His) sequence variant was seen in one index case but did not segregate with affected status in the respective family. In a heterologous expression system, the p.Arg423His mutation exhibited dominant-negative properties. The p.Arg420His mutation, which results in a nonfunctional channel subunit, was recurrent and associated with late-onset progressive ataxia. In two families the p.Arg423His mutation was associated with early-onset slow-progressive ataxia. Despite a phenotype reminiscent of the p.Phe448Leu mutation, segregating in a large early-onset French pedigree, the p.Arg423His mutation resulted in a nonfunctional subunit with a strong dominant-negative effect.
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    ABSTRACT: Kv4.3, with its complex open- and closed-state inactivation (CSI) characteristics, is a primary contributor to early cardiac repolarization. The two alternatively spliced forms, Kv4.3-short (Kv4.3-S) and Kv4.3-long (Kv4.3-L), differ by the presence of a 19-amino acid insert downstream from the sixth transmembrane segment. The isoforms are similar kinetically; however, the longer form has a unique PKC phosphorylation site. To test the possibility that inactivation is differentially regulated by phosphorylation, we expressed the Kv4.3 isoforms in Xenopus oocytes and examined changes in their inactivation properties after stimulation of PKC activity. Whereas there was no difference in open-state inactivation, there were profound differences in CSI. In Kv4.3-S, PMA reduced the magnitude of CSI by 24% after 14.4 s at -50 mV. In contrast, the magnitude of CSI in Kv4.3-L increased by 25% under the same conditions. Mutation of a putatively phosphorylated threonine (T504) to aspartic acid within a PKC consensus recognition sequence unique to Kv4.3-L eliminated the PMA response. The change in CSI was independent of the intervention used to increase PKC activity; identical results were obtained with either PMA or injected purified PKC. Our previously published 11-state model closely simulated our experimental data. Our data demonstrate isoform-specific regulation of CSI by PKC in Kv4.3 and show that the carboxy terminus of Kv4.3 plays an important role in regulation of CSI.
    Preview · Article · Sep 2009 · AJP Cell Physiology
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