Abstract
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 beta1-subunit. Sequence analysis revealed two potential caveolin-binding motifs along the Slo1 C terminus, one equivalent, 1007YNMLCFGIY1015, and another mirror image, 537YTEYLSSAF545, to the consensus sequence, varphiXXXXvarphiXXvarphi. Deletion of 1007YNMLCFGIY1015 caused approximately 80% loss of Slo1-caveolin-1 association while preserving channel normal folding and overall Slo1 and caveolin-1 intracellular distribution patterns. 537YTEYLSSAF545 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 1007YNMLCFGIY1015 motif obliterated channel surface expression. The results suggest 1007YNMLCFGIY1015 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 downsizing 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.
Highlights
Three caveolin proteins have been identified, caveolin-1, -2, and -3
Co-migration of Slo1 and Caveolin-1 in Sucrose Density Fractions—We investigated whether Slo1 can share with caveolin-1 similar membranous structures that could be identified by their sedimentation properties
Quantitative analysis of protein proximity in freshly isolated myocytes revealed that there may be at least two populations of cells characterized by their degree of proximity between Slo1 and caveolin-1 with ϳ30% of cells showing a high degree of protein proximity index (PPI) (Ն0.9)
Summary
MaxiK, large conductance, voltage- and Ca2ϩactivated Kϩ channel; Slo1, ␣-subunit of MaxiK channel; Cav-1, caveolin-1; SD, scaffolding domain; WB, Western blot; IP, immunoprecipitation; MES, 2-[N-morpholino]ethanesulfonic acid; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Ab, antibody; PPI, protein proximity index; CC, correlation coefficient; FPo, fractional open probability
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