The effect of calcium ions on the cGMPactivated current of outer segment membrane was examined by the excised-patch technique. Changes in the extracellular calcium concentration had marked effects on the cGMPactivated current, while changes in intracellular calcium concentration were ineffective. Changes in calcium concentration in the absence of cGMP had little, if any, effect on membrane conductance. These results suggest that both intracellular cGMP and extracellular calcium can directly affect the conductance underlying the light response in rod cells. The pharmacological agent l-cis-diltiazem reversibly inhibited the cGMP-activated current when applied to the intracellular side of an excised patch. When superfused over intact rod cells, l-cis-diltiazem reversibly blocked much of the normal light response. The isomer, d-cis-diltiazem, did not significantly affect either patches or intact rod cells. Thus, the lightregulated conductance has binding sites for both calcium and cGMP that may interact during the normal light response in rod cells and a site specific for l-cis-diltiazem that can be used to identify and further study the conductance mechanism.The hyperpolarizing response to light of the rod photoreceptor cell is caused by a decrease in the transmembrane current that enters its outer segment in the dark. This coupling of light to the membrane conductance of photoreceptors is likely to be mediated by intracellular messenger(s) (1). Experiments designed to decide between the two main intracellular messenger candidates, calcium (see ref.2) and cyclic GMP (see ref.3), have been complicated by the finding that the activities of these two putative messengers are interrelated (4). This has raised an important question about the phototransduction process: which internal messengers directly affect the membrane conductance that underlies the light response and which serve a regulatory role?Recent electrophysiological and biochemical experiments indicate that cGMP directly increases the cation permeability of the rod membrane. Fesenko et al. (5), using the excisedpatch technique (6), described a direct effect of cGMP on the membrane conductance of frog rod outer segments. This conductance had an ion selectivity resembling that of the conductance underlying the response to light of the rod cell, and its activation was relatively independent of the calcium concentration at the intracellular side of the membrane. This result suggested that cGMP is the internal messenger of visual transduction in the rod cell (5). However, while it is clear that cGMP can directly affect the outer segment conductance, the mechanism of cGMP action is unknown. For example, is it sufficient for cGMP to interact with the intracellular side of the membrane to activate the conductance or are coregulators involved? Of particular interest is the role of extracellular calcium ions. Yoshikami and Hagins (2) first described a pronounced increase of the light-regulated current upon lowering the concentration of external calcium. This ef...

One cytoplasmic aspect of the junctional membrane between coupled pairs of Fundulus blastomeres was perfused with solutions of known H and Ca ion concentrations. Conductance of junctional membrane was decreased by either ion. The sensitivity to H ions was about 10,000 times greater than that to Ca ions. The results suggest that junctional conductance can be modulated by changes in H ion concentration near physiological pH, but that unphysiologically high concentrations of Ca ion, such as would be reached only on cell death, are required for comparable changes in junctional conductance.The cells ofmany tissues are connected by arrays ofintercellular channels identified morphologically as gap junctions (1). Gap junctional channels permit electrolytes and small molecules to flow between coupled cells. Abundant circumstantial evidence suggests that this form of intercellular communication is important in development and in organized functioning of tissues (2). Factors that regulate the conductance of gap junctions may therefore play a central role in these important cellular processes.On the basis of experiments in which cells were uncoupled by treatments which presumably increased intracellular free calcium ions (Cai) it was proposed that increased Cai reduces junctional conductance (ga) (3). Subsequent experiments in which Cai was monitored by aequorin luminescence during these treatments supported this hypothesis (4). However, the relationship between Cai and gj has been evaluated only semiquantitatively to date (5, 6).Recently it was reported that acidification of the cytoplasm ofelectrotonically coupled embryonic cells reversibly abolished the coupling (7-9). In experiments on isolated pairs of coupled cells in which the intracellular pH (pH,) and gj were directly measured, the relationship between pHi and g. was shown to be a simple sigmoid curve (10). The relationship was well fit by a Hill plot with an apparent pKH of 7.3, only 0.4 pH unit below the normal pHi (pH 7.7) of these cells.Because at least some intracellular buffering systems interchange H and Ca ions (cf. ref. 11), an experimentally produced increase in the concentration of either ion might cause a significant secondary increase of the other. Consequently, several recent studies measured the levels ofboth H and Ca ions during experimental treatments that uncoupled cells. Low pHi was shown to decrease g without increasing Cai, as measured with aequorin in teleost {Fundulus) blastomeres (9) and with intracellular Ca-sensitive electrodes in amphibian (Xenopus) blastulae (12) and mammalian (sheep) cardiac muscle (13). Insect (Chironomus) salivary gland cells could be uncoupled by treatments that increased Cai without decrease in pHi. Conversely, treatments that lowered pHi could uncouple these cells without increased aequorin luminescence (14), supporting the independent action of pHi on g, observed in the other studies cited.However, in this report the absence of increased aequorin luminescence during uncoupling by low pHi was ascribed to p...

A B S T R A C T Removing the glial cells that encaseLimulus ventral photoreceptors allows direct observation of the cell surface. Light microscopy of denuded photoreceptors reveals a subdivision of the cell body into lobes. Often one lobe, but sometimes several, is relatively clear and translucent (the R lobes). The lobe adjacent to the axon (the A lobe) has a textured appearance. Scanning electron microscopy shows that microvilli cover the surface ot R lobes and are absent from the surface of A lobes. When a dim spot of light is incident on the R lobe, the probability of evoking a single photon response is two to three orders of magnitude higher than when the same spot is incident on the A lobe. We conclude that the sensitivity of the cell to light is principally a function of the R lobe.

Phototransduction in rod cells is likely to involve an intracellular messenger system that links the absorption of light by rhodopsin to a change in membrane conductance. The direct effect of guanosine 3',5'-monophosphate (cGMP) on excised patches of rod outer segment membrane strongly supports a role for cGMP as an intracellular messenger in phototransduction. It is reported here that magnesium and calcium directly affect the conductance of excised patches of rod membrane in the absence of cGMP and that magnesium, applied to intact rod cells, blocks a component of the cellular light response. The divalent cation-suppressed conductance in excised patches showed outward rectification and cation-selective permeability resembling those of the light-suppressed conductance measured from the intact rod cell. The divalent cation-suppressed conductance was partly blocked by a concentration of the pharmacological agent L-cis-diltiazem that blocked all of the cGMP-activated conductance. Divalent cations may act, together with cGMP, as an intracellular messenger system that mediates the light response of the rod photoreceptor cell.

The internal dialysis technique has been applied to Limutus ventral photoreceptors. This method potentially allows quantitative control of the concentration of diffusible molecules within living cells. During dialysis, Limulus photoreceptors retained their ability to respond to light. Under conditions of dim illumination, responses were normal for close to an hour. In bright light, abnormalities developed more rapidly. The reversible effects of raising the dialysate Mg2+ concentration and the entrance of rhodamine-labeled albumin into cells shows that the dialysis method is useful for assaying the effects ofsmall or large molecules on visual transduction. This method has been used to examine the effects of nucleotide triphosphates and cyclic nucleotides. The results show that nucleotide triphosphates (5-10 mM) are required to maintain a low rate of spontaneous quantum bumps. The importance ofcyclic nucleotides in transduction is less clear; the light response was reduced by either cGMP or cAMP but only at very high concentrations (10 mM). METHODSDialysis System. Limulus ventral photoreceptors were prepared by using standard procedures (7). The apparatus for denuding cells, recording their electrical properties, and dialyzing their cytoplasm is shown schematically in Fig. 1. Glia surrounding a cell were removed by using a suction pipette (inside diameter, 20 am) to provide a naked plasma membrane (8). The denuded photoreceptor was gently sucked onto the same firepolished glass pipette. This sealed the pipette interior from the external bath by up to 60-Mfl total resistance. Next, the membrane patch plugging the pipette orifice was disrupted, making the pipette and cell interior continuous. To disrupt the membrane, the patch was aspirated by using a smaller suction electrode placed inside the first. Alternatively, a large briefcurrent was used. Passing a 3-pum electrode through the patch membrane disrupted it, but usually resulted in resealing. The data presented in this paper are from electrically punctured cells. Puncture by aspiration resulted in a smaller series resistance (rs in Fig. 1, 100-300 kfl) than did electrical puncture (400-700 kfQ). In either case, the series resistance had only a minor effect on the quality of the voltage clamp for small (<3 nA) slowly changing currents such as a quantum bump (the response to a single photon). To obtain a good voltage clamp for larger currents, a separate microelectrode was used to measure membrane voltage, as described in Fig. 4. All experiments used a 3 M KCl/Ag/AgCl bridge in the pipette and an artificial seawater/Ag/AgCl bridge in the bath. Except for the experiment in Fig. 4, the cell and pipette interior was held at virtual ground by using a current-to-voltage transducer, and the bath was at high impedance from ground. Current from the voltage clamp passed into the bath and through the membrane and was measured in the pipette. The pipette-cell seal (r1) was taken to be the measured resistance before puncture of the patch membrane; usually r1 was 10-...