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

Abstract

A B S T RA C T The receptor potential of rhabdomeric photoreceptors is mediated primarily by a Na influx, but other ions must also permeate through light-dependent channels to account for some properties of the photoresponse. We examined ion conduction in macroscopic and single-channel light-induced currents of Lima and Pecten photoreceptors. In the absence of Na, a fivefold change in extracellular K shifted the reversal voltage of the photocurrent (V~v) by ~ 27 mV. Because the dependency of Vre v on [K] o was sub-Nernstian, and Vre v in each condition was more positive than EK, some other ion(s) with a positive equilibrium potential must be implicated, in addition to K. We assessed the participation of calcium, an important candidate because of its involvement in light adaptation. Three strategies were adopted to minimize the impairments to cytosolic Ca homeostasis and loss of responsiveness that normally result from the required ionic manipulations: (a) Internal dialysis with Na-free solutions, to prevent reverse operation of the Na/Ca exchanger. (b) Rapid solution changes, temporally limiting exposure to potentially detrimental ionic conditions. (c) Single-channel recording, exposing only the cell-attached patch of membrane to the test solutions. An inward whole-cell photocurrent could be measured with Ca as the only extracellular charge carrier. Decreasing the [Ca]o to 0.5 mM reduced the response by 43% and displaced the reversal potential by -4.3 mV; the shift was larger (AVr~v = -44 mV) when intracellular permeant cations were also removed. In all cases, however, the current carried by Ca was < 5% of that measured with normal [Na]o. Unitary light-activated currents were reduced in a similar way when the pipette contained only divalent cations, indicating a substantial selectivity for Na over Ca. The fall kinetics of the photoresponse was slower when external Ca was replaced by Ba, or when the membrane was depolarized; however, dialysis with 10 mM BAPTA failed to antagonize this effect, suggesting that mechanisms other than the Ca influx participate in the modulation of the time course of the photocurrent.

Light-transduction in melanopsin-expressing photoreceptors of Amphioxus

“…Detailed analysis of selective permeation would require examining the ionic dependency of the reversal potential, but the strong inward rectification of the photocurrent did not allow such measurements. Nonetheless, the differential role for Na and Ca is reminiscent of light-dependent ion channels in various invertebrate rhabdomeric photoreceptors (35,36). Membrane current fluctuations elicited by dim light were rapid and only a few picoamperes in amplitude; on the assumption that these waves represent quantal responses, the transduction gain would appear to be only severalfold greater than in mouse ipRGCs (37); the much larger overall light sensitivity of Amphioxus (2-3 orders of magnitude) may, thus, be due chiefly to higher expression levels of melanopsin.…”

Light Transduction in Invertebrate Hyperpolarizing Photoreceptors: Possible Involvement of a Go-Regulated Guanylate Cyclase

“…E xtracellular chemical stimulation was accomplished by a puffer pipette (tip outer diameter of 3-4 m) positioned ϳ30 -50 m from the target cell. Application of pressurized nitrogen (1-3 psi) to the pipette via a solenoidoperated valve permitted local solution exchange in Ͻ400 msec (Gomez and Nasi, 1996). Patch electrodes were fabricated with thin-wall borosilicate capillary tubing (7052, Garner Glass, C laremont, CA), fire-polished, and filled with an "intracellular" solution containing (in mM) 100 KC l, 200 K-aspartate or K-glutamate, 5 Na 2 ATP, 12 NaC l, 6 MgC l 2 , 10 H EPES, 1 EGTA, 0.2 GTP and 300 sucrose, pH 7.3.…”

Membrane Current Induced by Protein Kinase C Activators in Rhabdomeric Photoreceptors: Implications for Visual Excitation

“…To apply test substances extracellularly, we relied on a local perf usion technique consisting of a puffer micropipette positioned in the vicinity of the cell that could pressure eject a stream of test solution on activation of a solenoidoperated valve. Previous measurements obtained with a fluorescent dye included in the puffer pipette demonstrated that ϳ95% of solution exchange is attained within 200 -400 msec, and that the ejection plume completely engulfs the region occupied by the target cell, as visualized with an image-intensified CCD camera (Gomez and Nasi, 1996a). Alternatively, in some experiments, test compounds were administered intracellularly by dialysis via the patch electrode.…”