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ABSTRACT: cAMP, the classical second messenger, regulates many diverse cellular functions. The primary effector of cAMP signals, protein kinase A, differentially phosphorylates hundreds of cellular targets. Little is known, however, about the spatial and temporal nature of cAMP signals and their information content. Thus, it is largely unclear how cAMP, in response to different stimuli, orchestrates such a wide variety of cellular responses. Previously, we presented evidence that cAMP is produced in subcellular compartments near the plasma membrane, and that diffusion of cAMP from these compartments to the bulk cytosol is hindered. Here we report that a uniform extracellular stimulus initiates distinct cAMP signals within different cellular compartments. By using cyclic nucleotide-gated ion channels engineered as cAMP biosensors, we found that prostaglandin E(1) stimulation of human embryonic kidney cells caused a transient increase in cAMP concentration near the membrane. Interestingly, in the same time frame, the total cellular cAMP rose to a steady level. The decline in cAMP levels near the membrane was prevented by pretreatment with phosphodiesterase inhibitors. These data demonstrate that spatially and temporally distinct cAMP signals can coexist within simple cells.
Proceedings of the National Academy of Sciences 12/2001; 98(23):13049-54. · 9.68 Impact Factor
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Science 10/2001; 293(5538):2204-5. · 31.20 Impact Factor
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ABSTRACT: Phosphodiesterases (PDEs) catalyze the hydrolysis of the second messengers cAMP and cGMP. However, little is known about how PDE activity regulates cyclic nucleotide signals in vivo because, outside of specialized cells, there are few methods with the appropriate spatial and temporal resolution to measure cyclic nucleotide concentrations. We have previously demonstrated that adenovirus-expressed, olfactory cyclic nucleotide-gated channels provide real-time sensors for cAMP produced in subcellular compartments of restricted diffusion near the plasma membrane (Rich, T.C., K.A. Fagan, H. Nakata, J. Schaack, D.M.F. Cooper, and J.W. Karpen. 2000. J. Gen. Physiol. 116:147-161). To increase the utility of this method, we have modified the channel, increasing both its cAMP sensitivity and specificity, as well as removing regulation by Ca(2)+-calmodulin. We verified the increased sensitivity of these constructs in excised membrane patches, and in vivo by monitoring cAMP-induced Ca(2)+ influx through the channels in cell populations. The improved cAMP sensors were used to monitor changes in local cAMP concentration induced by adenylyl cyclase activators in the presence and absence of PDE inhibitors. This approach allowed us to identify localized PDE types in both nonexcitable HEK-293 and excitable GH4C1 cells. We have also developed a quantitative framework for estimating the K(I) of PDE inhibitors in vivo. The results indicate that PDE type IV regulates local cAMP levels in HEK-293 cells. In GH4C1 cells, inhibitors specific to PDE types I and IV increased local cAMP levels. The results suggest that in these cells PDE type IV has a high K(m) for cAMP, whereas PDE type I has a low K(m) for cAMP. Furthermore, in GH4C1 cells, basal adenylyl cyclase activity was readily observable after application of PDE type I inhibitors, indicating that there is a constant synthesis and hydrolysis of cAMP in subcellular compartments near the plasma membrane. Modulation of constitutively active adenylyl cyclase and PDE would allow for rapid control of cAMP-regulated processes such as cellular excitability.
The Journal of General Physiology 08/2001; 118(1):63-78. · 3.84 Impact Factor
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ABSTRACT: Cyclic nucleotide-gated channels contain four ligand-binding subunits, and they are directly activated by the binding of cGMP or cAMP. Channels with different combinations of subunits are known to have different sensitivities to the two nucleotides. However, the consequences of mixed occupancy by cGMP and cAMP are not well understood, and may have important implications for understanding the functions of these channels in different cell types. We studied the activation of homomeric and heteromeric retinal rod cyclic nucleotide-gated channels with the four ligand-binding sites occupied by different combinations of cGMP (a strong agonist) and cAMP (a weak agonist). Control of occupancy was obtained by covalently tethering different numbers of cGMP moieties using the photoaffinity analogue 8-p-azidophenacylthio-cGMP; the remaining sites were then saturated with cAMP, or cGMP, for comparison. The fractional current activated by cAMP increased dramatically as the number of tethered cGMP moieties increased. In homomeric channels comprised of the alpha subunit, cAMP became an effective agonist only after three of the four sites were occupied by tethered cGMP moieties. In contrast, in heteromeric channels comprised of two alpha and two beta subunits, cAMP caused significant activation after two sites were occupied by tethered cGMP moieties. In agreement with earlier work, a single residue on the beta subunit, N1201, accounted for much of the increased efficacy of cAMP on heteromeric channels. The results are consistent with significant interactions between subunits, including the two types of subunits in heteromeric channels.
Biochemistry 02/2001; 40(1):286-95. · 3.42 Impact Factor
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ABSTRACT: Cyclic AMP is a ubiquitous second messenger that coordinates diverse cellular functions. Current methods for measuring cAMP lack both temporal and spatial resolution, leading to the pervasive notion that, unlike Ca(2+), cAMP signals are simple and contain little information. Here we show the development of adenovirus-expressed cyclic nucleotide-gated channels as sensors for cAMP. Homomultimeric channels composed of the olfactory alpha subunit responded rapidly to jumps in cAMP concentration, and their cAMP sensitivity was measured to calibrate the sensor for intracellular measurements. We used these channels to detect cAMP, produced by either heterologously expressed or endogenous adenylyl cyclase, in both single cells and cell populations. After forskolin stimulation, the endogenous adenylyl cyclase in C6-2B glioma cells produced high concentrations of cAMP near the channels, yet the global cAMP concentration remained low. We found that rapid exchange of the bulk cytoplasm in whole-cell patch clamp experiments did not prevent the buildup of significant levels of cAMP near the channels in human embryonic kidney 293 (HEK-293) cells expressing an exogenous adenylyl cyclase. These results can be explained quantitatively by a cell compartment model in which cyclic nucleotide-gated channels colocalize with adenylyl cyclase in microdomains, and diffusion of cAMP between these domains and the bulk cytosol is significantly hindered. In agreement with the model, we measured a slow rate of cAMP diffusion from the whole-cell patch pipette to the channels (90% exchange in 194 s, compared with 22-56 s for substances that monitor exchange with the cytosol). Without a microdomain and restricted diffusional access to the cytosol, we are unable to account for all of the results. It is worth noting that in models of unrestricted diffusion, even in extreme proximity to adenylyl cyclase, cAMP does not reach high enough concentrations to substantially activate PKA or cyclic nucleotide-gated channels, unless the entire cell fills with cAMP. Thus, the microdomains should facilitate rapid and efficient activation of both PKA and cyclic nucleotide-gated channels, and allow for local feedback control of adenylyl cyclase. Localized cAMP signals should also facilitate the differential regulation of cellular targets.
The Journal of General Physiology 09/2000; 116(2):147-61. · 3.84 Impact Factor
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Methods in Enzymology 02/2000; 315:755-72. · 2.04 Impact Factor
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ABSTRACT: Retinal rod cyclic nucleotide-gated channels are composed of alpha and beta subunits. We have explored possible subunit arrangements by expressing tandemly linked dimers of both subunits and examining their responses to three different modulating agents. Channels formed from either alpha-beta or beta-alpha heterodimers had functional properties similar to those formed from coexpressed alpha and beta monomers and to native channels. These results point to an alpha-beta-alpha-beta arrangement. To ensure that heterodimers had not flipped around, we coexpressed alpha-alpha dimers with an excess of either beta monomers or beta-beta dimers. Our data indicate that heteromultimers do not form efficiently in an alpha-alpha-beta-beta arrangement. Thus, we propose that native rod cyclic nucleotide-gated channels are arranged with like subunits diagonally opposed: alpha-beta-alpha-beta.
Proceedings of the National Academy of Sciences 02/2000; 97(2):895-900. · 9.68 Impact Factor
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ABSTRACT: Cyclic nucleotide-gated channels contain four subunits, each with a C-terminal binding site for cGMP or cAMP. The dose-response relation for activation is usually fit with the Hill equation, I/I(max) = [cGMP]n/([cGMP]n + K(1/2)n, where I/I(max) is the fraction of maximal current, K(1/2) is the concentration of cGMP that gives a half-maximal current, and n is the Hill coefficient, taken as the minimum number of ligands required for significant activation. The dose-response relations in multichannel patches are often fit with Hill coefficients of </=2.0, even though other lines of evidence indicate that these channels contain four binding sites and that the binding of three or four ligands is required for significant opening. We have measured dose-response relations for a large number of single cyclic nucleotide-gated channels composed of the bovine rod alpha subunit. We find that the single-channel Hill coefficient is consistently higher than 2.5, with an average of 3.0 +/- 0.37 over 16 patches. In both multichannel and single-channel patches, large variations in K(1/2) have been observed, and are thought to arise from modifications such as phosphorylation. Here we show that mixtures of single channels with high Hill coefficients and variable K(1/2) values will give rise to shallow macroscopic dose-response relations with anomalously low Hill coefficients. This is because activation occurs over a broad range of cGMP concentrations. Thus, dose-response relations from multichannel patches should be interpreted with caution, particularly when detailed mechanistic issues such as cooperativity are being investigated.
Biochemistry 09/1999; 38(33):10642-8. · 3.42 Impact Factor
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ABSTRACT: Cyclic nucleotide-gated channels contain four subunits, each with a binding site for cGMP or cAMP in the cytoplasmic COOH-terminal domain. Previous studies of the kinetic mechanism of activation have been hampered by the complication that ligands are continuously binding and unbinding at each of these sites. Thus, even at the single channel level, it has been difficult to distinguish changes in behavior that arise from a channel with a fixed number of ligands bound from those that occur upon the binding and unbinding of ligands. For example, it is often assumed that complex behaviors like multiple conductance levels and bursting occur only as a consequence of changes in the number of bound ligands. We have overcome these ambiguities by covalently tethering one ligand at a time to single rod cyclic nucleotide-gated channels (Ruiz, ML., and J.W. Karpen. 1997. Nature. 389:389-392). We find that with a fixed number of ligands locked in place the channel freely moves between three conductance states and undergoes bursting behavior. Furthermore, a thorough kinetic analysis of channels locked in doubly, triply, and fully liganded states reveals more than one kinetically distinguishable state at each conductance level. Thus, even when the channel contains a fixed number of bound ligands, it can assume at least nine distinct states. Such complex behavior is inconsistent with simple concerted or sequential allosteric models. The data at each level of liganding can be successfully described by the same connected state model (with different rate constants), suggesting that the channel undergoes the same set of conformational changes regardless of the number of bound ligands. A general allosteric model, which postulates one conformational change per subunit in both the absence and presence of ligand, comes close to providing enough kinetically distinct states. We propose an extension of this model, in which more than one conformational change per subunit can occur during the process of channel activation.
The Journal of General Physiology 07/1999; 113(6):873-95. · 3.84 Impact Factor
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ABSTRACT: Previous studies have established that Ca2+-sensitive adenylyl cyclases, whether endogenously or heterologously expressed, are preferentially regulated by capacitative Ca2+ entry, compared with other means of elevating cytosolic Ca2+ (Chiono, M., Mahey, R., Tate, G., and Cooper, D. M. F. (1995) J. Biol. Chem. 270, 1149-1155; Fagan, K. A., Mahey, R., and Cooper, D. M. F. (1996) J. Biol. Chem. 271, 12438-12444; Fagan, K. A., Mons, N., and Cooper, D. M. F. (1998) J. Biol. Chem. 273, 9297-9305). These findings led to the suggestion that adenylyl cyclases and capacitative Ca2+ entry channels were localized in the same functional domain of the plasma membrane. In the present study, we have asked whether a heterologously expressed Ca2+-permeable channel could regulate the Ca2+-inhibitable adenylyl cyclase of C6-2B glioma cells. The cDNA coding for the rat olfactory cyclic nucleotide-gated channel was inserted into an adenovirus construct to achieve high levels of expression. Electrophysiological measurements confirmed the preservation of the properties of the expressed olfactory channel. Stimulation of the channel with cGMP analogs yielded a robust elevation in cytosolic Ca2+, which was associated with an inhibition of cAMP accumulation, comparable with that elicited by capacitative Ca2+ entry. These findings not only extend the means whereby Ca2+-sensitive adenylyl cyclases may be regulated, they also suggest that in tissues where they co-exist, cyclic nucleotide-gated channels and Ca2+-sensitive adenylyl cyclases may reciprocally modulate each other's activity.
Journal of Biological Chemistry 05/1999; 274(18):12445-53. · 4.77 Impact Factor
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ABSTRACT: One approach to drug design involves determination of the structure of binding sites on target proteins to provide templates for ligand construction. Alternatively, random combinations of chemical groups can be used to generate diverse molecules for screening in the search for effective compounds. Here we report a strategy for developing potent ligands for proteins with multiple binding sites, which combines elements of both approaches: 'polymer-linked ligand dimers', in which two ligands are joined by a polymer chain of variable length. We find that polymer-linked ligand dimers containing two cyclic GMP moieties are up to a thousand times more potent than cyclic GMP in activating cyclic-nucleotide-gated channels and cGMP-dependent protein kinase. Each target protein responds optimally to a polymer-linked ligand dimer with a different average polymer length, even though their cyclic-nucleotide-binding sites are conserved. The tuning of polymer-linked ligand dimers indicates that each protein has a unique spacing of binding sites and provides an estimate of the distance between these sites. As optimal ligands are selected empirically, the polymer-linked ligand dimer strategy enables potent and selective agents to be identified without requiring previous structural information about the target proteins.
Nature 11/1998; 395(6703):710-3. · 36.28 Impact Factor
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Advances in second messenger and phosphoprotein research 02/1998; 32:23-51.
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ABSTRACT: Cyclic nucleotide-gated (CNG) channels are directly activated by the binding of several ligands. For these channels as well as for other allosteric proteins, the functional effects of each ligand-binding event have been difficult to assess because ligands continuously bind and unbind at each site. Furthermore, in retinal rod photoreceptors the low cytoplasmic concentration of cyclic GMP means that channels exist primarily in partially liganded states, so it is important to determine how such channels behave. Previous studies of single channels have suggested that they occasionally open to subconducting states at low cGMP, but the significance of these states and how they arise is poorly understood. Here we combine the high resolution of single-channel recording with the use of a photoaffinity analogue of cGMP that tethers cGMP moieties covalently to their binding sites to show single retinal CNG channels can be effectively locked in four distinct ligand-bound states. Our results indicate that channels open more than they would spontaneously when two ligands are bound (approximately 1% of the maximum current), significantly more with three ligands bound (approximately 33%), and open maximally with four ligands bound. In each ligand-bound state, channels opened to two or three different conductance states. These findings place strong constraints on the activation mechanism of CNG channels.
Nature 10/1997; 389(6649):389-92. · 36.28 Impact Factor
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J W Karpen
Biophysical Journal 04/1997; 72(3):986-8. · 3.65 Impact Factor
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ABSTRACT: Ion channels gated by the binding of multiple ligands play a critical role in synaptic transmission and sensory transduction. It has been difficult to resolve the contribution of individual binding events to channel gating because ligands are continuously binding and unbinding at each site. In examining the allosteric mechanism of retinal rod cGMP-gated channels, we have circumvented this problem by making use of a cGMP derivative, 8-p-azidophenacylthio-cGMP (APT-cGMP), that can be covalently tethered to the binding sites in the presence of long-wavelength UV light. In excised membrane patches, a population of channels was isolated that contained covalently-attached ligands at all but one site. Activation of these channels by cGMP revealed a previously unknown heterogeneity in the ligand-binding sites. The dose-response relations were much shallower than predicted by single-site activation models, but were well described by models in which there are two populations of sites, in roughly equal proportion, that bind cGMP with apparent affinities that differ by a factor of 25. The two apparent affinities, incorporated into a four-site model of the channel, provided an accurate description of the patch's original dose-response relation. A comparison of results on native and expressed channels suggests that the heterogeneity in the native channel arises at least in part from the presence of two different cGMP-binding subunits.
The Journal of General Physiology 03/1996; 107(2):169-81. · 3.84 Impact Factor
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ABSTRACT: Ion channels from retinal rods and a variety of other cells are directly gated by cyclic nucleotides. The rod channel is known to contain a 63-kDa subunit, and there is molecular genetic evidence for the existence, in human retina, of a second subunit with a deduced molecular mass of about 100 kDa. When purified from bovine rods, the channel consists of the 63-kDa subunit and a 240-kDa associated protein that has been shown recently to contain a version of the cloned second subunit as part of a larger complex. We had previously shown that a photoaffinity analog of cGMP, 8-(p-azidophenacylthio)-[32P]cGMP, specifically labels both the 63- and 240-kDa proteins. Here the analog was used to identify cGMP-binding regions and amino acid contact points within these proteins. The specific labeling of the 63-kDa subunit was localized to a 66 amino acid fragment (Tyr-515-Met-580) that is contained entirely within a 110 amino acid region proposed to be the cGMP-binding site on the basis of homology with other cyclic nucleotide-binding proteins. Within this fragment, amino acid residues Val-524, Val-525, and Ala-526 were found to contain label. These residues are part of a larger hydrophobic cluster that appears to line the binding pocket. The results also indicate that the 240-kDa protein contains a similar cGMP-binding site. Sequencing of a specifically labeled 8-kDa fragment through 16 amino acid residues indicated that the fragment was derived from the portion of the 240-kDa complex that contains the second subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 08/1995; 34(26):8365-70. · 3.42 Impact Factor
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ABSTRACT: Adenylyl cyclase is the prototypical second messenger generator. Nearly all of the eight cloned adenylyl cyclases are regulated by one or other arm of the phospholipase C pathway. Functional and ultrastructural investigations have shown that adenylyl cyclases are intimately associated with sites of calcium ion entry into the cell. Oscillations in cellular cyclic AMP levels are predicted to arise because of feedback inhibition of adenylyl cyclase by Ca2+. Such findings inextricably intertwine cellular signalling by cAMP and internal Ca2+ and extend the known regulatory modes available to cAMP.
Nature 04/1995; 374(6521):421-4. · 36.28 Impact Factor
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ABSTRACT: Analogs of cGMP bearing diverse substituents at the C8 position of the guanine ring system have been shown to activate the cGMP-activated channel of retinal rods at concentrations lower than cGMP itself. In an effort to understand this behavior, we synthesized eight novel C8-substituted derivatives and tested their ability to activate channels in excised patches from salamander rod outer segments. We express the effectiveness of each analog as a ratio (in brackets) of the concentration required to open half of the channels in a patch to that required of 8-Br-cGMP, previously shown to be about 10 times more effective than cGMP. Five of the derivatives contained a thio substitution at C8: n-propylthio-cGMP [0.61], sulfoethylthio-cGMP [0.90], carboxyethylthio-cGMP [0.97], aminoethylthio-cGMP [2.8], and (trimethylamino)ethylthio-cGMP [8.5]. Three of the derivatives contained an amino substitution at C8: carboxyethylamino-cGMP [22], n-propylamino-cGMP [25], and aminoethylamino-cGMP [230]. The results indicate that thio-substitution at C8 produces more effective analogs than does amino-substitution, regardless of the chemical nature of the terminal functional group. Derivatives containing neutral and apolar tails opened channels at much lower concentrations than their positively-charged counterparts with the same C8 substituent. Analogs having negatively-charged tails were also more effective than those with positive charge but not quite as effective as those with neutral tails.(ABSTRACT TRUNCATED AT 250 WORDS)
Biochemistry 10/1993; 32(38):10089-95. · 3.42 Impact Factor
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ABSTRACT: Cyclic nucleotide-gated ion channels play a key role in visual excitation and a variety of other signaling pathways. The photoaffinity probe 8-p-azidophenacylthio-cGMP (APT-cGMP) has been developed for structural and functional studies of the cGMP-activated channel of retinal rod outer segments. Using this analog, we have demonstrated both specific labeling of the channel in a partially purified biochemical preparation from bovine rod outer segments and permanent activation of the channel current in excised membrane patches from salamander outer segments. After UV illumination, a 32P-labeled version of APT-cGMP was shown by SDS/PAGE and autoradiography to be covalently attached to the 63-kDa channel subunit. This incorporation was significantly reduced by 8-Br-cGMP but was not reduced by 5'-GMP. In patch-clamp experiments APT-cGMP was a potent activator of the channel; APT-cGMP typically opened half of the channels in a patch at a 10-fold lower concentration than cGMP. Exposure of membrane patches to UV light in the presence of APT-cGMP resulted in a persistent current observed in the absence of bath-applied nucleotide. This current increased with repeated exposure of the patch to both UV light and fresh APT-cGMP, approaching the maximum current originally evoked by saturating (500 microM) cGMP. At this point, addition of 500 microM cGMP caused a negligible increase in current. The persistent current had several other properties expected of current through cGMP-activated channels: it was outwardly rectifying; outward current was blocked > 90% by 2 mM internal Mg2+, whereas inward current was blocked much less efficiently; a low concentration of cGMP caused a larger increase in current atop a half-maximal persistent current than it did originally. We conclude that the persistent current was caused by the covalent tethering of cGMP moieties to channel binding sites, resulting in irreversible channel activation. APT-cGMP should prove useful for further studies of these and similar cGMP-binding sites and in the identification of unknown cGMP-binding proteins.
Proceedings of the National Academy of Sciences 06/1993; 90(11):5369-73. · 9.68 Impact Factor
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ABSTRACT: The effects of divalent cations on the gating of the cGMP-activated channel, and the effects of gating on the movement of divalent cations in and out of the channel's pore were studied by recording macroscopic currents in excised membrane patches from salamander retinal rods. The fractional block of cGMP-activated Na+ currents by internal and external Mg2+ as well as internal Ca2+ was nearly independent of cGMP concentration. This indicates that Mg2+ and Ca2+ bind with similar affinity to open and closed states of the channel. In contrast, the efficiency of block by internal Cd2+ or Zn2+ increased in proportion to the fraction of open channels, indicating that these ions preferentially occupy open channels. The kinetics of block by internal Ni2+, which competes with Mg2+ but blocks more slowly, were found to be unaffected by the fraction of channels open. External Ni2+, however, blocked and unblocked much more rapidly when channels were mostly open. This suggests that within the pore a gate is located between the binding site(s) for ions and the extracellular mouth of the channel. Micromolar concentrations of the transition metal divalent cations Ni2+, Cd2+, Zn2+, and Mn2+ applied to the cytoplasmic surface of a patch potentiated the response to subsaturating concentrations of cGMP without affecting the maximum current induced by saturating cGMP. The concentration of cGMP that opened half the channels was often lowered by a factor of three or more. Potentiation persisted after the experimental chamber was washed with divalent-free solution and fresh cGMP was applied, indicating that it does not result from an interaction between divalent cations and cGMP in solution; 1 mM EDTA or isotonic MgCl2 reversed potentiation. Voltage-jump experiments suggest that potentiation results from an increase in the rate of cGMP binding. Lowering the ionic strength of the bathing solution enhanced potentiation, suggesting that it involves electrostatic interactions. The strong electrostatic effect on cGMP binding and absence of effect on ion permeation through open channels implies that the cGMP binding sites on the channel are well separated from the permeation pathway.
The Journal of General Physiology 02/1993; 101(1):1-25. · 3.84 Impact Factor
