ACTIVATION OF CA2+-DEPENDENT CL- CURRENTS IN CULTURED RAT SENSORY NEURONS BY FLASH-PHOTOLYSIS OF DM-NITROPHEN

1. Voltage-gated Ca2+ currents (I-Ca) and Ca2+-activated Cl- currents (I-Cl(Ca)) were recorded from cultured mt dorsal root ganglion (DRG) neurones using the whole-cell configuration of the patch clamp technique. Intracellular photorelease of Ca2+ by flash photolysis of DM-nitrophen elicited transient inward currents only in those cells which possessed Ca2+ activated Cl- tail currents following I-Ca. The reversal potential of the flash responses was hyperpolarized when extracellular Cl- was replaced by SCN-. The flash responses and the Ca2+-activated Cl- tail currents were inhibited by the Cl- channel blockers niflumic acid (10-100 mu m) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)(10 mu M). 2. After activation by I-Ca, the Ca2+-activated Cl- current could be reactivated during its decay by photorelease of caged Ca2+. Experiments carried out on neurones held at 0 mV demonstrated that I-Cl(Ca) could be chronically activated due to residual Ca2+ influx. These data directly demonstrated that the decay of I-Cl(Ca) is not due to inactivation but rather to deactivation as a result of removal of the Ca2+ load from the cell cytoplasm. 3. Photorelease of caged inositol 1,4,5-trisphosphate (IP3) failed to activate any Ca2+-dependent current responses in cultured DRG neurones, although application of caffeine elicited transient inward currents, and responses to photoreleased IP3 could be obtained from freshly dissociated smooth muscle cells. 4. Photorelease of Ca2+ provides a useful method for investigating the properties of I-Cl(CA) independently from other physiological parameters. In addition, we have directly demonstrated that I-Cl(Ca) in DRG: neurones does not inactivate, and so may continue to modulate membrane excitability as long as the intracellular Ca2+ concentration ([Ca2+](i)) close to the cell membrane is elevated. This property also makes I-Cl(Ca) a useful physiological index of elevated [Ca2+](i) in studies of intracellular Ca2+ release and homeostasis.