Intracellular Cl(−) Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl(−). The purpose of the present study was to determine (a) whether Cl(−) plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl(−)](i) for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl(−)- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na(+) and 0–194 mM Cl(−). The artificial seawater bathing the BMF initially contained no Na(+). After dialysis was halted, adding 50 mM Na(+) to the artificial seawater caused an amiloride-sensitive pH(i) increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J (Na-H)) increased with increasing [Cl(−)](i) under both normo- and hypertonic conditions, with similar apparent K (m) values (∼120 mM). However, the maximal J (Na-H )increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pH(i) requires Cl(−) under both normo- and hypertonic conditions, but at any given [Cl(−)](i), J (Na-H) is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl(−)](i) is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl(−)-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl(−)-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl(−), dialyzing with GTPγS or AlF(3), or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl(−)-depleted fibers, the Cl(−)-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF(3) indicates that the G protein is a heterotrimeric G protein.