The effects of mitochondrial inhibitors on Ca2+ signalling and electrical conductances required for pacemaking in interstitial cells of Cajal in the mouse small intestine

Abstract

Interstitial cells of Cajal (ICC-MY) are pacemakers that generate and propagate electrical slow waves in gastrointestinal (GI) muscles. Slow waves appear to be generated by the release of Ca2+ from intracellular stores and activation of Ca2+-activated Cl− channels (Ano1). Conduction of slow waves to smooth muscle cells coordinates rhythmic contractions. Mitochondrial Ca2+ handling is currently thought to be critical for ICC pacemaking. Protonophores, inhibitors of the electron transport chain (FCCP, CCCP or antimycin) or mitochondrial Na+/Ca2+ exchange blockers inhibited slow waves in several GI muscles. Here we utilized Ca2+ imaging of ICC in small intestinal muscles in situ to determine the effects of mitochondrial drugs on Ca2+ transients in ICC. Muscles were obtained from mice expressing a genetically encoded Ca2+ indicator (GCaMP3) in ICC. FCCP, CCCP, antimycin, a uniporter blocker, Ru360, and a mitochondrial Na+/Ca2+ exchange inhibitor, CGP-37157 inhibited Ca2+ transients in ICC-MY. Effects were not due to depletion of ATP, as oligomycin did not affect Ca2+ transients. Patch-clamp experiments were performed to test the effects of the mitochondrial drugs on key pacemaker conductances, Ano1 and T-type Ca2+ (CaV3.2), in HEK293 cells. Antimycin blocked Ano1 and reduced CaV3.2 currents. CCCP blocked CaV3.2 current but did not affect Ano1 current. Ano1 and Cav3.2 currents were inhibited by CGP-37157. Inhibitory effects of mitochondrial drugs on slow waves and Ca2+ signalling in ICC can be explained by direct antagonism of key pacemaker conductances in ICC that generate and propagate slow waves. A direct obligatory role for mitochondria in pacemaker activity is therefore questionable.