Local Ca2+ rise near store operated Ca2+ channels inhibits cell coupling during capacitative Ca2+ influx.
Using a new fluorescence imaging technique, LAMP, we recently reported that Ca(2+) influx through store operated Ca(2+) channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH(i)) and Ca(2+)([Ca(2+)](i)) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca(2+) influx, there was a modest decline of pH(i) measured by BCECF. Decreasing pH(i) below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH(i) drop with thioacetate and bulk [Ca(2+)(i) rise with ionomycin was much less effective in inhibiting cell coupling than Ca(2+) influx. Moreover, clamping pH(i) with a weak acid and a weak base during Ca(2+) influx largely suppressed bulk pH(i) drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca(2+)(i) with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca(2+) influx. We concluded that local Ca(2+) elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca(2+) influx. To assess how Ca(2+) influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca(2+) influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca(2+) influx may be a more general phenomenon.
Dakin
Kenneth
K
Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, 75390-9039, USA.
Li
Wen-Hong
WH
D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002450:Cell Communication; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D000072259:Connexin 26; D017630:Connexins; D003600:Cytosol / Q000378:metabolism*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D017629:Gap Junctions / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007365:Intercellular Junctions; D015640:Ion Channel Gating; D008856:Microscopy, Fluorescence / Q000379:methods; D018408:Patch-Clamp Techniques
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D017630:Connexins; C000607042:GJB2 protein, human; D000072259:Connexin 26; D004533:Egtazic Acid; D000252:Calcium-Transporting ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium