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

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 first generated and investigated 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. Results demonstrated that 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. The present 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.