Ca ions play an essential role in hair cell functioning, since localized intracellular Ca changes modulate the transduction process and regulate the amplitude of the receptor potential. This, in turn, activates Ca and K basolateral conductances, which ultimately control transmitter release at the cell's cytoneural junction. Several studies have been recently reported on Ca entry via the voltage-gated Ca channels of hair cells, and on Ca extrusion at the level of the stereocilia mediated by highly packed Ca pumps. On the other hand, the mechanism of Ca clearance at the cell basolateral pole is less known, therefore the Ca extrusion via putative Na:Ca or Na:Ca,K exchanger was investigated in hair cells ìsolated from frog semicircular canals. Cells concurrently imaged during whole celi recordings using a fluorescent dye selective for Ca showed that the voltage-gated Ca entry occurred at selected sites (hotspots) located at the cell synaptic pole. Since the rate of Ca extrusion by any exchanger working in the forward mode is speeded up by cell hyperpolarization, voltage dependence of Ca clearance dynamics was studied following a standard 160 ms depolarization pulse to -20 mV to elicit Ca entry. In any hotspot and in any of the three cell types found in crista ampullaris (club-like, pear-like and cylinder-like), hyperpolarization did not accelerate at all Ca clearance kinetics. Reverse exchange was probed in patch-clamped cells, dialyzed with a Ca-, K-free solution containing 115 Na (concentrations in mM), upon switching from Ca-free, 115 Li external solution to 4 Ca + 115 Li or to 4 Ca + 20 K + 95 Li solution. The K currents were blocked with external 15 TEA and 5 Cs; the holding potential was -70 mV to block the Ca current. After correcting for the leak change upon switching from the external Ca-free solution to 4 Ca, no attual current changes were evoked by the solutions tested. In lizard rod photoreceptors in the same conditions, peak exchange currents exceeding 100 pA were recorded in the Ca + K external solution. The forward exchange mode was probed by rapidly increasing the intracellular Ca concentration by means of flash photolysis of two novel caged Ca complexes of 4-(2-nitrophenyl)-3,6-dioxaoctanedioic acid and 4-(4,5-dimethoxy-2-nitrophenyl)-3,6-dioxaoctanedioic acid in the presence of internal K and external Na. No currents were evoked by the Ca jump, whereas in rod photoreceptors, forward exchange currents up to 50 pA were recorded in the same conditions. It was concluded that neither Na:Ca nor Na:Ca,K electrogenic exchanger exists in this hair celi type.
Ca2+ Extrusion in Frog Semicircular Canal Hair Cells Probed with Flash Photolysis of Novel Ca-caged Compounds and Fast Calcium Imaging
RISPOLI, Giorgio;MARTINI, Marta;FARINELLI, Federica;ROSSI, Marialisa
2002
Abstract
Ca ions play an essential role in hair cell functioning, since localized intracellular Ca changes modulate the transduction process and regulate the amplitude of the receptor potential. This, in turn, activates Ca and K basolateral conductances, which ultimately control transmitter release at the cell's cytoneural junction. Several studies have been recently reported on Ca entry via the voltage-gated Ca channels of hair cells, and on Ca extrusion at the level of the stereocilia mediated by highly packed Ca pumps. On the other hand, the mechanism of Ca clearance at the cell basolateral pole is less known, therefore the Ca extrusion via putative Na:Ca or Na:Ca,K exchanger was investigated in hair cells ìsolated from frog semicircular canals. Cells concurrently imaged during whole celi recordings using a fluorescent dye selective for Ca showed that the voltage-gated Ca entry occurred at selected sites (hotspots) located at the cell synaptic pole. Since the rate of Ca extrusion by any exchanger working in the forward mode is speeded up by cell hyperpolarization, voltage dependence of Ca clearance dynamics was studied following a standard 160 ms depolarization pulse to -20 mV to elicit Ca entry. In any hotspot and in any of the three cell types found in crista ampullaris (club-like, pear-like and cylinder-like), hyperpolarization did not accelerate at all Ca clearance kinetics. Reverse exchange was probed in patch-clamped cells, dialyzed with a Ca-, K-free solution containing 115 Na (concentrations in mM), upon switching from Ca-free, 115 Li external solution to 4 Ca + 115 Li or to 4 Ca + 20 K + 95 Li solution. The K currents were blocked with external 15 TEA and 5 Cs; the holding potential was -70 mV to block the Ca current. After correcting for the leak change upon switching from the external Ca-free solution to 4 Ca, no attual current changes were evoked by the solutions tested. In lizard rod photoreceptors in the same conditions, peak exchange currents exceeding 100 pA were recorded in the Ca + K external solution. The forward exchange mode was probed by rapidly increasing the intracellular Ca concentration by means of flash photolysis of two novel caged Ca complexes of 4-(2-nitrophenyl)-3,6-dioxaoctanedioic acid and 4-(4,5-dimethoxy-2-nitrophenyl)-3,6-dioxaoctanedioic acid in the presence of internal K and external Na. No currents were evoked by the Ca jump, whereas in rod photoreceptors, forward exchange currents up to 50 pA were recorded in the same conditions. It was concluded that neither Na:Ca nor Na:Ca,K electrogenic exchanger exists in this hair celi type.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
