Abstract
A chloride current, I(Cl,H), slowly activating on hyperpolarisation was investigated in Drosophila melanogaster larval muscles using the two-electrode voltage clamp. Sizeable currents were observed after the intracellular chloride concentration ([Cl-]i) had been elevated by diffusion of Cl- from the electrodes. The time course of I(Cl,H) was rather variable and required two exponentials to be accurately described. The reversal potential, -40 to -20 mV in Cl- -loaded fires, shifted on lowering external [Cl-] in the positive direction. Steady-state activation of I(Cl,H) was characterised by V0.5 of approximately -120 mV and a slope factor, k, of approximately 10 mV at a [Cl-]i approximately 35 mmol l(-1). Raising [Cl-]i to approximately 50 mmol l(-1) caused a negative shift of V0.5 equivalent to the change of E(Cl) and led to a nearly threefold increase in maximal steady-state conductance. I(Cl,H) was resistant to 10 mmol l(-1) Zn2+ and 1 mmol l(-1) Cd2+ but was greatly reduced by 1 mmol l(-1) 9-anthracenecarboxylic acid (9-AC). I(Cl,H) was affected by changes of extracellular pH and increased on lowering extracellular osmolality. 9-AC also decreased muscle fibre resting conductance by approximately 20% and increased muscle contractions. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis confirmed the expression of all three ClC genes in muscle, and immunohistochemistry indicated location of Drosophila melanogaster chloride channel-2 (DmClC-2) at the Z-lines. We conclude that DmClC-2 accounts for the channels underlying I(Cl,H), and in part for the resting chloride conductance. DmClC-2 may serve general homeostatic mechanisms such as pH- and osmo-regulation or may support muscle function on high motor activity or during a particular neurohormonal state of the animal.
