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

Abstract

The relationship among intracellular pH (pH_i), −log₁₀ intracellular Ca²⁺ concentration (pCa_i) and gap junctional conductance, the participation of Ca²⁺ stores, and the role of calmodulin in channel regulation have been studied inXenopus oocytes, expressing the native connexin (Cx38), exposed to external solutions bubbled with 100% CO₂. The time courses of pH_i [measured with 2′,7′-bis(2-carboxyethyl)-5,6-carboxylluorscein (BCECF)], (pCa_i) (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 (pCa_i) than with pH_i. 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-pH_i-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 5 h. 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 ≈60% in 24 h, by ≈76% in 48 h and by ≈93% in 72 h. Oocytes that had lost gating sensitivity to CO₂. partially recovered gating competency following calmodulin injection. The data suggest that lowered pH_i uncouples gap junctions by a Ca²⁺-calmodulin-mediated mechanism.