Slo1 caveolin-binding motif, a mechanism of caveolin-1-Slo1 interaction regulating Slo1 surface expression

The large conductance, voltage- and Ca2+- activated potassium (MaxiK, BK) channel and caveolin-1 play important roles in regulating vascular contractility. Here, we hypothesized that the MaxiK alpha-subunit (Slo1) and caveolin-1 may interact with each other. Slo1 and caveolin-1 physiological association in native vascular tissue is strongly supported by (i) detergent-free purification of caveolin-1-rich domains demonstrating a pool of aortic Slo1 co-migrating with caveolin-1 to light density sucrose fractions, (ii) reverse co-immunoprecipitation, and (iii) double immunolabeling of freshly isolated myocytes revealing caveolin-1 and Slo1 proximity at the plasmalemma. In HEK293T cells, Slo1-caveolin-1 association was unaffected by the smooth muscle MaxiK beta 1-subunit. Sequence analysis revealed two potential caveolin-binding motifs along the Slo1 C terminus, one equivalent, (1007)YNMLCFGIY(1015), and another mirror image, (537)YTEYLSSAF(545), to the consensus sequence, phi XXXX phi XX phi. Deletion of 1007YNMLCFGIY1015 caused similar to 80% loss of Slo1-caveolin-1 association while preserving channel normal folding and overall Slo1 and caveolin-1 intracellular distribution patterns. (537)YTEYLSSAF(545) deletion had an insignificant dissociative effect. Interestingly, caveolin-1 coexpression reduced Slo1 surface and functional expression near 70% without affecting channel voltage sensitivity, and deletion of (1007)YNMLCFGIY(1015) motif obliterated channel surface expression. The results suggest (1007)YNMLCFGIY(1015) possible participation in Slo1 plasmalemmal targeting and demonstrate its role as a main mechanism for caveolin-1 association with Slo1 potentially serving a dual role: (i) maintaining channels in intracellular compartments down sizing their surface expression and/or (ii) serving as anchor of plasma membrane resident channels to caveolin-1-rich membranes. Because the caveolin-1 scaffolding domain is juxtamembrane, it is tempting to suggest that Slo1-caveolin-1 interaction facilitates the tethering of the Slo1 C-terminal end to the membrane.