| Voltage-gated sodium channels play a crucial role in the upstroke of action potentials in electrically excitable cells. Since action potentials are at the core of electrical signaling in a multitude of processes in neuronal, cardiac, and skeletal muscle cells, it is not difficult to imagine that mutations targeting the sodium channel family result in a host of inherited diseases of hypo- and hyperexcitability. The fundamental role of the sodium channel raises provocative questions about how its structure relates to and supports its function. Since the discovery of the sodium channel protein over two decades ago, much has been learned about its molecular and electrophysiological properties. Yet, several basic questions are still unanswered. Sometimes, long after a biophysical property is well documented, the responsible structure remains elusive. Fast inactivation during prolonged depolarization, for example, is a hallmark property of voltage-gated sodium channels. Fast inactivation is thought to involve closure of an intracellular inactivation gate formed by a cytoplasmic loop connecting domains 3 and 4. What is not known is the structural identity of the receptor that engages the closure of this putative inactivation gate. On the other hand, sometimes details at the structural level have been well studied, yet the structural conservation apparently only serves to intrigue scientists with its raison d'être. For example, the S3 transmembrane segment, found throughout the superfamily of voltage-gated ion channels, contains several highly conserved structural features, including a negatively charged residue and a kink. Yet, its contributions to ion channel function are poorly understood. Our ambition is to answer these basic questions. Using site-directed mutagenesis, cysteine scanning with sulfhydryl reagents, and photocrosslinking, we examined macroscopic sodium currents from rat or human adult skeletal muscle sodium channels expressed heterologously in a mammalian cell line. We present here evidence suggesting that four residues in the cytoplasmic S4-S5 loop of domain 4 form the receptor site for the inactivation gate. We further describe the voltage-dependent movement and hydrophilic crevice around the segment S3 of domain 4. |
