α-Subunit Tyrosine Phosphorylation Is Required for Activation of the Large Conductance Ca2+-Activated Potassium Channel in the Rabbit Sphincter of Oddi

Abstract

Large conductance Ca²⁺-activated potassium (BK_Ca) channels are regulated by intracellular free Ca²⁺ concentrations ([Ca²⁺]i) and channel protein phosphorylation. In hypercholesterolemia (HC), motility impairment of the sphincter of Oddi (SO) is associated with abnormal [Ca²⁺]i accumulation in smooth muscle cells of the rabbit SO (RSOSMCs), which is closely related to BK_Ca channel activity. However, the underlying mechanisms regulating channel activity remain unclear. In this study, an HC rabbit model was generated and used to investigate BK_Ca channel activity of RSOSMCs via SO muscle tone measurement in vitro and manometry in vivo, electrophysiological recording, intracellular calcium measurement, and Western blot analyses. BK_Ca channel activity was decreased, which correlated with [Ca²⁺]i overload and reduced tyrosine phosphorylation of the BK_Ca α-subunit in the HC group. The abnormal [Ca²⁺]i accumulation and decreased BK_Ca channel activity were partially restored by Na₃VO₄ pretreatment but worsened by genistein in RSOSMCs in the HC group. This study suggests that α-subunit tyrosine phosphorylation is required for [Ca²⁺]i to activate BK_Ca channels, and there is a negative feedback between the BK_Ca channel and the L-type voltage-dependent Ca²⁺ channel that regulates [Ca²⁺]i. This study provides direct evidence that tyrosine phosphorylation of BK_Ca α-subunits is required for [Ca²⁺]i to activate BK_Ca channels in RSOSMCs, which may be the underlying physiological and pathologic mechanism regulating the activity of BK_Ca channels in SO cells.