Abstract
KChIP proteins regulate Shal, Kv4.x, channel expression by binding to a conserved sequence at the N terminus of the subunit. The binding of KChIP facilitates a redistribution of Kv4 protein to the cell surface, producing a large increase in current along with significant changes in channel gating kinetics. Recently we have shown that mutants of Kv4.2 lacking the ability to bind an intersubunit Zn(2+) between their T1 domains fail to form functional channels because they are unable to assemble to tetramers and remain trapped in the endoplasmic reticulum. Here we find that KChIPs are capable of rescuing the function of Zn(2+) site mutants by driving the mutant subunits to assemble to tetramers. Thus, in addition to known trafficking effects, KChIPs play a direct role in subunit assembly by binding to monomeric subunits within the endoplasmic reticulum and promoting tetrameric channel assembly. Zn(2+)-less Kv4.2 channels expressed with KChIP3 demonstrate several distinct kinetic changes in channel gating, including a reduced time to peak and faster entry into the inactivated state as well as extending the time to recover from inactivation by 3-4 fold.
Highlights
Folding events, and reveal the processes by which Kv channel complexes form
KChIP3 Rescues Function of Zn2ϩ Site Mutant—The interaction of KChIP auxiliary subunits with Kv4.2 ␣-subunits is an important step in the formation of functional A-type Kv channels [3, 11]
We were interested in testing if the intersubunit interactions of KChIPs might have a direct role in channel assembly beyond their established role in redistributing channels onto the cell surface
Summary
Voltage-gated potassium; EGFP, enhanced green fluorescent protein; CHO, Chinese hamster ovary; SEC, size exclusion chromatography; FPLC, fast protein liquid chromatography; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; ANOVA, analysis of variance. We found that mutation to any of the Zn2ϩ binding site amino acids caused a block of functional channel expression by disrupting subunit tetramerization. This disruption traps the protein within the endoplasmic reticulum and produces a shift in the migration of solubilized subunits on size exclusion chromatography to monomeric molecular weights [8]. Because functional channels could not be formed without the intact Zn2ϩ site, we sought to determine if it was possible to rescue these mutant channels by co-expression with KChIP auxiliary subunit proteins that are known to enhance Shal channel expression by 10 –50 times [3]. We have tested whether KChIP3 binding promotes assembly of the Zn2ϩ site mutant Kv4.2 subunit proteins by testing for the rescue of channel tetramerization and cell surface function by KChIP3. We characterized the resultant complexes as to their assembly and biophysical properties in comparison to wild-type Kv4.2 co-expressed with KChIP3
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