Light-activated ion channels in solitary photoreceptors of the scallop Pecten irradians.

Abstract

A B S T R A C T Retinas from the scallopPecten irradians were enzymatically dispersed, yielding a large number of isolated photoreceptors suitable for tight-seal recording. Whole-cell voltage clamp measurements demonstrated that the phototransducing machinery remained intact: quantum bumps could be elicited by dim illumination, while brighter flashes produced larger, smooth photocurrents. Single-channel currents specifically activated by light were recorded in cell-attached patches, and were almost exclusively confined to the rhabdomeric region. Their density is sufficiendy high to account for the macroscopic photoresponse. Channel activation is graded with stimulus intensity in a range comparable to that of the whole-cell response, and can be recorded with illumination sufficiently dim to evoke only quantum bumps. Light-dependent channel openings are very brief, on average 1 ms or less at 20-22°C, apparently not because of blockage by extracellular divalent cations. The mean open time does not change substantially with stimulus intensity. In particular, since dwell times are in the millisecond range even with the dimmest lights, the channel closing rate does not appear to be the rate-limiting step for the decay kinetics of discrete waves. The latency of the first opening after light onset is inversely related to light intensity, and the envelope of channel activity resembles the time course of the whole-cell photocurrent. Unitary currents are inward at resting potential, and have a reversal voltage similar to that of the macroscopic light response. Voltage modulates the activity of light-sensitive channels by increasing the opening rate and also by lengthening the mean open times as the patch is depolarized. The unitary conductance of the predominant class of events is ~ 48 pS, but at least one additional category of smaller-amplitude openings was observed. The relative incidence of large and small events does not appear to be related in a simple way to the state of adaptation of the cell.

Ion permeation through light-activated channels in rhabdomeric photoreceptors. Role of divalent cations.

“…Enzymatically dissociated rhabdomeric photoreceptors from Lima scabra and Pecten irradians were obtained using the protocols previously described (Nasi, 1991a;Nasi and Gomez, 1992). Macroscopic light-evoked currents recorded under whole-cell voltage clamp (Nasi, 1991c) were low-pass filtered at 500-1,500 Hz with a Bessel four-pole filter.…”

“…Macroscopic light-evoked currents recorded under whole-cell voltage clamp (Nasi, 1991c) were low-pass filtered at 500-1,500 Hz with a Bessel four-pole filter. For cell-attached recordings of lightdependent single-channel currents (Nasi and Gomez, 1992), the cutoff frequency ranged from 2 to 5 KHz. Recordings were digitized on-line (5-15 KHz sampling rate, 2821 12-bit analog interface board; Data Translation, Marlboro, MA) and stored on Bernoulli cartridges (Iomega, South Roy, UT) for subsequent analysis.…”

“…The extent of Ca contribution to the light-dependent current of rhabdomeric photoreceptors is difficult to assess, not only because the required ionic manipulations can affect the transduction cascade, but also because of other complexities, such as the intrinsic voltage dependency of the gating process of light-sensitive channels (Bacigalupo et al, 1986;Nasi and Gomez, 1992). In the present report, ionic permeation was examined in enzymatically isolated photoreceptors from the mollusks Lima scabra and Pecten irradians.…”

“…Because these cells are glia-free and present an exposed rhabdomeric lobe, the composition of the extracellular medium can be accurately controlled; their small size also permits efficient dialysis of the cytosol via a patch pipette, so that uncertainties on the intracellular ionic environment are reduced. The structural features and functional properties of retinal cells from the two species are extremely similar (Nasi, 1991b, c;Nasi and Gomez, 1992;Gomez and Nasi 1994). The advantage of Pecten rhabdomeric cells is that light-dependent singlechannel currents can be routinely recorded (Nasi and Gomez, 1992), whereas Lima photoreceptors prove more resistant for whole-cell voltage-clamp recording and survive prolonged experimental protocols involving multiple solution changes.…”

“…The structural features and functional properties of retinal cells from the two species are extremely similar (Nasi, 1991b, c;Nasi and Gomez, 1992;Gomez and Nasi 1994). The advantage of Pecten rhabdomeric cells is that light-dependent singlechannel currents can be routinely recorded (Nasi and Gomez, 1992), whereas Lima photoreceptors prove more resistant for whole-cell voltage-clamp recording and survive prolonged experimental protocols involving multiple solution changes.…”