Abstract 17600: Membrane Trafficking of Large Conductance, Ca2+-and Voltage-Activated K+ (bk) Channels is Modulated by Rab4GTPase
The large-conductance Ca2+-activated K+ (BK) channels are important ion channels, linking the metabolic state and cellular Ca2+ homeostasis with cellular excitability. The BK channel provides an important physiological mechanism to limit excessive vasoconstriction and mediate vasodilation. Thus, this channel plays a central role in the regulation of blood pressure. However, the molecular mechanism that regulates BK channel cell surface trafficking remains unclear. The Rab GTPase controls a variety of important cellular processes, such as endocytosis, trafficking, endosome fusion and exocytosis. We hypothesize that BK channel trafficking is modulated by Rab4, which plays a critical role in early recycling of proteins back to cell membrane. We found that internalized BK channel α subunits could be recycled to the cell surface, detected by immunofluorescent staining of the cell surface BK channel proteins. In addition, endogenous BK channels were co-immunoprecipitated with Rab4 both in rat aortas and in human coronary smooth muscle cells. Double labeling of BK channels and Rab4 with different immunofluorescent probes showed that the two proteins were partially co-localized in a distinct punctate pattern. Using surface protein biotinylation and immunostaining techniques, we found that co-expression of GDP-locked Rab4S22N dominant-negative mutant in HEK293 cells stably expressing the BK-α channel decreased the steady-state levels of BK channel surface expression, whereas co-expression of the constitutively active GTPase-deficient Rab4Q67L mutant increased the steady-state levels of BK channel surface expression. In addition, whole-cell BK currents were significantly reduced when co-expressed with the dominant-negative forms of Rab4 in HEK cells. These data suggest that Rab4 is associated with BK channel trafficking and alterations in Rab GTPase may contribute to changes in the cell surface targeting of functional BK channel proteins. Therefore, strategies designed to manipulate specific BK trafficking pathways may provide a new approach for the treatment of BK channel related vascular channelopathies.
- © 2012 by American Heart Association, Inc.