Mitochondria Act Within InsP3R Clusters To Maintain Ca2+ Release In Smooth Muscle

Abstract

Many smooth muscle activities including contraction, transcription, growth and apoptosis are regulated by transient inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). InsP3 binds to receptors (InsP3R) present on the sarcoplasmic reticulum to evoke Ca2+ release. InsP3R exist in clusters and Ca2+ released from one receptor may activate nearby InsP3R within this cluster in a CICR-like process to evoke a ‘puff’. Ca2+ released may also diffuse to adjacent clusters to trigger further Ca2+ release and generate a Ca2+ rise throughout the cell. Mitochondrial Ca2+ uptake limits a negative feedback process operative on InsP3R to maintain Ca2+ release. Inhibition of mitochondrial Ca2+ uptake decreases InsP3-mediated Ca2+ waves by ≥50%. We addressed whether mitochondria act to maintain release by operating within or between InsP3R clusters. Ca2+ puffs were evoked by localized photolysis of InsP3 in single voltage-clamped colonic smooth muscle cells in which [Ca2+]c and ΔΨM were measured simultaneously. EGTA, a slow Ca2+ buffer, was used to functionally uncouple puff sites to prevent the formation of Ca2+ waves. EGTA was used at a concentration ([300 μM]) which does not affect the magnitude or kinetics of Ca2+ puffs. InsP3-evoked Ca2+ puffs had amplitudes of 0.5-5.0 F/F0 and durations of ∼200 ms at half-maximum amplitude. Puffs were abolished by the InsP3R inhibitor 2-APB. The protonophore CCCP and the mitochondrial inhibitor rotenone, each used with oligomycin, depolarized the mitochondrial membrane potential (ΔΨM) and prevented mitochondrial Ca2+ uptake. Depolarizing ΔΨM with CCCP attenuated Ca2+ puffs by ∼65% while rotenone inhibited them by ∼60%. These results indicate mitochondrial Ca2+ uptake occurs quickly enough to influence InsP3R communication at the intra-cluster level. Supported by the Wellcome Trust and British Heart Foundation.