Adam Raes

A K+ Channel Splice Variant Common in Human Heart Lacks a C-terminal Domain Required for Expression of Rapidly Activating Delayed Rectifier Current

We have cloned HERGUSO, a C-terminal splice variant of the human ether-à-go-go-related gene (HERG), the gene encoding the rapid component of the delayed rectifier (IKr), from human heart, and we find that its mRNA is ∼2-fold more abundant than that for HERG1(the originally described cDNA). After transfection of HERGUSO in Ltk − cells, no current was observed. However, coexpression of HERGUSO with HERG1 modified IKr by decreasing its amplitude, accelerating its activation, and shifting the voltage dependence of activation 8.8 mV negative. As with HERGUSO, HERGΔC (a HERG1 construct lacking the C-terminal 462 amino acids) also produced no current in transfected cells. However, IKr was rescued by ligation of 104 amino acids from the C terminus of HERG1 to the C terminus of HERGΔC, indicating that the C terminus of HERG1 includes a domain (≤104 amino acids) that is critical for faithful recapitulation of IKr. The lack of this C-terminal domain not only explains the finding that HERGUSO does not generate IKr but also indicates a similar mechanism for hitherto-uncharacterized long QT syndrome HERG mutations that disrupt the splice site or the C-terminal. We suggest that the amplitude and gating of cardiac IKrdepends on expression of both HERG1 and HERGUSO.

Kv channel gating requires a compatible S4-S5 linker and bottom part of S6, constrained by non-interacting residues.

Voltage-dependent K+ channels transfer the voltage sensor movement into gate opening or closure through an electromechanical coupling. To test functionally whether an interaction between the S4-S5 linker (L45) and the cytoplasmic end of S6 (S6T) constitutes this coupling, the L45 in hKv1.5 was replaced by corresponding hKv2.1 sequence. This exchange was not tolerated but could be rescued by also swapping S6T. Exchanging both L45 and S6T transferred hKv2.1 kinetics to an hKv1.5 background while preserving the voltage dependence. A one-by-one residue substitution scan of L45 and S6T in hKv1.5 further shows that S6T needs to be α-helical and forms a “crevice” in which residues I422 and T426 of L45 reside. These residues transfer the mechanical energy onto the S6T crevice, whereas other residues in S6T and L45 that are not involved in the interaction maintain the correct structure of the coupling.

The S4-S5 Linker of KCNQ1 Channels Forms a Structural Scaffold with the S6 Segment Controlling Gate Closure

In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac potassium current (IKs). Structurally, they share their topology with other Kv channels and consist out of six transmembrane helices (S1–S6) with the S1–S4 segments forming the voltage-sensing domain (VSD). The opening or closure of the intracellular channel gate, which localizes at the bottom of the S6 segment, is directly controlled by the movement of the VSD via an electromechanical coupling. In other Kv channels, this electromechanical coupling is realized by an interaction between the S4-S5 linker (S4S5L) and the C-terminal end of S6 (S6T). Previously we reported that substitutions for Leu353 in S6T resulted in channels that failed to close completely. Closure could be incomplete because Leu353 itself is the pore-occluding residue of the channel gate or because of a distorted electromechanical coupling. To resolve this and to address the role of S4S5L in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5L. The residues with a “high impact” on channel gating (when mutated) clustered on one side of the S4S5L α-helix. Hence, this side of S4S5L most likely contributes to the electromechanical coupling and finds its residue counterparts in S6T. Accordingly, substitutions for Val254 resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu353 in S6T. Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

Kv2.1 and silent Kv subunits underlie the delayed rectifier K current in cultured small mouse DRG neurons

Silent voltage-gated K(+) (K(v)) subunits interact with K(v)2 subunits and primarily modulate the voltage dependence of inactivation of these heterotetrameric channels. Both K(v)2 and silent K(v) subunits are expressed in the mammalian nervous system, but little is known about their expression and function in sensory neurons. This study reports the presence of K(v)2.1, K(v)2.2, and silent subunit K(v)6.1, K(v)8.1, K(v)9.1, K(v)9.2, and K(v)9.3 mRNA in mouse dorsal root ganglia (DRG). Immunocytochemistry confirmed the protein expression of K(v)2.x and K(v)9.x subunits in cultured small DRG neurons. To investigate if K(v)2 and silent K(v) subunits are underlying the delayed rectifier K(+) current (I(K)) in these neurons, K(v)2-mediated currents were isolated by the extracellular application of rStromatoxin-1 (ScTx) or by the intracellular application of K(v)2 antibodies. Both ScTx- and anti-K(v)2.1-sensitive currents displayed two components in their voltage dependence of inactivation. Together, both components accounted for approximately two-thirds of I(K). A comparison with results obtained in heterologous expression systems suggests that one component reflects homotetrameric K(v)2.1 channels, whereas the other component represents heterotetrameric K(v)2.1/silent K(v) channels. These observations support a physiological role for silent K(v) subunits in small DRG neurons.

Substitution Scan of the S4-S5 Linker Region in KCNQ1 Channel: Structural Scaffold for Critical Protein Interactions

KCNQ1 α-subunits are composed out of six transmembrane segments (S1-S6) that tetramerize into a functional channel. In vivo, KCNQ1 α-subunits associate with the β-subunit KCNE1 to generate the slowly activating cardiac IKs and consequently mutations in KCNQ1 are linked to the congenital LQT1 syndrome. Similar to other Kv channels, the S1-S4 segments form the voltage sensing domain that senses the membrane potential and that controls the opening or closure of the channel gate through an electromechanical coupling. In other channels a direct interaction between the S4-S5 linker and bottom part of S6 has been shown to constitute this electromechanical coupling. We previously identified residues in the C-terminal part of S6 that are critical for KCNQ1 gating. To investigate if these residues interact with the S4-S5 linker, we performed an alanine/tryptophan substitution scan of the S4-S5 linker sequence. Based on their impact on channel gating, we categorized these substitutions as either “high” or “low impact”. The pattern of “high impact” positions was consistent with an α-helical configuration and clustered on one side of the S4-S5 linker. Since substitutions at these positions markedly impaired channel gating, they are good candidates to contact residues in the bottom part of S6. Indeed, replacing valine 254 in the S4-S5 linker by a leucine resulted in channels that were partially constitutively open but channel closure could be rescued by combining V254L with the S6 mutation L353A that by itself displayed a similar phenotype as V254L. The observation that all known LQT1 mutations in the S4-S5 linker map on the “high impact” side further strengthens the proposal that this face of the S4-S5 linker contacts the C-terminal S6 segment and constitutes part of the electromechanical coupling in KCNQ1 channels.