Dominant-negative control of cAMP-dependent IKs upregulation in human long-QT syndrome type 1.

RATIONALE: The mutation A341V in the S6 transmembrane segment of KCNQ1, the α-subunit of the slowly activating delayed-rectifier K(+) (I(Ks)) channel, predisposes to a severe long-QT1 syndrome with sympathetic-triggered ventricular tachyarrhythmias and sudden cardiac death. OBJECTIVE: Several genetic risk modifiers have been identified in A341V patients, but the molecular mechanisms underlying the pronounced repolarization phenotype, particularly during ß-adrenergic receptor stimulation, remain unclear. We aimed to elucidate these mechanisms and provide new insights into control of cAMP-dependent modulation of I(Ks). METHODS AND RESULTS: We characterized the effects of A341V on the I(Ks) macromolecular channel complex in transfected Chinese hamster ovary cells and found a dominant-negative suppression of cAMP-dependent Yotiao-mediated I(Ks) upregulation on top of a dominant-negative reduction in basal current. Phosphomimetic substitution of the N-terminal position S27 with aspartic acid rescued this loss of upregulation. Western blot analysis showed reduced phosphorylation of KCNQ1 at S27, even for heterozygous A341V, suggesting that phosphorylation defects in some (mutant) KCNQ1 subunits can completely suppress I(Ks) upregulation. Functional analyses of heterozygous KCNQ1 WT:G589D and heterozygous KCNQ1 WT:S27A, a phosphorylation-inert substitution, also showed such suppression. Immunoprecipitation of Yotiao with KCNQ1-A341V (in the presence of KCNE1) was not different from wild-type. CONCLUSIONS: Our results indicate the involvement of the KCNQ1-S6 region at/or around A341 in cAMP-dependent stimulation of I(Ks), a process that is under strong dominant-negative control, suggesting that tetrameric KCNQ1 phosphorylation is required. Specific long-QT1 mutations, including heterozygous A341V, disable this regulation.

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 ß-subunit KCNE1 to generate the slowly activating cardiac potassium current (I(Ks)). 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 (S4S5(L)) and the C-terminal end of S6 (S6(T)). Previously we reported that substitutions for Leu(353) in S6(T) resulted in channels that failed to close completely. Closure could be incomplete because Leu(353) 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 S4S5(L) in KCNQ1 channel gating, we performed an alanine/tryptophan substitution scan of S4S5(L). The residues with a "high impact" on channel gating (when mutated) clustered on one side of the S4S5(L) α-helix. Hence, this side of S4S5(L) most likely contributes to the electromechanical coupling and finds its residue counterparts in S6(T). Accordingly, substitutions for Val(254) resulted in channels that were partially constitutively open and the ability to close completely was rescued by combination with substitutions for Leu(353) in S6(T). Double mutant cycle analysis supported this cross-talk indicating that both residues come in close contact and stabilize the closed state of the channel.

The rate-dependent biophysical properties of the LQT1 H258R mutant are counteracted by a dominant negative effect on channel trafficking.

The long QT syndrome (LQTS) is a cardiac disorder caused by a prolonged ventricular repolarization. The co-assembly of the pore-forming human KCNQ1 alpha-subunits with the modulating hKCNE1 beta-subunits generates I(Ks)in vivo, explaining why mutations in the hKCNQ1 gene underlie the LQT1 form of congenital LQT. Here we describe the functional defects of the LQT1 mutation H258R located in the S4-S5 linker, a segment important for channel gating. Mutant subunits with this arginine substitution generated no or barely detectable currents in a homotetrameric condition, but did generate I(Ks)-like currents in association with hKCNE1. Compared to the WT hKCNQ1/hKCNE1 complex, the H258R/hKCNE1 complex displayed accelerated activation kinetics, slowed channel closure and a hyperpolarizing shift of the voltage-dependence of activation, thus predicting an increased K(+) current. However, current density analysis combined with subcellular localization indicated that the H258R subunit exerted a dominant negative effect on channel trafficking to the plasma membrane. The co-expression hKCNQ1/H258R/hKCNE1, mimicking the heterozygous state of a patient, displayed similar properties. During repetitive stimulation the mutant yielded more current compared to WT at 1 Hz but this effect was counteracted by the trafficking defect at faster frequencies. These rate-dependent effects may be relevant given the larger contribution of I(Ks) to the "repolarization reserve" at higher action potential rates. The combination of complex kinetics that counteract the trafficking problem represents a particular mechanism underlying LQT1.

Role of the S6 C-terminus in KCNQ1 channel gating.

Co-assembly of KCNQ1 alpha-subunits with KCNE1 beta-subunits results in the channel complex underlying the cardiac IKs current in vivo. Like other voltage-gated K+ channels, KCNQ1 has a tetrameric configuration. The S6 segment of each subunit lines the ion channel pore with the lower part forming the activation gate. To determine residues involved in protein-protein interactions in the C-terminal part of S6 (S6T), alanine and tryptophan perturbation scans were performed from residue 348-362 in the KCNQ1 channel. Several residues were identified to be relevant in channel gating, as substitutions affected the activation and/or deactivation process. Some mutations (F351A and V355W) drastically altered the gating characteristics of the resultant KCNQ1 channel, to the point of mimicking the IKs current. Furthermore, mutagenesis of residue L353 to an alanine or a charged residue impaired normal channel closure upon hyperpolarization, generating a constitutively open phenotype. This indicates that the L353 residue is essential for stabilizing the closed conformation of the channel gate. These findings together with the identification of several LQT1 mutations in the S6 C-terminus of KCNQ1 underscore the relevance of this region in KCNQ1 and IKs channel gating.

Functional effects of a KCNQ1 mutation associated with the long QT syndrome.

OBJECTIVE: Long QT syndrome (LQTS) is an inherited disorder of ventricular repolarization caused by mutations in cardiac ion channel genes, including KCNQ1. In this study the electrophysiological properties of a LQTS-associated mutation in KCNQ1 (Q357R) were characterized. This mutation is located near the C-terminus of S6, a region that is important for the gate structure. METHODS AND RESULTS: Co-assembly of KCNE1 with the mutant Q357R elicited a current displaying slower activation compared to the wild-type KCNQ1/KCNE1 channels. The voltage dependence of activation of Q357R was shifted to more positive potentials. Moreover, a strong reduction in current density was observed that was partially attributed to the altered voltage dependence and kinetics of activation. The reduced current amplitude was also caused by intracellular retention of Q357R/KCNE1 channels as was shown by confocal microscopy. It indicated that the Q357R mutation disturbed protein expression by a trafficking or assembly problem of the Q357R/KCNE1 complex. To mimic the patient status KCNQ1, Q357R and KCNE1 were co-expressed, which revealed a dominant negative effect on current density and activation kinetics. CONCLUSION: The effects of the Q357R mutation on the activation of the channel together with a reduced expression at the membrane would lead to a reduction in I(Ks) and thus in "repolarization reserve" under physiological circumstances. As such it explains the long QT syndrome observed in these patients.