Inhibition of calmodulin expression prevents low-pH-induced gap junction uncoupling inXenopus oocytes

Abstract

The relationship among intracellular pH (pHi), -log10 intracellular Ca2+ concentration (pCai) and gap junctional conductance, the participation of Ca2+ stores, and the role of calmodulin in channel regulation have been studied in Xenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO2. The time courses of pHi [measured with 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorscein (BCECF)], pCai (measured with the membrane-associated fura-C18) and junctional conductance (measured with a double voltage-clamp protocol) were compared. The data obtained confirm previous evidence for a closer relationship of junctional conductance with pCai than with pHi. Evidence for an inhibitory effect of intracellularly injected ruthenium red or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) on CO2-induced uncoupling, coupled to negative results with Ca2+-free external solutions, point to a low-pHi -induced Ca2+ release from internal stores, likely to be primarily mitochondria. The hypothesis proposing a participation of calmodulin in channel gating was tested by studying the effects of calmodulin expression inhibition by intracellular injection of oligonucleotides antisense to the two calmodulin mRNAs expressed in the oocytes. Calmodulin mRNA was permanently eliminated in 5h. The oocytes injected with the antisense nucleotides progressively lost the capacity to uncouple with CO2 within 72 h. The effect of CO2 on junctional conductance was reduced by approximately 60% in 24 h, by approximately 76% in 48 h and by approximately 93% in 72 h. Oocytes that had lost gating sensitivity to CO2 partially recovered gating competency following calmodulin injection. The data suggest that lowered pHi uncouples gap junctions by a Ca2+- calmodulin-mediated mechanism.

Innexins form two types of channels

“…This is in contrast to channels formed by connexins, which either are non-responsive to increased cytoplasmic calcium or are closed, depending on their protein composition [35][36][37][38].…”

Granulosa cells regulate intracellular pH of the murine growing oocyte via gap junctions: development of independent homeostasis during oocyte growth

“…Although it is known that pH i can modulate gap junction permeability (Morley et al, 1996;Peracchia et al, 1996;Saez et al, 2005;Sosinsky and Nicholson, 2005), there is little available information about their role in synchronising pH i in connected cells. One recent study showed that microinjection of acid into cardiomyocytes causes detectable pH i decreases in adjacent cells (Zaniboni et al, 2003).…”

Regulation of Connexin Channels by pH

“…The molecular mechanisms that underlie this modulation of connexin channel activity are unclear and may differ among connexin isoforms and cell types. It has been proposed that the modulation is due to direct protonation of connexin (8), changes in ionized calcium concentration (11), and activation of calmodulin (12)(13)(14). For connexin-43 and for connexin-32/connexin-38 chimerae, recent work strongly indicates a pH-dependent interaction between segments of the C-terminal domain and the single cytoplasmic loop that inhibits channel activity (3,(15)(16)(17)(18)(19)(20)(21).…”