The description of a novel, de novo gain of function mutation in KCNQ1, responsible for atrial fibrillation and short QT syndrome in utero indicates that some of these cases may have a genetic basis and confirms a previous hypothesis that gain of function mutations in KCNQ1 channels can shorten the duration of ventricular and atrial action potentials.

HERG (human ether-a-go-go-related gene) encodes a delayed rectifier K ؉ channel vital to normal repolarization of cardiac action potentials. Attenuation of repolarizing K ؉ current caused by mutations in HERG or channel block by common medications prolongs ventricular action potentials and increases the risk of arrhythmia and sudden death. The critical role of HERG in maintenance of normal cardiac electrical activity derives from its unusual gating properties. Opposite to other voltage-gated K ؉ channels, the rate of HERG channel inactivation is faster than activation and appears to be intrinsically voltage dependent. To investigate voltage sensor movement associated with slow activation and fast inactivation, we characterized HERG gating currents. When the cut-open oocyte voltage clamp technique was used, membrane depolarization elicited gating current with fast and slow components that differed 100-fold in their kinetics. Unlike previously studied voltage-gated K ؉ channels, the bulk of charge movement in HERG was protracted, consistent with the slow rate of ionic current activation. Despite similar kinetic features, fast inactivation was not derived from the fast gating component. Analysis of an inactivation-deficient mutant HERG channel and a Markov kinetic model suggest that HERG inactivation is coupled to activation.voltage clamp ͉ Xenopus ͉ ion channel ͉ EAG H ERG (human ether-a-go-go-related gene) is one of many voltage-gated K ϩ channels expressed in the heart and brain that act in concert to mediate repolarization of action potentials. Naturally occurring mutations in HERG or channel block by common medications cause long QT syndrome, a disorder of myocellular repolarization that predisposes affected individuals to life-threatening arrhythmias (1). HERG gating is unusual because of its mechanism of inward rectification that reduces outward current at depolarized membrane potentials. Unlike typical inward rectifier K ϩ channels, where outward current is blocked by intracellular polyamines and Mg 2ϩ (2), rectification of HERG is caused by a combination of voltage-dependent gating processes: rapid inactivation and slow activation (3, 4). Like C-type inactivation in Shaker channels, mutations in the outer mouth of the pore influence inactivation in HERG channels. However, unlike Shaker, inactivation of HERG is Ϸ100 times faster than activation, and appears to be intrinsically voltage dependent and not coupled to activation. For example, a pore mutation (S631A) in HERG selectively shifts the voltage dependence of inactivation by ϩ100 mV without influencing activation (5, 6), and a double mutation (G628C͞S631C) eliminates inactivation (3). These findings suggest that the voltage dependency of HERG channel inactivation and activation may be derived from distinct voltage-sensing mechanisms.Protein rearrangements associated with HERG gating have been monitored by using a fluorescent probe covalently attached to the outer segment of the S4 domain (7). Two voltagedependent components of fluorescence were describe...

A key unresolved question regarding the basic function of voltage-gated ion channels is how movement of the voltage sensor is coupled to channel opening. We previously proposed that the S4-S5 linker couples voltage sensor movement to the S6 domain in the human ether-a'-go-go-related gene (hERG) K ؉ channel. The recently solved crystal structure of the voltage-gated Kv1.2 channel reveals that the S4-S5 linker is the structural link between the voltage sensing and pore domains. In this study, we used chimeras constructed from hERG and ether-a'-go-go (EAG) channels to identify interactions between residues in the S4-S5 linker and S6 domain that were critical for stabilizing the channel in a closed state. To verify the spatial proximity of these regions, we introduced cysteines in the S4-S5 linker and at the C-terminal end of the S6 domain and then probed for the effect of oxidation. The D540C-L666C channel current decreased in an oxidizing environment in a state-dependent manner consistent with formation of a disulfide bond that locked the channel in a closed state. Disulfide bond formation also restricted movement of the voltage sensor, as measured by gating currents. Taken together, these data confirm that the S4-S5 linker directly couples voltage sensor movement to the activation gate. Moreover, rather than functioning simply as a mechanical lever, these findings imply that specific interactions between the S4-S5 linker and the activation gate stabilize the closed channel conformation.A fundamental property of all voltage-gated ion channels is the ability to open or close in response to changes in membrane potential. In voltage-gated K ϩ channels, the transmembrane domains are partitioned into distinct functional modules: a voltage-sensing module (S1-S4) and an ion-conducting module (S5-S6). Changes in the transmembrane electrical field exert a force on the highly charged S4 domain to initiate a process that culminates in the opening of the activation gate (1). The activation gate is formed by crisscrossing of the C-terminal portions of the S6 ␣-helices to form a narrow aperture near the cytoplasmic interface (2). Channel opening is proposed to involve splaying of the S6 helices at a conserved glycine, thereby widening the aperture to allow passage of ions (3, 4). Although substantial progress has been made in defining the structural basis of the voltage sensor and activation gate, the mechanism of coupling voltage sensing to channel opening, "electromechanical coupling" (5), remains poorly defined. Electromechanical coupling may involve global rearrangements between large domains within the channel complex, for example, outward movement of S4 coupled to rearrangements in S5 that are transmitted to S6. Alternatively, the coupling mechanism might involve discreet interactions between specific residues in more localized regions. The intracellular S4-S5 linker is ideally suited to function as an electromechanical coupler given that it is physically tethered to the S4 and, as such, could function to transduce voltag...

Neuronal restricted precursors (NRPs) () can generate multiple neurotransmitter phenotypes during maturation in culture. Undifferentiated E-NCAM+ (embryonic neural cell adhesion molecule) immunoreactive NRPs are mitotically active and electrically immature, and they express only a subset of neuronal markers. Fully mature cells are postmitotic, process-bearing cells that are neurofilament-M and synaptophysin immunoreactive, and they synthesize and respond to different subsets of neurotransmitter molecules. Mature neurons that synthesize and respond to glycine, glutamate, GABA, dopamine, and acetylcholine can be identified by immunocytochemistry, RT-PCR, and calcium imaging in mass cultures. Individual NRPs also generate heterogeneous progeny as assessed by neurotransmitter response and synthesis, demonstrating the multipotent nature of the precursor cells. Differentiation can be modulated by sonic hedgehog (Shh) and bone morphogenetic protein (BMP)-2/4 molecules. Shh acts as a mitogen and inhibits differentiation (including cholinergic differentiation). BMP-2 and BMP-4, in contrast, inhibit cell division and promote differentiation (including cholinergic differentiation). Thus, a single neuronal precursor cell can differentiate into multiple classes of neurons, and this differentiation can be modulated by environmental signals.