The Role of S4 Charges in Voltage-dependent and Voltage-independent KCNQ1 Potassium Channel Complexes

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 α subunits to generate the voltage-dependent IKs channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium “leak” currents in gastrointestinal epithelia. Other Kv α subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2

The role of S4 charges in voltage-dependent and voltage-independent KCNQ1 potassium channel complexes.

Voltage-gated potassium (Kv) channels extend their functional repertoire by coassembling with MinK-related peptides (MiRPs). MinK slows the activation of channels formed with KCNQ1 alpha subunits to generate the voltage-dependent I(Ks) channel in human heart; MiRP1 and MiRP2 remove the voltage dependence of KCNQ1 to generate potassium "leak" currents in gastrointestinal epithelia. Other Kv alpha subunits interact with MiRP1 and MiRP2 but without loss of voltage dependence; the mechanism for this disparity is unknown. Here, sequence alignments revealed that the voltage-sensing S4 domain of KCNQ1 bears lower net charge (+3) than that of any other eukaryotic voltage-gated ion channel. We therefore examined the role of KCNQ1 S4 charges in channel activation using alanine-scanning mutagenesis and two-electrode voltage clamp. Alanine replacement of R231, at the N-terminal side of S4, produced constitutive activation in homomeric KCNQ1 channels, a phenomenon not observed with previous single amino acid substitutions in S4 of other channels. Homomeric KCNQ4 channels were also made constitutively active by mutagenesis to mimic the S4 charge balance of R231A-KCNQ1. Loss of single S4 charges at positions R231 or R237 produced constitutively active MinK-KCNQ1 channels and increased the constitutively active component of MiRP2-KCNQ1 currents. Charge addition to the CO2H-terminal half of S4 eliminated constitutive activation in MiRP2-KCNQ1 channels, whereas removal of homologous charges from KCNQ4 S4 produced constitutively active MiRP2-KCNQ4 channels. The results demonstrate that the unique S4 charge paucity of KCNQ1 facilitates its unique conversion to a leak channel by ancillary subunits such as MiRP2

Constitutively Activating GNAS Somatic Mutation in Right Ventricular Outflow Tract Tachycardia.

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Voltage-Dependent C-Type Inactivation in a Constitutively Open K+ Channel

Most voltage-gated potassium (Kv) channels undergo C-type inactivation during sustained depolarization. The voltage dependence and other mechanistic aspects of this process are debated, and difficult to elucidate because of concomitant voltage-dependent activation. Here, we demonstrate that MinK-KCNQ1 (IKs) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-IKs inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-IKs inactivation and recovery from inactivation were voltage-dependent with physiologic intracellular ion concentrations, and in the absence or presence of external K+, with an estimated gating charge, zi, of ∼1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-IKs) exhibited constitutive C-type inactivation. The results suggest that F340W-IKs channels exhibit voltage-dependent C-type inactivation involving S4, without the necessity for voltage-dependent opening, allosteric coupling to voltage-dependent S6 transitions occurring during channel opening, or voltage-dependent changes in ion occupancy. The data also identify F340 as a critical hub for KCNQ1 gating processes and their modulation by MinK, and present a unique system for further mechanistic studies of the role of coupling of C-type inactivation to S4 movement, without contamination from voltage-dependent activation

MinK-dependent internalization of the IKs potassium channel

KCNQ1-MinK potassium channel complexes (4alpha:2beta stoichiometry) generate IKs, the slowly activating human cardiac ventricular repolarization current. The MinK ancillary subunit slows KCNQ1 activation, eliminates its inactivation, and increases its unitary conductance. However, KCNQ1 transcripts outnumber MinK transcripts five to one in human ventricles, suggesting KCNQ1 also forms other heteromeric or even homomeric channels there. Mechanisms governing which channel types prevail have not previously been reported, despite their significance: normal cardiac rhythm requires tight control of IKs density and kinetics, and inherited mutations in KCNQ1 and MinK can cause ventricular fibrillation and sudden death. Here, we describe a novel mechanism for this control