TY - JOUR
T1 - Local Ca2+ rise near store operated Ca2+ channels inhibits cell coupling during capacitative Ca2+ influx
AU - Dakin, Kenneth
AU - Li, Wen Hong
N1 - Funding Information:
This research was supported by a research grant (I-1510) from the Welch Foundation and a career development award from the American Diabetes Association to W.-H. Li. We thank Dr. Frederick Grinnell for providing primary human fibroblasts and Dr. Xi Lin for providing Cx26-IRES-DsRed plasmid.
PY - 2006/1
Y1 - 2006/1
N2 - Using a new fluorescence imaging technique, LAMP, we recently reported that Ca2+ influx through store operated Ca2+ 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 (pHi) and Ca2+ ([Ca2+]i) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca2+ influx, there was a modest decline of pHi measured by BCECF. Decreasing pHi below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pHi drop with thioacetate and bulk [Ca2+ rise with ionomycin was much less effective in inhibiting cell coupling than Ca2+ influx. Moreover, clamping pHi with a weak acid and a weak base during Ca2+ influx largely suppressed bulk pHi drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca2+]i with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca2+ influx. We concluded that local Ca2+ elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca2+ influx. To assess how Ca2+ influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca2+ influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca2+ influx may be a more general phenomenon.
AB - Using a new fluorescence imaging technique, LAMP, we recently reported that Ca2+ influx through store operated Ca2+ 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 (pHi) and Ca2+ ([Ca2+]i) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca2+ influx, there was a modest decline of pHi measured by BCECF. Decreasing pHi below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pHi drop with thioacetate and bulk [Ca2+ rise with ionomycin was much less effective in inhibiting cell coupling than Ca2+ influx. Moreover, clamping pHi with a weak acid and a weak base during Ca2+ influx largely suppressed bulk pHi drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca2+]i with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca2+ influx. We concluded that local Ca2+ elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca2+ influx. To assess how Ca2+ influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca2+ influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca2+ influx may be a more general phenomenon.
KW - Capacitative Ca influx
KW - Connexin 43
KW - Fluorescence imaging
KW - Gap junction coupling
KW - LAMP assay
KW - Photoactivatable fluorophores
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U2 - 10.1080/15419060600631425
DO - 10.1080/15419060600631425
M3 - Article
C2 - 16613778
AN - SCOPUS:33646095990
SN - 1061-5385
VL - 13
SP - 29
EP - 39
JO - Cell Communication and Adhesion
JF - Cell Communication and Adhesion
IS - 1-2
ER -