Light-dependent channels from excised patches of Limulus ventral photoreceptors are opened by cGMP.

Abstract

The identity of the second messenger that directly activates the light-dependent conductance in invertebrate photoreceptors remains unclear; the available evidence provides some support for cGMP and Ca2'. To resolve this issue we have applied these second messengers to membrane patches excised from the light-sensitive lobe of Limulus ventral photoreceptors. Our results show that these patches contain channels that can be opened by cGMP, but not by Ca2+. These cGMP-activated channels closely resemble the channels activated by light in cell-attached patches. This evidence suggests that cGMP is the messenger that opens the light-dependent channel in invertebrate photoreceptors.In photoreceptors, large numbers (102-103) of membrane channels are affected by the absorption of a single photon (1, 2). This amplification is produced by a second messenger cascade (3). In vertebrate rods and cones (4-8) the final step of this cascade involves the direct control of ion channels by cGMP. Efforts to establish which second messenger controls the light-dependent channels in invertebrate photoreceptors have given conflicting results (9). Injection of inositol trisphosphate (IP3) (10, 11) or Ca2+ (12) into Limulus ventral photoreceptors leads to the activation of the light-dependent conductance. This evidence, together with biochemical evidence that the inositol phospholipid pathway (11,(13)(14)(15) METHODS To apply the patch clamp technique to Limulus ventral photoreceptors, it is first necessary to remove the glial cells and connective tissue that surround the photoreceptor cell that is to be studied. This was done mechanically by using a suction pipette while observing the cell through a x 100 compound microscope, as described (17). Gigaohm seals were made on the light-sensitive microvillar membrane ofthe photoreceptor (18) by using 1-to 2-Mfl electrodes filled with artificial seawater (ASW) and coated with Sylgard (Dow). After obtaining the seals, patches were excised in the insideout configuration into an "internal" solution (described below) resembling the ionic composition ofthe cytoplasm. As described in Results, it was important to prevent exposure of the excised patch to ASW. In our initial work, we achieved this by exposing the region of the cell-attached patch to internal solution delivered from a separate pipette. We found, however, that this procedure was cumbersome and that obtaining stable excised patches was rare. We then switched to a different strategy that gave a higher yield of stable excised patches. In this procedure we perfused the entire bath with internal solution before making the seals (depolarizing the membrane voltage to near zero). The patch could therefore be excised directly into the bath without exposure to ASW. To apply different solutions to the excised patch, the pipette tip was placed near the mouth of one of a series of parallel glass capillaries through which different solutions Abbreviations: IP3, inositol trisphosphate; ASW, artificial seawater. 7938The publication costs of this a...

Antagonists of the cGMP‐gated conductance of vertebrate rods block the photocurrent in scallop ciliary photoreceptors.

“…Considering that cyclic nucleotides also function as internal messengers for chemoreception in the ciliary neurons of the olfactory epithelium, and that in those cells the cyclic nucleotide-gated channels are also blocked by diltiazem, by 3',4'-dichlorobenzamil and by amiloride (Kolesnikov, Zhainazorov & Kosolapov, 1990;Frings, Lynch & Lindemann, 1992), an extension of this proposition is that a similar cyclic nucleotides-based signalling cascade has evolved in a variety of sensory cells of ciliary origin. This, of course, does not exclude the possibility that some elements of this pathway may also be found in other cells; excitatory effects of cGMP have been reported in patches of Limulus rhabdomeric membrane (Bacigalupo, Johnson, Vergara & Lisman, 1991), and a cGMP-activated channel expressed in the eyes and antennae of Drosophila has been cloned (Baumann, Frings, Godde, Seifert & Kaupp, 1994). A functional link between these channels and the light-triggered phospholipase C cascade, however, has not been demonstrated, so that their participation in phototransduction remains to be elucidated.…”

Multiple conductance states of the light-activated channel of Limulus ventral photoreceptors. Alteration of conductance state during light.

“…The second messenger reponsible for opening the lightactivated channels of invertebrate photoreceptors may be cGMP (Johnson et al, 1986;Bacigalupo et al, 1990Bacigalupo et al, , 1991. Previous work showed that light activates channels having a conductance of 40 pS.…”

Rapid Coupling of Calcium Release to Depolarization inLimulus polyphemusVentral Photoreceptors as Revealed by Microphotolysis and Confocal Microscopy

“…If, as is indicated by experiments on excised patches (Bacigalupo et al, 1991), calcium does not bind directly to and open ion channels in the microvillar membrane, then the intermediate steps that couple calcium to the activation of ion channels impose a delay of only 1-3 msec. The significance of this result for visual excitation depends on when calcium is released during the light response.…”

“…However, it has not been possible so far to verify that released calcium can activate ion channels sufficiently rapidly to contribute to the generation of inward current during the light response. In excised patches of light-sensitive membrane, cyclic guanosine monophosphate (cGMP), but not calcium ions, activates ion channels (Bacigalupo et al, 1991). Calcium-activated production of cGMP has, therefore, been proposed to couple the elevation of Ca i to the activation of ion channels (Shin et al, 1993).…”