Ca2+ depletion in the ER causes store-operated Ca2+ entry via the TRPC6 channel in mouse brown adipocytes

Abstract

Abstract Mitochondrial uncoupling by β3-adrenergic activation or an uncoupler (FCCP) causes Ca2+ release from the mitochondria and subsequent Ca2+ release from the endoplasmic reticulum (ER), evoking store-operated Ca2+ entry (SOCE) due to Ca2+ depletion from the ER in rodent brown adipocytes. In this study, we investigated how Ca2+ depletion from the ER elicits SOCE in mouse brown adipocytes using fluorometry of intracellular Ca2+ concentration ([Ca2+]i). The application of cyclopiazonic acid (CPA), a reversible sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) pump blocker in the ER, caused an increase in [Ca2+]i. Moreover, CPA-induced SOCE was suppressed by the application of a Ca2+-free Krebs solution and the transient receptor potential canonical 6 (TRPC6) blockers, which were 2-aminoethoxydiphenyl borate (2-APB), ML-9, and GsMTx-4. Application of TRPC6 channel analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) and flufenamic acid elicited Ca2+ entry. Moreover, our RT-PCR analyses detected mRNAs for TRPC6, STIM1, and Orai1 in brown adipose tissues. In addition, western blot analyses showed the expression of the TRPC6 protein. Thus, TRPC6 is one of the Ca2+ pathways involved in SOCE, and Ca2+ entry is directly linked to mitochondrial uncoupling, which is involved in the late phase of β3-adrenergic or FCCP-induced [Ca2+]i increases. These modes of Ca2+ entry provide the basis for heat production via activation of Ca2+-dependent dehydrogenase and the expression of uncoupling protein 1 (UCP1) proteins. Enhancing thermogenic metabolism in brown adipocytes may serve as broad therapeutic utility to reduce obesity and metabolic syndrome.