Caveolin-1 Facilitates the Direct Coupling between Large Conductance Ca2+-activated K+ (BKCa) and Cav1.2 Ca2+ Channels and Their Clustering to Regulate Membrane Excitability in Vascular Myocytes

Abstract

Background:The contribution of caveolae to physiological interaction between two major ion channels, large conductance Ca 2ϩ -activated K ϩ (BK Ca ) and Ca 2ϩ (Cav1.2) channels, is unknown in vascular myocytes. Results: The loss of caveola by caveolin-1 deficiency reduced BK Ca -Cav1.2 coupling, Cav1.2 clustering, and membrane excitability regulation. Conclusion: Caveolin-1 provides platform for BK Ca -Cav1.2 molecular complex. Significance: Caveolin-1-BK Ca -Cav1.2 in caveola forms a novel Ca 2ϩ signal domain for arterial tonus regulation.

Orai1–Orai2 complex is involved in store-operated calcium entry in chondrocyte cell lines

“…Orai1 and Orai2 molecules were counted in membranes of HEK293 cells by observing bleaching steps of GFP fused to these channels in a single particle, as described previously [27,28]. Because high endogenous expression of Orai in OUMS-27 cells maked subunit counting difficult, we used HEK293 cells in this series of experiments.…”

“…FRET efficiency (E FRET ) was evaluated based on the acceptor photobleaching method, in which the emission of the donor fluorophore is compared before and after photobleaching of the acceptor [27,28]. The fluorescence of YFP was photobleached using a mercury lamp (100 W, C-SHG1; Nikon) and a G-2A filter cube (Ex510−560/DM575/BA590; Nikon) for 1.5 min.…”

“…Single-molecule imaging was performed using a TIRF imaging system with objective lenses (CFI Plan Apo TIRF 60×/1.45 or 100×/1.49, oil immersion; Nikon), as described previously [27,28]. Data were collected with an electron multiplied-CCD camera and analyzed by AQUACOSMOS software (Hamamatsu Photonics).…”

“…Electrophysiological studies were performed using a wholecell voltage-clamp technique with a CEZ-2400 amplifier (Nihon Kohden, Tokyo, Japan), an analog-digital converter (DIGIDATA 1440; Molecular Devices, Sunnyvale, CA, USA) and a pCLAMP software (version 10.3; Molecular Devices), as described previously [28]. Since CRAC channel current (I CRAC ) in OUMS-27 cells was too small to detect, we used heterologous expression system in this series of experiments.…”

Potassium Channels in Regulation of Vascular Smooth Muscle Contraction and Growth

“…T. Nelson & Quayle, 1995; Perez, Bonev, & Nelson, 2001; Perez, Bonev, Patlak, & Nelson, 1999; Porter et al, 1998; Wellman et al, 2002; Wellman & Nelson, 2003). In contrast, Ca 2+ influx through VGCC may activate BK Ca channels in other vessels including hamster and mouse cremaster arterioles (Westcott, Goodwin, Segal, & Jackson, 2012; Westcott & Jackson, 2011), rabbit coronary arteries (Guia, Wan, Courtemanche, & Leblanc, 1999) and mouse mesenteric arteries (Y. Suzuki, Yamamura, Ohya, & Imaizumi, 2013).…”