PERMEATION AND GATING PROPERTIES OF PRESYNAPTIC CALCIUM CHANNELS IN HAIR CELLS OF RANA CATESBEIANA
The objective of this study was to determine the elementary properties of calcium channels in amphibian hair cells. Calcium channel currents were recorded in hair cells isolated from the bullfrog inner ear using the tight-seal patch clamp technique. As defined by the blocking effect of nimodipine on whole-cell and single-channel currents, L-type (~80%) and non L-type (~20%) components were present in these cells. Western blot of the sensory epithelia and confocal imaging with antibody and conotoxin GVIA-conjugated fluorophores confirmed the presence of Ca V1.3 (L-type) and Ca V2.2 (non L-type) calcium channel subunits in hair cells. To determine the permeation properties of single-channels, the divalent cations Ca ^2+ , Sr ^2+ and Ba ^2+ were used as charge carriers. By varying the concentration of the charge carriers (2-70 mM), it was possible to observe saturation of unitary conductance, and to estimate the apparent dissociation constant (K D , in mM) for L-type (Ba ^2+ = 7.4 +/- 1.0; Ca ^2+ = 7.1 +/- 2.2; Sr ^2+ = 4.0 +/- 2.4) and non L-type channels (Ba ^2+ = 5.3 +/- 3.2; Ca ^2+ = 2.0 +/- 1.0; Sr ^2+ = 2.0 +/- 1.5). To gain insight into the mechanism of ion permeation, mixtures of Ba ^2+ and Ca ^2+ at total concentrations above (70 mM) and close to (5 mM) the KD of the channels were used. There was no evidence for an anomalous mole fraction effect when the ion concentration was 70 mM. However at 5 mM, single-channel conductance showed minima at Ba ^2+ :Ca ^2+ ratio of 1:4. These results are consistent with the concept of a multi-ion pore, and suggest that ion-ion interactions are critical for permeation at low pore occupancy. Finally, it was determined that the concentration and species of charge carrier affect the activation of single L-type channel currents. I propose that permeation and gating, as measured from the kinetics of single-channel currents, are interacting processes. Overall, these results support the existence of different classes of calcium channels in hair cells, and provide the hallmarks of hair cell calcium channels as Ca ^2+ transporters and multi-ion pores.
School:University of Cincinnati
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:bullfrog sacculus hair cell calcium channel presynaptic
Date of Publication:01/01/2001