Ion channels are required to generate electrical activity that drives contractility in organs such as the gastrointestinal tract and the heart. In previous grant cycles we have shown that human small intestinal smooth muscle cells (SMC) express a voltage-sensitive Na+ channel, Nav1.5, the a subunit of which is encoded by SCN5A and that Nav1.5 is mechanosensitive. Mechano-regulation of Nav1.5 is highly relevant because of the steep voltage-sensitivity, with small changes in channel kinetics markedly affecting physiology. Nav1.5 is selectively expressed. It generates a Na+ current in the intestinal tract of humans, dogs and rats but not in several other species such as guinea pig and mouse. Mutations in Nav1.5 cause disease. The central hypothesis of this proposal is that mechanosensitivity of the Nav1.5 is due to physical changes in the voltage sensor(s), and that physiologically relevant mechanical stimuli markedly alter Nav1.5 function. We also hypothesize that in a subset of patients with irritable bowel syndrome (IBS), specific mutations in SCN5A result in altered electrophysiology and mechanosensitivity of Nav1.5 and that Nav1.5 regulates membrane potential and Ca2+ dynamics of human SMC. We will test the central hypothesis in 3 specific aims. In SA 1 we will determine the basic mechanisms that underlie ion channel mechanosensitivity. In SA 2 we will determine the physiological relevance of Nav1.5 mutations found in IBS. In SA 3 we will determine the physiological role of Nav1.5. The specific aims are supported by preliminary data which show that SCN5A mutations are found in approximately 3% of patients with IBS (over 1.35 million), that IBS SCN5A mutations change the electrophysiology of Nav1.5, that mutants and toxins modulate mechanosensitivity, that mechanosensitivity can also be modulated by FDA approved drugs, that knockdown and pharmacological block of Nav1.5 hyperpolarize human intestinal circular SMC membrane potential and change slow wave frequency, that Nav1.5 is clustered on the cell membrane and that Na+ entry through Nav1.5 sets local intracellular Na+ and regulates Ca2+ through Na+/Ca2+ exchanger (NCX). We will use patch clamp techniques, ultrafast pressure delivery, high resolution patch imaging, immunohistochemistry, Western blots, single cell PCR, quantitative PCR, lentivirus RNA knock down techniques, organotypic and single cell cultures, total internal reflection fluorescence (TIRF) imaging of proteins, Ca2+ and Na+ as well as microelectrode recordings to investigate the central hypothesis. Successful completion of the proposed studies has both basic significance and clinical impact. As a result of the work done in the previous grant cycles and the preliminary data presented in this proposal, we are now poised to significantly advance our understanding, at a sub- molecular level, of the fundamental mechanisms that underlie mechanosensitivity, of the role Nav1.5 plays in normal and abnormal human intestinal physiology and to establish a role of ion channelopathies in a subset of patients with IBS.