Disruption of the maxi-K-caveolin-1 interaction alters current expression in human myometrial cells.

Reprod Biol Endocrinol

Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.

Published: November 2009

Background: One determinant of the total K+ myometrial smooth muscle cell (MSMC) current is the large conductance, calcium- and voltage-activated potassium channel (maxi-K channel). This channel provides a repolarizing current in response to excitatory stimuli, most notably in response to increases in the levels of intracellular Ca2+, and blocking the channel by pharmacological means induces the depolarization of MSMCs and also enhances contraction strength. In MSMCs, maxi-K channels can reside in the caveolae, where they associate with the scaffolding protein caveolin-1 (cav-1). The aim of this study was to investigate the consequences of this interaction - more specifically, how disruption of the association between the maxi-K channel and cav-1 may influence the current expression and excitability of myometrial cells - with the aim of better understanding the mechanisms that underlie the regulation of normal and aberrant uterine function.

Methods: Myometrial biopsies were collected from women undergoing elective C-sections. From these samples, myometrial cells were isolated, cultured, infected with a virus containing either caveolin-1 (cav-1) siRNA or scrambled cav-1 siRNA, and finally subjected to patch-clamp analysis. Mutant caveolin-binding site maxi-K channel constructs were generated and transfected into mouse Ltk- fibroblasts. Channel activity, expression, association, and localization were examined by patch-clamping, Western blot, immunoprecipitation, and immunofluorescence, respectively.

Results: The caveolin-1 siRNA suppressed the total K+ current in human myometrial smooth muscle cells (hMSMC), as evident from comparison to the currents generated by both non-infected cells and cells infected with scrambled siRNA controls. The interaction between the maxi-K channel and caveolin depends on a region in the channel's C-terminal caveolin-binding site. Mutations of aromatic residues in this site (mutant F1012A, mutant Y1007A, F1012A and mutant Y1007A, F1012A, Y1015A) resulted in a decrease in K+ current compared to that produced by wild-type channels transfected into mouse Ltk- fibroblasts. However, mutation of all three aromatic amino acids (mutant Y1007A, F1012A, Y1015A) was necessary to disrupt the association between caveolin and the maxi-K channel, as visualized by immunofluorescence and immunoprecipitation.

Conclusion: Our results suggest that disruption of the caveolin-binding site interferes with the cav-1/maxi-K channel interaction, and that lack of the cav-1/maxi-K channel interaction in MSMCs attenuates the total K+ channel current of the cell.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2785819PMC
http://dx.doi.org/10.1186/1477-7827-7-131DOI Listing

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