Regulation of bradykinin-induced activation of volume-sensitive outwardly rectifying anion channels by Ca 2+ nanodomains in mouse astrocytes

Abstract

Volume-sensitive outwardly rectifying (VSOR) anion channels play a key role in a variety of essential cell functions including cell volume regulation, cell death induction and intercellular communications. We previously demonstrated that, in cultured mouse cortical astrocytes, VSOR channels are activated in response to an inflammatory mediator, bradykinin, even without an increase in cell volume. Here we report that this VSOR channel activation must be mediated firstly by 'nanodomains' of high [Ca 2+] i generated at the sites of both Ca 2+ release from intracellular Ca 2+ stores and Ca 2+ entry at the plasma membrane. Bradykinin elicited a [Ca 2+] i rise, initially caused by Ca 2+ release and then by Ca 2+ entry. Suppression of the [Ca 2+] i rise by removal of extracellular Ca 2+ and by depletion of Ca 2+ stores suppressed the VSOR channel activation in a graded manner. Quantitative RT-PCR and suppression of gene expression with small interfering RNAs indicated that Orai1, TRPC1 and TRPC3 channels are involved in the Ca 2+ entry and especially the entry through TRPC1 channels is strongly involved in the bradykinin-induced activation of VSOR channels. Moreover, Ca 2+-dependent protein kinases Cα and β were found to mediate the activation after the [Ca 2+] i rise through inducing generation of reactive oxygen species. Intracellular application of a slow Ca 2+ chelator, EGTA, at 10 mm or a fast chelator, BAPTA, at 1 mm, however, had little effect on the VSOR channel activation. Application of BAPTA at 10 mm suppressed significantly the activation to one-third. These suggest that the VSOR channel activation induced by bradykinin is regulated by Ca 2+ in the vicinity of individual Ca 2+ release and entry channels, providing a basis for local control of cell volume regulation and intercellular communications. © 2011 The Authors. Journal compilation © 2011 The Physiological Society.