title: Disruption of the maxi-K-caveolin-1 interaction alters current expression in human myometrial cells.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Molecular Physiology and Biophysics, Carver College of Medicine, University of Iowa, Iowa City, IA, USA. adambrainard@hotmail.com
authors.forename: Adam M
authors.initials: AM
authors.lastname: Brainard
authors.forename: Victoria P
authors.initials: VP
authors.lastname: Korovkina
authors.forename: Sarah K
authors.initials: SK
authors.lastname: England
mesh_terms: D020816:Amino Acid Motifs / Q000187:drug effects / Q000235:genetics; D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000235:genetics; D051242:Caveolin 1 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D050505:Mutant Proteins / Q000378:metabolism / Q000502:physiology; D009215:Myometrium / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011247:Pregnancy; D011485:Protein Binding / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D054730:Protein Interaction Domains and Motifs / Q000187:drug effects / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D051036:Large-Conductance Calcium-Activated Potassium Channels; D050505:Mutant Proteins; D034741:RNA, Small Interfering