Voltage-gated Naplus channels mediate the spread of electrical excitation in cardiac and neuronal tissues. Local anesthetics (LAs) primarily target Naplus channels, inhibit Naplus current, and thereby provide important clinical utility in suppressing cardiac arrhythmia s and in local anesthesia. Naplus channels are membrane-spanning proteins that undergo voltage -triggered conformational changes (gating). These changes underlie the opening and closing of a selective integral pore responsible for conducting an inward Naplus current flux. Phasic inhibition or use-dependence, a prominent feature of LA action, is the progressive depression of Naplus current during repetitive stimulation. Critical to this effect, are persistent, drug-blocked, channel states that accumulate from stimulus to stimulus. The molecular events leading to these states remain controversial. A theme common to many proposed mechanisms is that during gating, the channel visits a conformational state that preferentially binds LA, culminating in enduring channel block. The interdependence of binding and gating in this notion is striking. The capacity of many previous studies to discern the underlying molecular mechanisms, is limited by their inability to discriminate between binding and gating effects. Recent technical advances provide for improved separation of binding and gating. These include the direct observation of drug molecules binding in the pore of open channels with single-channel measurements, simplified gating through genetic engineering (mutagenesis), and drug presentation to selected states using ultra-rapid solution changes with cell-free membrane patches (less than 0.5 ms). These techniques combined with permanently charged LA analogs provide a unique experimental platform to determine the mechanisms involved in long-lived channel blockade. This proposal will concentrate on the role of an intrapore receptor, the open channel state, channel gates, and inactivated states in longlived blockade induced by LAs. Wild type and mutant Naplus channels with simplified gating will be expressed in Xenopus oocytes and studied using two- electrode voltage-clamp, patch clamp of inside-out cell-free membranes, and ultra-rapid solution changes. Two distinct mammalian Naplus channel isoforms will be investigated: rat skeletal muscle, RskM1; and rat brain IIA, RBSCIIA. Parallel findings in these isoforms will provide insight into general molecular mechanisms. Results specific to RBSCIIA will have clinical import since these cells are targeted in local anesthesia, and as some antioconvulsants block Naplus channels in a manner similar to LAs. This proposal promises a deeper understanding of the molecular pharmacology of a variety of clinical compounds that target and block Naplus channels including local anesthetics, cardiac anti-arrythmics, and anticonvulsants.
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