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Chapter 18 Modulation of Sodium Conductance in Photoreceptor Membranes by Calcium Ions and cGMP
CURRENT TOPICS IN MEMBRANES AND TRANSPORT, VOLUME 15
Chapter 18
Modulation of Sodium Conductance in Photoreceptor Membranes by Calcium Ions and cGMP ROBERT T . SORBI‘ Istituto di Fisologiu Umiinir Universitv of Purmu Purma, Ituly
It has been suggested (Hagins, 1972) that Ca2+ is the soluble transmitter needed to link the disk membranes with the plasma membrane of the rod outer segment (ROS) in the process of photoreceptor excitation (Baylor and Fuortes, 1970). According to this hypothesis, in the dark calcium ions are sequestered and maintained within the disk spaces by active Ca 2+ transport and plasma membrane permeability to Na+ is high, allowing a ‘“a+ dark current” to flow into the cytoplasm. When light bleaches disk membrane rhodopsin, disk permeability to Ca“ is enhanced, causing increased Ca2+ activity in the cytoplasm and closing the Na+ channels. The data obtained by either increasing or lowering the cytoplasmic Ca2+ activity strongly support the idea that Ca2+ can modulate plasma membrane permeability to Na+ (Hagins and Yoshikami, 1974, 1977; Brown et a / . , 1977; Oakley and Pinto, 1980). However, many experiments have been attempted in search of light-dependent Ca2+ release from the disk (for a review, see NOH et al., 1979; Schnetkamp, 1979), and still no direct evidence indicates that in vivo physiological levels of illumination are able to promote the release of Ca2+from the disks. Recently a light-induced increase of the calcium activity has been recorded in the extracellular fluid surrounding the outer segments in isolated retinas (Gold and Korenbrot, 1980; this volume, Chapter 17; Yoshikami et al., 1980). This observation raises the possibility but does not give direct proof, that ‘Present address: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
331
Copright 0 I981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 012-153315-8
332
ROBERT T. SORB1
the increase in the extracellular activity reflects a light-induced increase of the cytoplasmic activity of calcium ions in the outer segment of the rod. In our laboratory, light-irrduced effects on the ionic fluxes from the ROS were detected after bleaching less than I % of the rhodopsin, but no change was observed in calcium fluxes (Cavaggioni et al., 1973). We could not observe the dim light effects when the ROS were broken (Fig. IA), which suggests that broken membranes allow the leakage of some necessary endogenous factor (Sorbi and Cavaggioni, 1975). In the broken ROS preparation, however, a bright light bleaching more than 5% of the rhodopsin increased the effluxes of Na+, K+ , and Rb+, but still not those of Ca2+ (Fig. 1B). Since this effect of light was
ZL I
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I
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(A) Effect of white light, bleaching less than I % of the rhodopsin, on the rate of efflux of from isolated rods and isolated disks, (B) Effect of white light, bleaching more than 5% uf the rhodopsin, on the rate of efflux of *'Rb, "Na, 36C1,and 45Cafrom isolated disks. In both (A) and (B), the arrows indicate the onset and the end of illumination. (From Sorbi and Cavaggioni, 1975.) FIG. 1.
"Rb
18. Ca AND cGMP EFFECTS ON Na CONDUCTANCE
333
proportional to the amount of bleached rhodopsin, it might be due to nonspecific changes in disk membrane properties induced by the modification of such a large proportion of the proteins within the membrane and by the release of a large number of retinal molecules. In 1971, a possible role for cyclic-nucleotide metabolism in visual excitation was proposed for the first time (Bitensky et al., 1971). Since then much work has been done in this area (Bitensky et al., this volume, Chapter 14), and currently cyclic guanosine 3' ,5'-monophosphate (cGMP) seems to be a viable candidate for the internal transmitter role (for a review, see Hubbell and Bownds, 1979; Pober and Bitensky, 1979; Bitensky et al., this volume, Chapter 14). The hypothesis involving cCMP in visual excitation suggests that in the dark plasma membrane permeability is high because of cGMP-dependent phosphorylation of some protein(s) related to membrane permeability to Na+ (Polans et al., 1979). In the presence of GTP, cGMP-phosphodiesterase (cGMP-PDE) is activated by bleached rhodopsin (Wheeler and Bitensky, 1977; Bignetti ef a f . , 1978) with a rate constant that is consistent with the time required for excitation (Yee and Liebman, 1978; Caretta e t a / . , 1979a). Consequently the internal cGMP concentration, [cGMPIi, falls with a rate that is compatible with its involvement in excitation (Woodruff et ul., 1977; Kilbride, 1980) and causes dephosphorylation of the protein(s) related to membrane permeability (Polans et al., 1979). A direct relationship between plasma membrane permeability to Na+ and the endogenous cGMP content of isolated ROS has been shown (Woodruff et al., 1977), and we have demonstrated that cCMP, in the micromolar range, can modulate the Na' permeability of the disk membrane (Fig. 2) (Caretta et al., 1979b). Thus the endogenous factor we lost in 1975 was probably cGMP. Moreover, iontophoretically injecting cGMP into the ROS causes depolarization of the plasma membrane and increases the latency of the photoresponse (Miller and Nicol, 1979). This evidence suggests that cGMP may affect the plasma membrane in the same way as the disk membranes; i.e., it increases the Na+ conductance. Since the effect of cGMP on Na' permeability is still present on thoroughly washed disk membranes, where proteins dephosphorylated by light in a cGMP-dependent way are presumed to be absent (Polans et ul., 1979), it is worthwhile to propose that a phosphorylation-dephosphorylation process may not be involved. One of the major criticisms that can be raised against a role of cGMP in visual excitation is the relative inefficiency of a negative transmitter. In fact, it seems to be less efficient for the plasma membrane to sense a decrease in [cGMP], which in the dark is approximately 40-90 F M in the ROS (Woodruff et al., 1977; Robinson and Hagins, 1980), than to detect the presence of a few hundred calcium ions above the background which is postulated to be zero. However, the plasma membrane should be able to detect fast and pronounced decreases of [cGMP] in the microdomain of excitation if the recharge of the transmitter from
334
ROBERT T. SORB1
. 100cGMP
1WpM
.
*
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16 mln
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I
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15
aamples
FIG.2. Effect of cGMP on the uptake (A) and on the release (B) of 2ZNafrom disk vesicles. (A) Loading of"Na by the disk vesicles. Solid circles, in the presence of 1 mM c G M P open circles, in the absence of cCMP. The values are normali7ed i n order to have the point at 8 min (with cGMP) equal to 100, to make a comparison between different experiments possible. The values are the mean of the number of experiments indicated at each point, and the bars indicate the standard deviation. (B) Effect of 100 pM cGMP on the rate of efflux of "'Na from disk vesicles.
the surrounding nonexcited spaces is slow compared to the PDE activity. Measurements of the space constant of excitation in the rod do not rule out such a hypothesis (Yau et d., 1980; this volume, Chapter 2). The question now arises, How is it possible to combine the data that favor Ca" as the internal transmitter with those that favor cGMP. The finding that Ca" influences the cGMP content in the ROS (Cohen el d.,1978), presumably by acting on a guanylate cyclase (Pannbacker, 1973; Krishnan et al.. 1978; de Vries and Ferrendelli, 1980), could lead to the conclusion that calcium modulates the sodium conductance via cCMP. However, Yoshikami et af. (1980) have shown that Cast affects sodium conductance directly and without the time lag needed to enhance [cGMPli. Recently, we have reported that cGMP influences Ca2' movement to and from the disk membranes (Cavaggioni and Sorbi, 1980). At physiological concentrations, cGMP stimulates the release and inhibits the uptake of calcium ions by the disk vesicles (Fig. 3). Increasing the cGMP concentration in the perfusing solution (in the micromolar range) causes the effect to become larger. Both the Ca2' uptake and the cGMP effect on thc Ca2+ uptake seem completely ATP-independent (Fig. 3). Thus cGMP may affect the binding sites for Ca2+on the disk membranes, as has already been suggested for
335
18. Ca AND cGMP EFFECTS ON Na CONDUCTANCE
:
-f-
E
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FIG.3. Effect of cGMP on the uptake (A) and on the release (B) of 45Cafrom disk vesicles. (A) Squares, in the presence of 1 mM ATP; triangles, in the absence of ATP. Open symbols, in the presence of 400 p M c G M P solid symbols, in the absence of cGMP. The disk vesicles were from the same stock. (B) Effect of 100 p M cGMP on the rate of 45Ca release from disk vesicles.
other systems (Weler and Laing, 1979). Since this is the case, it is not surprising that a light-induced increase in the internal Ca2+ activity has been so difficult to detect. These new data suggest, on the contrary, a light-induced decrease in the Ca2+activity inside the ROS as a function of cGMP modulation. As a consequence, a model may now be proposed in which both Ca2+ and cGMP are coprimary and complementary factors in the process of visual excitation, if we suppose that the Ca2+-bindingsites affected by cGMP are functional gates for the Na+ channels. Stieve and Bruns (1978) have suggested that in invertebrate photoreceptors the sodium channels are transmembrane proteins with
336
ROBERT T. SORB1
binding sites for which Ca2+ and Na+ may compete antagonistically. When Na+ is bound to these sites, the channels are open for Nat conductance. When Ca2+ is bound to these sites, the channels are closed for Na+ conductance. Similar arguments have been presented by Schnetkamp (1 980) for the Ca2 -translocating system in the plasma membrane of vertebrate photoreceptors. When the abovementioned hypothesis is considered in conjunction with the cGMP effect on Ca2+-binding sites, 1 feel it likely that, when cCMP dccreases the binding site affinity for CaZt, it may favor the binding of Na+ at the same sites. Thus, in the dark, when the cGMP concentration is elevated, Na+ binds to and opens the channels. In the light. the cGMP concentration in the microdomain falls because of the light-activated cGMP-PDE, and Ca2+ binding to the channels is favored. Such calcium binding switches the channel to the closed conformation. Moreover, since the guanylate cyclase seems to be Ca2+-modulated, when the Ca2+ activity is diminished, this enzyme is stimulated in order to restore the normal cGMP concentration. It is possible to develop this model in order to deal with sensitivity regulation, taking into account the idea that the Ca2+-binding sites on the disk membrane are probably ineffective in modulating the Na+ dark current (Bitensky et u l . , this volume, Chapter 14). +
REFERENCES Baylor, D., and Fuortes, M. G.F. (1970). Electrical responses of single cones in the retina of the turtle. J . Phy.riol. (Lottdott) 207, 77-92, Bignetti, E., Cavaggioni, A., and Sorbi, R. T. (1978). Light-activatcd hydrolysis of GTP and cyclic GMP in the rod outer segments. J. Physiol. (London) 279, 55-69. Bitensky, M . W., Gorman, R. E., and Miller, W. H. (1971). Adcnyl cyclase: A link between photon capture and changes in membrane permeability of frog photoreceptors. Prnc. N a t l . A c i d . Sci. U.S.A. 63, 561-562. Brown, J . E . , Coles, J. A , , and Pinto, L. H. (1977). Effect of injections of calcium and EGTA into the outer segments of retinal rods of Bufo murinus. J . G m . Physiol. 269, 707-722. Caretta, A , , Cavaggioni, A,, and Sorbi. R. T. (1979a). Phosphodiestcrase and GTPase in rod outer segments: Kinetics in vitro. Eiochim. Bophys. Aclu 583, 1-13. Caretta, A . . Cavaggioni, A , , and Sorhi. R. T. (1979b). Cyclic GMP and permeahility of the disc membrane of the photoreceptors. J . Physiol. (London) 295, 171-178. Cavaggioni, A,, and Sorbi, R. T. (1980). Cyclic GMP affects calcium movements in the disc membrane of photoreceptors. Proc. Niitl. Arad. 6 - i . U.S.A. 78, 3961-3968. Cavaggioni. A , , Sorbi, R . T., and Turini, S. (1973). Efflux of potassium from isolated rod outer segments: A photic effect. J. Physiol. (London) 232, 609-620. Cohen, A. I., Hall, I . A,, and Ferrendelli, J . A. (1978). Calcium and cyclic nucleotide regulation in incubated mouse retinas. J. Gen. Physiol. 71, 595-612. de Vries, G. W., and Ferrendelli, J. A . (1980). Evidence lor multiple guanylate cyclase in a vertebrate photoreceptor. ARVO Abstr. in Invesl. Ophthalmol. Visual Sri. 19, 2I . Gold, G. H . , and Korenbrot, J . I. (1980). Light-induced calcium release by intact retinal rods. Proc. Nall. Acud. Sci. U . S . A . 77. 5557-5561.
18. Ca AND cGMP EFFECTS ON Na CONDUCTANCE
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Hagins, W. A. (1972). The visual process: Excitatory mechanism in the primary receptor cells. Annir. R e v . Biophjs. B i o m g . 1 , 131-159. Hagins, W . A,, and Yoshikami, S . (1974). A role for Ca” in excitation of retinal rods and cones. Exp. Eye Res. 18, 299-305. Hagins, W . A,. and Yoshikami, S . (1977). lntracellular transmission of visual excitation in photoreceptors: Electrical effects of chelating agents introduced into rods by vesicles fusion. In “Vetebrate photoreception” (H. D. Barlow and P. Fatt, eds.), pp. 97-138. Academic Press, New York. Hubbell, W. L., and Bownds, M. D. (1979). Visual transduction in vertebrate photoreceptors. Annu. Rev. Nrurosci. 2, 17-34. Kilbride, P. (1980). Calcium effects on frog retinal cGMP levels and their light-initiated rate of decay. .I. G m . Phvsiol. 75, 457-465. Krishnan, N . , Fletcher, R. T., Chader, G . J . , and Krishna, G. (1978). Characterization of guanylate cyclase of rod outer segments of the bovine retina. Biuchim. Biophys. Acia 523, 506-515. Miller, W. H., and Nicol. G. D. (1979). Evidence that cyclic GMP regulates membrane potential in rod photoreceptors. Nature (London) 280, 64-66. Noll, G . , Stieve, H., and Winterhager, J . (1979). Interaction of bovine rhodopsin with calcium. 11. Calcium release in bovine rod outer segments upon bleaching. Biophy.~.Strucr. Mech. 5, 43-53. Oakley, B . , 11, and Pinto, L. H. (1980). [Ca2+],modulates membrane sodium conductance in rod outer segments. ARVO Abstr. in Invest. Ophthalmol. Visual Sci. 19, 102. Pannbacker, R. G. (1973). Control of guanylate cyclase activity in the rod outer segment. Science 182, 1138-1 140. Pober, J . S . , and Bitensky, M. W. (1979). Light-regulated enzymes of vertebrate retinal rods. Adv. Cyclic Nucleotide Res. 11, 265-301. Polans, A. S . , Hermolin, J . , and Bownds, M. D. (1979). Light-induced dephosphorylation of two proteins in frog rod outer segments. J . Gen. Physiol. 74, 595-613. Robinson, W. E., and Hagins, W. A. (1980). Bound and free nucleotides in rod outer segments. Biophys. Soc. Abstr. in Fed. Pruc., Fed. Am. Soc. Exp. Biol. 39, 2067. Schnetkamp, P. P. M. (1979). Calcium translocation and storage of isolated intact cattle outer segments in darkness. Biochim. Biophys. Actu 554, 441 -459. Schnetkamp, P. P. M. (1980). Ion selectivity of the cation transport system of isolated intact cattle rod outer segments: Evidence for a direct communication between the rod plasma membrane and the rod disk membranes. Biochim. Biophys. Actu 598, 66-90. Sorhi, R. T . , and Cavaggioni, A. (1975). Effect of strong illumination on the ion efflux from isolated discs of frog photoreceptors. Biochim. Biophys. Actu 394, 577-585. Stieve, H . , and Bruns, M. (1978). Extracellular calcium, magnesium and sodium ion competition in the conductance control of the photosensory membrane of Limulus ventral nerve photoreceptor. 2. Narurforsch.: Eiosci. 33C, 574-579. Weller, M . , and Laing, W . (1979). The effect of cyclic nucleotides and protein phosphorylation on calcium permeability and binding in the sarcoplasmic reticulum. Biochim. Biophys. Actu 551, 406-4 19. Wheeler, G. L.. and Bitensky, M. W. (1977). A light-activated GTPase in vertebrate photoreceptors: Regulation of light-activated cyclic GMP phosphodiesterase. Proc. Null. Acud. Sci. U.S.A . 74, 4238-4242. Woodruff, M. L., Bownds, M. D., Green, S . H., Morrissey, J . L., and Shedlovsky, A. (1977). Guanosine 3‘,5’-cyclic monophosphate and in vitro physiology of frog photoreceptor memGen. Physiol. 69, 667-679. branes. .I.
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Yau. K . W . , Baylor, D . , Matthews. G . . Lamb, T . , and McNaughton, P. (1980). Background adaptation in toad rods. A R V O Abstr. in I I I W S I . Uphthulrnul. Visual Sci. 19. 192. Yee, R . . and Licbman, P. A . (1978). Light-activated phosphodiesterase of the rod outer segment: Kinetics and parameters of activation and deactivation. J . B i d . Chem. 253, 8902-8909. Yoshikaini, S . , George, J . S . , and Hagins, W . A. (1980). Light-induced calcium fluxes from outer segment layer of vertebrate retinas. Narure (Lundun) 286. 395-398.
Chapter 18
Modulation of Sodium Conductance in Photoreceptor Membranes by Calcium Ions and cGMP ROBERT T . SORBI‘ Istituto di Fisologiu Umiinir Universitv of Purmu Purma, Ituly
It has been suggested (Hagins, 1972) that Ca2+ is the soluble transmitter needed to link the disk membranes with the plasma membrane of the rod outer segment (ROS) in the process of photoreceptor excitation (Baylor and Fuortes, 1970). According to this hypothesis, in the dark calcium ions are sequestered and maintained within the disk spaces by active Ca 2+ transport and plasma membrane permeability to Na+ is high, allowing a ‘“a+ dark current” to flow into the cytoplasm. When light bleaches disk membrane rhodopsin, disk permeability to Ca“ is enhanced, causing increased Ca2+ activity in the cytoplasm and closing the Na+ channels. The data obtained by either increasing or lowering the cytoplasmic Ca2+ activity strongly support the idea that Ca2+ can modulate plasma membrane permeability to Na+ (Hagins and Yoshikami, 1974, 1977; Brown et a / . , 1977; Oakley and Pinto, 1980). However, many experiments have been attempted in search of light-dependent Ca2+ release from the disk (for a review, see NOH et al., 1979; Schnetkamp, 1979), and still no direct evidence indicates that in vivo physiological levels of illumination are able to promote the release of Ca2+from the disks. Recently a light-induced increase of the calcium activity has been recorded in the extracellular fluid surrounding the outer segments in isolated retinas (Gold and Korenbrot, 1980; this volume, Chapter 17; Yoshikami et al., 1980). This observation raises the possibility but does not give direct proof, that ‘Present address: Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
331
Copright 0 I981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 012-153315-8
332
ROBERT T. SORB1
the increase in the extracellular activity reflects a light-induced increase of the cytoplasmic activity of calcium ions in the outer segment of the rod. In our laboratory, light-irrduced effects on the ionic fluxes from the ROS were detected after bleaching less than I % of the rhodopsin, but no change was observed in calcium fluxes (Cavaggioni et al., 1973). We could not observe the dim light effects when the ROS were broken (Fig. IA), which suggests that broken membranes allow the leakage of some necessary endogenous factor (Sorbi and Cavaggioni, 1975). In the broken ROS preparation, however, a bright light bleaching more than 5% of the rhodopsin increased the effluxes of Na+, K+ , and Rb+, but still not those of Ca2+ (Fig. 1B). Since this effect of light was
ZL I
0
I
I
10
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(A) Effect of white light, bleaching less than I % of the rhodopsin, on the rate of efflux of from isolated rods and isolated disks, (B) Effect of white light, bleaching more than 5% uf the rhodopsin, on the rate of efflux of *'Rb, "Na, 36C1,and 45Cafrom isolated disks. In both (A) and (B), the arrows indicate the onset and the end of illumination. (From Sorbi and Cavaggioni, 1975.) FIG. 1.
"Rb
18. Ca AND cGMP EFFECTS ON Na CONDUCTANCE
333
proportional to the amount of bleached rhodopsin, it might be due to nonspecific changes in disk membrane properties induced by the modification of such a large proportion of the proteins within the membrane and by the release of a large number of retinal molecules. In 1971, a possible role for cyclic-nucleotide metabolism in visual excitation was proposed for the first time (Bitensky et al., 1971). Since then much work has been done in this area (Bitensky et al., this volume, Chapter 14), and currently cyclic guanosine 3' ,5'-monophosphate (cGMP) seems to be a viable candidate for the internal transmitter role (for a review, see Hubbell and Bownds, 1979; Pober and Bitensky, 1979; Bitensky et al., this volume, Chapter 14). The hypothesis involving cCMP in visual excitation suggests that in the dark plasma membrane permeability is high because of cGMP-dependent phosphorylation of some protein(s) related to membrane permeability to Na+ (Polans et al., 1979). In the presence of GTP, cGMP-phosphodiesterase (cGMP-PDE) is activated by bleached rhodopsin (Wheeler and Bitensky, 1977; Bignetti ef a f . , 1978) with a rate constant that is consistent with the time required for excitation (Yee and Liebman, 1978; Caretta e t a / . , 1979a). Consequently the internal cGMP concentration, [cGMPIi, falls with a rate that is compatible with its involvement in excitation (Woodruff et ul., 1977; Kilbride, 1980) and causes dephosphorylation of the protein(s) related to membrane permeability (Polans et al., 1979). A direct relationship between plasma membrane permeability to Na+ and the endogenous cGMP content of isolated ROS has been shown (Woodruff et al., 1977), and we have demonstrated that cCMP, in the micromolar range, can modulate the Na' permeability of the disk membrane (Fig. 2) (Caretta et al., 1979b). Thus the endogenous factor we lost in 1975 was probably cGMP. Moreover, iontophoretically injecting cGMP into the ROS causes depolarization of the plasma membrane and increases the latency of the photoresponse (Miller and Nicol, 1979). This evidence suggests that cGMP may affect the plasma membrane in the same way as the disk membranes; i.e., it increases the Na+ conductance. Since the effect of cGMP on Na' permeability is still present on thoroughly washed disk membranes, where proteins dephosphorylated by light in a cGMP-dependent way are presumed to be absent (Polans et ul., 1979), it is worthwhile to propose that a phosphorylation-dephosphorylation process may not be involved. One of the major criticisms that can be raised against a role of cGMP in visual excitation is the relative inefficiency of a negative transmitter. In fact, it seems to be less efficient for the plasma membrane to sense a decrease in [cGMP], which in the dark is approximately 40-90 F M in the ROS (Woodruff et al., 1977; Robinson and Hagins, 1980), than to detect the presence of a few hundred calcium ions above the background which is postulated to be zero. However, the plasma membrane should be able to detect fast and pronounced decreases of [cGMP] in the microdomain of excitation if the recharge of the transmitter from
334
ROBERT T. SORB1
. 100cGMP
1WpM
.
*
3
50-
P i
0L
0
4
8
12
16 mln
I
0
I
5
10
15
aamples
FIG.2. Effect of cGMP on the uptake (A) and on the release (B) of 2ZNafrom disk vesicles. (A) Loading of"Na by the disk vesicles. Solid circles, in the presence of 1 mM c G M P open circles, in the absence of cCMP. The values are normali7ed i n order to have the point at 8 min (with cGMP) equal to 100, to make a comparison between different experiments possible. The values are the mean of the number of experiments indicated at each point, and the bars indicate the standard deviation. (B) Effect of 100 pM cGMP on the rate of efflux of "'Na from disk vesicles.
the surrounding nonexcited spaces is slow compared to the PDE activity. Measurements of the space constant of excitation in the rod do not rule out such a hypothesis (Yau et d., 1980; this volume, Chapter 2). The question now arises, How is it possible to combine the data that favor Ca" as the internal transmitter with those that favor cGMP. The finding that Ca" influences the cGMP content in the ROS (Cohen el d.,1978), presumably by acting on a guanylate cyclase (Pannbacker, 1973; Krishnan et al.. 1978; de Vries and Ferrendelli, 1980), could lead to the conclusion that calcium modulates the sodium conductance via cCMP. However, Yoshikami et af. (1980) have shown that Cast affects sodium conductance directly and without the time lag needed to enhance [cGMPli. Recently, we have reported that cGMP influences Ca2' movement to and from the disk membranes (Cavaggioni and Sorbi, 1980). At physiological concentrations, cGMP stimulates the release and inhibits the uptake of calcium ions by the disk vesicles (Fig. 3). Increasing the cGMP concentration in the perfusing solution (in the micromolar range) causes the effect to become larger. Both the Ca2' uptake and the cGMP effect on thc Ca2+ uptake seem completely ATP-independent (Fig. 3). Thus cGMP may affect the binding sites for Ca2+on the disk membranes, as has already been suggested for
335
18. Ca AND cGMP EFFECTS ON Na CONDUCTANCE
:
-f-
E
I
J
8-
4-
*
2-
01
0
10
5
0
I
20
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I
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FIG.3. Effect of cGMP on the uptake (A) and on the release (B) of 45Cafrom disk vesicles. (A) Squares, in the presence of 1 mM ATP; triangles, in the absence of ATP. Open symbols, in the presence of 400 p M c G M P solid symbols, in the absence of cGMP. The disk vesicles were from the same stock. (B) Effect of 100 p M cGMP on the rate of 45Ca release from disk vesicles.
other systems (Weler and Laing, 1979). Since this is the case, it is not surprising that a light-induced increase in the internal Ca2+ activity has been so difficult to detect. These new data suggest, on the contrary, a light-induced decrease in the Ca2+activity inside the ROS as a function of cGMP modulation. As a consequence, a model may now be proposed in which both Ca2+ and cGMP are coprimary and complementary factors in the process of visual excitation, if we suppose that the Ca2+-bindingsites affected by cGMP are functional gates for the Na+ channels. Stieve and Bruns (1978) have suggested that in invertebrate photoreceptors the sodium channels are transmembrane proteins with
336
ROBERT T. SORB1
binding sites for which Ca2+ and Na+ may compete antagonistically. When Na+ is bound to these sites, the channels are open for Nat conductance. When Ca2+ is bound to these sites, the channels are closed for Na+ conductance. Similar arguments have been presented by Schnetkamp (1 980) for the Ca2 -translocating system in the plasma membrane of vertebrate photoreceptors. When the abovementioned hypothesis is considered in conjunction with the cGMP effect on Ca2+-binding sites, 1 feel it likely that, when cCMP dccreases the binding site affinity for CaZt, it may favor the binding of Na+ at the same sites. Thus, in the dark, when the cGMP concentration is elevated, Na+ binds to and opens the channels. In the light. the cGMP concentration in the microdomain falls because of the light-activated cGMP-PDE, and Ca2+ binding to the channels is favored. Such calcium binding switches the channel to the closed conformation. Moreover, since the guanylate cyclase seems to be Ca2+-modulated, when the Ca2+ activity is diminished, this enzyme is stimulated in order to restore the normal cGMP concentration. It is possible to develop this model in order to deal with sensitivity regulation, taking into account the idea that the Ca2+-binding sites on the disk membrane are probably ineffective in modulating the Na+ dark current (Bitensky et u l . , this volume, Chapter 14). +
REFERENCES Baylor, D., and Fuortes, M. G.F. (1970). Electrical responses of single cones in the retina of the turtle. J . Phy.riol. (Lottdott) 207, 77-92, Bignetti, E., Cavaggioni, A., and Sorbi, R. T. (1978). Light-activatcd hydrolysis of GTP and cyclic GMP in the rod outer segments. J. Physiol. (London) 279, 55-69. Bitensky, M . W., Gorman, R. E., and Miller, W. H. (1971). Adcnyl cyclase: A link between photon capture and changes in membrane permeability of frog photoreceptors. Prnc. N a t l . A c i d . Sci. U.S.A. 63, 561-562. Brown, J . E . , Coles, J. A , , and Pinto, L. H. (1977). Effect of injections of calcium and EGTA into the outer segments of retinal rods of Bufo murinus. J . G m . Physiol. 269, 707-722. Caretta, A , , Cavaggioni, A,, and Sorbi. R. T. (1979a). Phosphodiestcrase and GTPase in rod outer segments: Kinetics in vitro. Eiochim. Bophys. Aclu 583, 1-13. Caretta, A . . Cavaggioni, A , , and Sorhi. R. T. (1979b). Cyclic GMP and permeahility of the disc membrane of the photoreceptors. J . Physiol. (London) 295, 171-178. Cavaggioni, A,, and Sorbi, R. T. (1980). Cyclic GMP affects calcium movements in the disc membrane of photoreceptors. Proc. Niitl. Arad. 6 - i . U.S.A. 78, 3961-3968. Cavaggioni. A , , Sorbi, R . T., and Turini, S. (1973). Efflux of potassium from isolated rod outer segments: A photic effect. J. Physiol. (London) 232, 609-620. Cohen, A. I., Hall, I . A,, and Ferrendelli, J . A. (1978). Calcium and cyclic nucleotide regulation in incubated mouse retinas. J. Gen. Physiol. 71, 595-612. de Vries, G. W., and Ferrendelli, J. A . (1980). Evidence lor multiple guanylate cyclase in a vertebrate photoreceptor. ARVO Abstr. in Invesl. Ophthalmol. Visual Sri. 19, 2I . Gold, G. H . , and Korenbrot, J . I. (1980). Light-induced calcium release by intact retinal rods. Proc. Nall. Acud. Sci. U . S . A . 77. 5557-5561.
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