KChIP3 Rescues the Functional Expression of Shal Channel Tetramerization Mutants

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.The formation of voltage-gated potassium (Kv) 1 channels is a multistep process with many different interactions and folding events required to form the completed channel (1). The common functional core of all Kv channels assembles as a tetramer of pore-forming ␣-subunits. This tetramer is the core of the future ion channel signal transduction complex, but additional folding steps as well as interactions with auxiliary proteins occur before the final functional channel complex at the cell surface is formed. Many auxiliary subunit proteins that bind to Kv ␣-subunits have been identified, but precisely when these interactions occur during channel complex formation and what role these interactions play in helping the channels to assemble, traffic, and function are topics of great interest (1-4). Through the use of heterologous expression systems and mutagenesis studies, we can expose many of these important interactions and folding events, and reveal the processes by which Kv channel complexes form. A comparison of channel expression and functional properties with and without specific auxiliary proteins reveals how these different processes contribute to the formation and function of ion channel complexes.An early step in Kv channel formation involves the tetramerization of the ␣-subunit T1 domains at the cytoplasmic N terminus of the protein (5-7). For Kv4.2 channels, a critical component of the T1 domain interaction involves the coordination of an intersubunit Zn 2ϩ ion found on non-Shaker type Kv channel T1 domains (8 -10). Although Zn 2ϩ binding sites are common in proteins, intersubunit Zn 2ϩ binding sites, as found in the T1 domain, are relatively rare. To determine what functions might be regulated by the T1 intersubunit Zn 2ϩ site, we generated a series of mutations to the Zn 2ϩ coordination residues and tested them for cell surface expression (8). We found that mut...

Three-dimensional structure of the KChIP1–Kv4.3 T1 complex reveals a cross-shaped octamer

“…1a). The contacts with two T1 subunits seen in this arrangement are consistent with the report that, in full-length channels, KChIP binding can overcome T1 assembly defects 26 and may endow the KChIP-Kv4 T1 complex with cooperative properties that affect gating.…”

LQT1-associated Mutations Increase KCNQ1 Proteasomal Degradation Independently of Derlin-1

“…In addition, although more contradictory, previous studies also reported increased trafficking of KCNQ1 when co-expressed with KCNE1 (31). Finally, the chaperone role that we propose for KCNE1 is consistent with previous publications showing that ␤-subunits stabilize and enhance the trafficking of other channels (32,33). Together our data assign a new role to the KCNE1 subunit in channel stability and biogenesis.…”

Augmentation of Kv4.2-encoded Currents by Accessory Dipeptidyl Peptidase 6 and 10 Subunits Reflects Selective Cell Surface Kv4.2 Protein Stabilization

“…3F). Also consistent with previous findings (17,26), co-expression with KChIP3 significantly (p Ͻ 0.01) increases Kv4.2 current densities (not illustrated).…”

“…In contrast to DPP6/10, the decay phases of the Kv4.2-encoded currents were dramatically prolonged in cells co-expressing KChIP3 (data not shown) reflecting an increase in the percentage of current that inactivates slowly rather than a change in the inactivation time constant (24,26). Consistent with this suggestion, Kv4.2-encoded current inactivation in cells co-expressing KChIP3 was best characterized by a single exponential with a mean Ϯ S.E.…”

“…The observation that DPP6 expression is unaffected by the loss of Kv4.2 ␣ subunits contrasts strikingly with recent findings demonstrating marked reductions in the expression of the accessory KChIP2, KChIP3, and KChIP4 subunits in Kv4.2 Ϫ/Ϫ and Kv4.3 Ϫ/Ϫ cortices and the virtual elimination of all three KChIP proteins in cortices from animals lacking both Kv4.2 and Kv4.3 (36), suggesting that different mechanisms underlie KChIP-and DPP-mediated increases in Kv4-encoded current densities (15,17,25,26,29,31). Interestingly, consistent with this hypothesis, DPP6-YFP appears to be predominately localized at or near the plasma membrane when expressed (in HEK-293 cells) alone ( Fig.…”