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

[Figure: see text].

Regulation of the Kv2.1 potassium channel by MinK and MiRP1.

Kv2.1 is a voltage-gated potassium (Kv) channel alpha-subunit expressed in mammalian heart and brain. MinK-related peptides (MiRPs), encoded by KCNE genes, are single-transmembrane domain ancillary subunits that form complexes with Kv channel alpha-subunits to modify their function. Mutations in human MinK (KCNE1) and MiRP1 (KCNE2) are associated with inherited and acquired forms of long QT syndrome (LQTS). Here, coimmunoprecipitations from rat heart tissue suggested that both MinK and MiRP1 form native cardiac complexes with Kv2.1. In whole-cell voltage-clamp studies of subunits expressed in CHO cells, rat MinK and MiRP1 reduced Kv2.1 current density three- and twofold, respectively; slowed Kv2.1 activation (at +60 mV) two- and threefold, respectively; and slowed Kv2.1 deactivation less than twofold. Human MinK slowed Kv2.1 activation 25%, while human MiRP1 slowed Kv2.1 activation and deactivation twofold. Inherited mutations in human MinK and MiRP1, previously associated with LQTS, were also evaluated. D76N-MinK and S74L-MinK reduced Kv2.1 current density (threefold and 40%, respectively) and slowed deactivation (60% and 80%, respectively). Compared to wild-type human MiRP1-Kv2.1 complexes, channels formed with M54T- or I57T-MiRP1 showed greatly slowed activation (tenfold and fivefold, respectively). The data broaden the potential roles of MinK and MiRP1 in cardiac physiology and support the possibility that inherited mutations in either subunit could contribute to cardiac arrhythmia by multiple mechanisms.

A KCNE2 mutation in a patient with cardiac arrhythmia induced by auditory stimuli and serum electrolyte imbalance.

AIMS: Auditory stimulus-induced long QT syndrome (LQTS) is almost exclusively linked to mutations in the hERG potassium channel, which generates the I Kr ventricular repolarization current. Here, a young woman with prior episodes of auditory stimulus-induced syncope presented with LQTS and ventricular fibrillation (VF) with hypomagnesaemia and hypocalcaemia after completing a marathon, followed by subsequent VF with hypokalaemia. The patient was found to harbour a KCNE2 gene mutation encoding a T10M amino acid substitution in MiRP1, an ancillary subunit that co-assembles with and functionally modulates hERG. Other family members with the mutation were asymptomatic, and the proband had no mutations in hERG or other LQTS-linked cardiac ion channel genes. The T10M mutation was absent from 578 unrelated, ethnically matched control chromosomes analysed here and was previously described only once-in an LQTS patient-but not functionally characterized. METHODS AND RESULTS: T10M-MiRP1-hERG currents were assessed using whole-cell voltage clamp of transfected Chinese Hamster ovary cells. T10M-MiRP1-hERG channels showed

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 (I(Ks)) channels with an S6-domain mutation, F340W in KCNQ1, exhibit constitutive activation but voltage-dependent C-type inactivation. F340W-I(Ks) inactivation was sensitive to extracellular cation concentration and species, and it altered ion selectivity, suggestive of pore constriction. The rate and extent of F340W-I(Ks) 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, z(i), of approximately 1. Finally, double-mutant channels with a single S4 charge neutralization (R231A,F340W-I(Ks)) exhibited constitutive C-type inactivation. The results suggest that F340W-I(Ks) 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.

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.

The impact of ancillary subunits on small-molecule interactions with voltage-gated potassium channels.

Voltage-gated potassium channels (Kv channels) are the major determinants of cellular repolarization in excitable cells--they open in response to depolarization and facilitate selective efflux of potassium ions across the plasma membrane. Because of the importance of exquisitely timed cellular repolarization in controlling action potential morphology and duration, Kv channels are attractive therapeutic targets, particularly for drugs aimed at controlling aberrant electrical excitability such as is observed in cardiac arrhythmia and epilepsy. While the pore-forming alpha subunits of Kv channels are sufficient to form functional channels, a host of cytoplasmic and transmembrane ancillary subunits modulate their trafficking, function and regulation in vivo. Here, we consider the impact of ancillary subunits on Kv channel pharmacology, and discuss how increased understanding of the roles of ancillary subunits in native Kv channel complexes will lead to development of safer, more specific and more efficacious therapeutic small molecules.

MinK-related peptide 2 modulates Kv2.1 and Kv3.1 potassium channels in mammalian brain.

Delayed rectifier potassium current diversity and regulation are essential for signal processing and integration in neuronal circuits. Here, we investigated a neuronal role for MinK-related peptides (MiRPs), membrane-spanning modulatory subunits that generate phenotypic diversity in cardiac potassium channels. Native coimmunoprecipitation from rat brain membranes identified two novel potassium channel complexes, MiRP2-Kv2.1 and MiRP2-Kv3.1b. MiRP2 reduces the current density of both channels, slows Kv3.1b activation, and slows both activation and deactivation of Kv2.1. Altering native MiRP2 expression levels by RNAi gene silencing or cDNA transfection toggles the magnitude and kinetics of endogenous delayed rectifier currents in PC12 cells and hippocampal neurons. Computer simulations predict that the slower gating of Kv3.1b in complexes with MiRP2 will broaden action potentials and lower sustainable firing frequency. Thus, MiRP2, unlike other known neuronal beta subunits, provides a mechanism for influence over multiple delayed rectifier potassium currents in mammalian CNS via modulation of alpha subunits from structurally and kinetically distinct subfamilies.

An RNA polymerase mutant deficient in DNA melting facilitates study of activation mechanism: application to an artificial activator of transcription.

Transcription initiation is a major target for the regulation of gene expression in all organisms. Transcription activators can stimulate different steps in the initiation process including the initial binding of RNA polymerase (RNAP) to the promoter and a subsequent promoter-melting step. Typically, kinetic assays are required to determine whether an activator exerts its effect on the initial binding of RNAP or on the promoter-melting step. Here we take advantage of a mutant Escherichia coli RNAP that is deficient in promoter melting to assess the ability of an activator to stabilize the initial binding of RNAP to the promoter. For the well-characterized activator CRP, we show that this RNAP mutant can be used to distinguish between effects on initial binding and promoter melting; these results provide an independent confirmation of the results of kinetic analysis. We then employ the melting-deficient RNAP mutant to demonstrate an effect of an artificial activator of transcription on the initial binding of RNAP. Our findings demonstrate that a melting-deficient RNAP mutant can be used to trap a normally unstable intermediate in transcription initiation, thus providing a novel tool for probing activation mechanism.

MinK-dependent internalization of the IKs potassium channel.

AIMS: 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. METHODS AND RESULTS: Whole-cell patch-clamping, confocal immunofluorescence microscopy, antibody feeding, biotin feeding, fluorescent transferrin feeding, and protein biochemistry techniques were applied to COS-7 cells heterologously expressing KCNQ1 with wild-type or mutant MinK and dynamin 2 and to native IKs channels in guinea-pig myocytes. KCNQ1-MinK complexes, but not homomeric KCNQ1 channels, were found to undergo clathrin- and dynamin 2-dependent internalization (DDI). Three sites on the MinK intracellular C-terminus were, in concert, necessary and sufficient for DDI. Gating kinetics and sensitivity to XE991 indicated that DDI decreased cell-surface KCNQ1-MinK channels relative to homomeric KCNQ1, decreasing whole-cell current but increasing net activation rate; inhibiting DDI did the reverse. CONCLUSION: The data redefine MinK as an endocytic chaperone for KCNQ1 and present a dynamic mechanism for controlling net surface Kv channel subunit composition-and thus current density and gating kinetics-that may also apply to other alpha-beta type Kv channel complexes.

Interaction of KCNE subunits with the KCNQ1 K+ channel pore.

KCNQ1 alpha subunits form functionally distinct potassium channels by coassembling with KCNE ancillary subunits MinK and MiRP2. MinK-KCNQ1 channels generate the slowly activating, voltage-dependent cardiac IKs current. MiRP2-KCNQ1 channels form a constitutively active current in the colon. The structural basis for these contrasting channel properties, and the mechanisms of alpha subunit modulation by KCNE subunits, are not fully understood. Here, scanning mutagenesis located a tryptophan-tolerant region at positions 338-340 within the KCNQ1 pore-lining S6 domain, suggesting an exposed region possibly amenable to interaction with transmembrane ancillary subunits. This hypothesis was tested using concomitant mutagenesis in KCNQ1 and in the membrane-localized 'activation triplet' regions of MinK and MiRP2 to identify pairs of residues that interact to control KCNQ1 activation. Three pairs of mutations exerted dramatic effects, ablating channel function or either removing or restoring control of KCNQ1 activation. The results place KCNE subunits close to the KCNQ1 pore, indicating interaction of MiRP2-72 with KCNQ1-338; and MinK-59,58 with KCNQ1-339, 340. These data are consistent either with perturbation of the S6 domain by MinK or MiRP2, dissimilar positioning of MinK and MiRP2 within the channel complex, or both. Further, the results suggest specifically that two of the interactions, MiRP2-72/KCNQ1-338 and MinK-58/KCNQ1-340, are required for the contrasting gating effects of MinK and MiRP2.

RNA interference reveals that endogenous Xenopus MinK-related peptides govern mammalian K+ channel function in oocyte expression studies.

The physiological properties of most ion channels are defined experimentally by functional expression of their pore-forming alpha subunits in Xenopus laevis oocytes. Here, we cloned a family of Xenopus KCNE genes that encode MinK-related peptide K(+) channel beta subunits (xMiRPs) and demonstrated their constitutive expression in oocytes. Electrophysiological analysis of xMiRP2 revealed that when overexpressed this gene modulates human cardiac K(+) channel alpha subunits HERG (human ether-a-go-go-related gene) and KCNQ1 by suppressing HERG currents and removing the voltage dependence of KCNQ1 activation. The ability of endogenous levels of xMiRP2 to contribute to the biophysical attributes of overexpressed mammalian K(+) channels in oocyte studies was assessed next. Injection of an xMiRP2 sequence-specific short interfering RNA (siRNA) oligo reduced endogenous xMiRP2 expression 5-fold, whereas a control siRNA oligo had no effect, indicating the effectiveness of the RNA interference technique in Xenopus oocytes. The functional effects of endogenous xMiRP2 silencing were tested using electrophysiological analysis of heterologously expressed HERG channels. The RNA interference-mediated reduction of endogenous xMiRP2 expression increased macroscopic HERG current as much as 10-fold depending on HERG cRNA concentration. The functional effects of human MiRP1 (hMiRP1)/HERG interaction were also affected by endogenous xMiRP2. At high HERG channel density, at which the effects of endogenous xMiRP2 are minimal, hMiRP1 reduced HERG current. At low HERG current density, hMiRP1 paradoxically up-regulated HERG current, a result consistent with hMiRP1 rescuing HERG from suppression by endogenous xMiRP2. Thus, endogenous Xenopus MiRP subunits contribute to the base-line properties of K(+) channels like HERG in oocyte expression studies, which could explain expression level- and expression system-dependent variation in K(+) channel function.