GATING AND SELECTIVITY OF CA2+ PERMEABLE CHANNELS OF SENSORY CELLS

Ca2+-permeable channels are ubiquitous and are gated by the binding of a ligand to a specific site or by a change in the membrane potential. In the present work, the cGMP gated channel of the vertebrate photoreceptors and the voltage gated Ca2+ channel of the hair cells of the frog semicircular canal are analyzed. The cGMP channels are crucial in many cellular processes, and in particular they mediate also taste and olfactory transduction in vertebrates. The rod cGMP channel is a symmetric heterotetramer composed of two alpha and two beta subunits, all having binding sites for cGMP, which open the channel in less than 1 msec (Hill coefficient of 1.5-3, half saturation observed at 5-50 micromoles of cGMP). The channel is weakly voltage dependent, and the current-voltage (I/V) relations can be fitted by the Goldman-Hodgkin-Katz equation in the case that only a single ionic species carried the current, whereas under bi-ionic conditions it fits the I/V only if Na+ and NH4+ are the permeant ions. The pore discriminate poorly among monovalent cations, whereas it is very permeable to Ca2+, and it is sensitive to some Ca2+ channel blockers. However, Ca2+ (as well as Mg2+) blocks the pore while permeating through it (ion permeation occurs in a "single file" mode), thus Ca2+ carries a smaller portion of the dark current with respect to Na+ (15% vs. 85%) and reduces the channel conductance to about 100 fS (in the absence of divalent cations the conductance is 25-60 pS). The Ca2+ permeability is also modulated by the intracellular guanosine level, possibly via a binding site belonging to the channel itself. The Ca2+ channels of the hair cells of the semicircular canals are poorly known from an electrophysiological and a molecular point of view. As many other Ca2+-permeable channels, the largest current (up to 300 pA) is attained at -20 mV, the reversal potential is smaller (about +20 mV) than the one predicted by the Nernst potential, and the current inactivates to some extent during depolarizing steps (10-280 ms duration). With time, the current becomes progressively smaller and the gating kinetics progressively slower (run-down), up to a point that no more functional channels are present. Previous data suggest that a single Ca2+-channel, of the L-type, exists in the hair cell membrane. However, now we present evidence that only in few cells, irrespective of their gross morphological aspect: 1) the substitution of the extracellular Ca2+ (4 mM) with an equimolar amount of Ba2+ produces a two fold increase in the inward current, 2) Bay K 8644 (5 micromoles), applied either extra- or intracellularly increases the Ca2+-current and/or produces a small negative shift in the I-V curve. These results point to the presence of two different populations of Ca2+-channels or to a different modulatory state of a single channel type