Fraction of the dark current carried by Ca(2+) through cGMP-gated ion channels of intact rod and cone photoreceptors

The selectivity for Ca(2+) over Na(+), PCa/PNa, is higher in cGMP-gated (CNG) ion channels of retinal cone photoreceptors than in those of rods. To ascertain the physiological significance of this fact, we determined the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact rods and cones. We activated CNG channels by suddenly (<5 ms) increasing free 8Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+)-binding dye, Fura-2, also loaded into the cells. The ratio of changes in fura-2 fluorescence and the integral of the membrane current, under a restricted set of experimental conditions, is a direct measure of the fractional Ca(2+) flux. Under normal physiological salt concentrations, the fractional Ca(2+) flux is higher in CNG channels of cones than in those of rods, but it differs little among cones (or rods) of different species. Under normal physiological conditions and for membrane currents 相似文献   

Voltage-dependence of ion permeation in cyclic GMP-gated ion channels is optimized for cell function in rod and cone photoreceptors

The kinetics of the photocurrent in both rod and cone retinal photoreceptors are independent of membrane voltage over the physiological range (-30 to -65 mV). This is surprising since the photocurrent time course is regulated by the influx of Ca(2+) through cGMP-gated ion channels (CNG) and the force driving this flux changes with membrane voltage. To understand this paradigm, we measured Pf, the fraction of the cyclic nucleotide-gated current specifically carried by Ca(2+) in intact, isolated photoreceptors. To measure Pf we activated CNG channels by suddenly increasing free 8-Br-cGMP in the cytoplasm of rods or cones loaded with a caged ester of the cyclic nucleotide. Simultaneous with the uncaging flash, we measured the cyclic nucleotide-dependent changes in membrane current and fluorescence of the Ca(2+) binding dye, Fura-2, also loaded into the cells. We determined Pf under physiological solutions at various holding membrane voltages between -65 and -25 mV. Pf is larger in cones than in rods, but in both photoreceptor types its value is independent of membrane voltage over the range tested. This biophysical feature of the CNG channels offers a functional advantage since it insures that the kinetics of the phototransduction current are controlled by light, and not by membrane voltage. To explain our observation, we developed a rate theory model of ion permeation through CNG channels that assumes the existence of two ion binding sites within the permeation pore. To assign values to the kinetic rates in the model, we measured experimental I-V curves in membrane patches of rods and cones over the voltage range -90 to 90 mV in the presence of simple biionic solutions at different concentrations. We optimized the fit between simulated and experimental data. Model simulations describe well experimental photocurrents measured under physiological solutions in intact cones and are consistent with the voltage-independence of Pf, a feature that is optimized for the function of the channel in photoreceptors.     

In intact mammalian photoreceptors, Ca2+-dependent modulation of cGMP-gated ion channels is detectable in cones but not in rods

In the mammalian retina, cone photoreceptors efficiently adapt to changing background light intensity and, therefore, are able to signal small differences in luminance between objects and backgrounds, even when the absolute intensity of the background changes over five to six orders of magnitude. Mammalian rod photoreceptors, in contrast, adapt very little and only at intensities that nearly saturate the amplitude of their photoresponse. In search of a molecular explanation for this observation we assessed Ca2+-dependent modulation of ligand sensitivity in cyclic GMP-gated (CNG) ion channels of intact mammalian rods and cones. Solitary photoreceptors were isolated by gentle proteolysis of ground squirrel retina. Rods and cones were distinguished by whether or not their outer segments bind PNA lectin. We measured membrane currents under voltage-clamp in photoreceptors loaded with Diazo-2, a caged Ca2+ chelator, and fixed concentrations of 8Br-cGMP. At 600 nM free cytoplasmic Ca2+ the midpoint of the cone CNG channels sensitivity to 8BrcGMP, 8BrcGMPK1/2, is approximately 2.3 microM. The ligand sensitivity is less in rod than in cone channels. Instantly decreasing cytoplasmic Ca2+ to <30 nM activates a large inward membrane current in cones, but not in rods. Current activation arises from a Ca2+ -dependent modulation of cone CNG channels, presumably because of an increase in their affinity to the cyclic nucleotide. The time course of current activation is temperature dependent; it is well described by a single exponential process of approximately 480 ms time constant at 20-21 degrees C and 138 ms at 32 degrees C. The absence of detectable Ca2+-dependent CNG current modulation in intact rods, in view of the known channel modulation by calmodulin in-vitro, affirms the modulation in intact rods may only occur at low Ca2+ concentrations, those expected at intensities that nearly saturate the rod photoresponse. The correspondence between Ca2+ dependence of CNG modulation and the ability to light adapt suggest these events are correlated in photoreceptors.     

Time course and Ca(2+) dependence of sensitivity modulation in cyclic GMP-gated currents of intact cone photoreceptors

We determined the Ca(2+) dependence and time course of the modulation of ligand sensitivity in cGMP-gated currents of intact cone photoreceptors. In electro-permeabilized single cones isolated from striped bass, we measured outer segment current amplitude as a function of cGMP or 8Br-cGMP concentrations in the presence of various Ca(2+) levels. The dependence of current amplitude on nucleotide concentration is well described by the Hill function with values of K(1/2), the ligand concentration that half-saturates current, that, in turn, depend on Ca(2+). K(1/2) increases as Ca(2+) rises, and this dependence is well described by a modified Michaelis-Menten function, indicating that modulation arises from the interaction of Ca(2+) with a single site without apparent cooperativity. (Ca)K(m), the Michaelis-Menten constant for Ca(2+) concentration is 857 +/- 68 nM for cGMP and 863 +/- 51 for 8Br-cGMP. In single cones under whole-cell voltage clamp, we simultaneously measured changes in membrane current and outer segment free Ca(2+) caused by sudden Ca(2+) sequestration attained by uncaging diazo-2. In the presence of constant 8Br-cGMP, 15 micro, Ca(2+) concentration decrease was complete within 50 ms and membrane conductance was enhanced 2.33 +/- 0.95-fold with a mean time to peak of 1.25 +/- 0.23 s. We developed a model that assumes channel modulation is a pseudo-first-order process kinetically limited by free Ca(2+). Based on the experimentally measured changes in Ca(2+) concentration, model simulations match experimental data well by assigning the pseudo-first-order time constant a mean value of 0.40 +/- 0.14 s. Thus, Ca(2+)-dependent ligand modulation occurs over the concentration range of the normal, dark-adapted cone. Its time course suggests that its functional effects are important in the recovery of the cone photoresponse to a flash of light and during the response to steps of light, when cones adapt.     

Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit

Phototransduction relies on the precise balance of speed and sensitivity to achieve optimal performance. The cyclic nucleotide-gated (CNG) ion channels, with their Ca(2+) permeability, high sensitivity to changes in cytosolic cGMP, rapid gating kinetics, and Ca(2+)-calmodulin modulation, are beautifully optimized for their role in light detection. Many of these specializations come about from the heteromeric composition of the native channel, comprised of CNGA1 and CNGB1 subunits. However, the stoichiometry and arrangement of these subunits is unknown. Here we have used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channel in the surface membrane. We find, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. These results have implications for CNG channel function in particular and assembly of membrane proteins in general.     

The limit of photoreceptor sensitivity: molecular mechanisms of dark noise in retinal cones

Detection threshold in cone photoreceptors requires the simultaneous absorption of several photons because single photon photocurrent is small in amplitude and does not exceed intrinsic fluctuations in the outer segment dark current (dark noise). To understand the mechanisms that limit light sensitivity, we characterized the molecular origin of dark noise in intact, isolated bass single cones. Dark noise is caused by continuous fluctuations in the cytoplasmic concentrations of both cGMP and Ca(2+) that arise from the activity in darkness of both guanylate cyclase (GC), the enzyme that synthesizes cGMP, and phosphodiesterase (PDE), the enzyme that hydrolyzes it. In cones loaded with high concentration Ca(2+) buffering agents, we demonstrate that variation in cGMP levels arise from fluctuations in the mean PDE enzymatic activity. The rates of PDE activation and inactivation determine the quantitative characteristics of the dark noise power density spectrum. We developed a mathematical model based on the dynamics of PDE activity that accurately predicts this power spectrum. Analysis of the experimental data with the theoretical model allows us to determine the rates of PDE activation and deactivation in the intact photoreceptor. In fish cones, the mean lifetime of active PDE at room temperature is approximately 55 ms. In nonmammalian rods, in contrast, active PDE lifetime is approximately 555 ms. This remarkable difference helps explain why cones are noisier than rods and why cone photocurrents are smaller in peak amplitude and faster in time course than those in rods. Both these features make cones less light sensitive than rods.     

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.     

cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice

Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca²⁺ channel inositol 1,4,5-trisphosphate receptor 1 (IP₃R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP₃R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3^−/−/Nrl^−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP₃R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca²⁺ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency.     

cAMP modulates the excitability of immortalized H=hypothalamic (GT1) neurons via a cyclic nucleotide gated channel

GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca(2+)](i), as well as pulsatile release of GNRH: We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca(2+) inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca(2+) oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca(2+) signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca(2+) oscillations. The effects of Sp-cAMPS or NE on Ca(2+) signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca(2+) reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca(2+) oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.     

Functional activity of photoreceptor cyclic nucleotide-gated channels is dependent on the integrity of cholesterol- and sphingolipid-enriched membrane domains

Rod and cone photoreceptor cyclic nucleotide-gated (CNG) channels play pivotal roles in phototransduction. This work investigates the functional significance of photoreceptor CNG channel association with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The primary subunits of cone and rod CNG channels, CNGA3 and CNGA1, respectively, were heterologously expressed in HEK 293 cells, and channel activity was determined by ratiometric measurement of [Ca (2+)] i in response to cyclic guanosine monophosphate (cGMP) stimulation. CNGA3 was found to be largely insoluble following Triton X-100 extraction and cofractionationed with biochemically isolated membrane domains enriched in caveolin-1. Cofractionation of both natively expressed CNGA3 and CNGB1 (the modulatory subunit of the rod CNG channel) with the low buoyant density, caveolin-1-enriched membranes was also confirmed in mouse retinas. The functional significance of this association was established by the observed negative effects of depletion of raft lipids on the channel activity. Treatment with the cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), significantly inhibited CNGA3 and CNGA1 activation in response to cGMP stimulation. MCD treatment lowered cellular cholesterol levels by approximately 45% without altering fatty acid composition, suggesting that the inhibition of channel activity by MCD treatment is not due to perturbation of other membrane lipids. Treatment with the sphingolipid biosynthesis inhibitor myriocin resulted in impaired activation and cytosolic redistribution of CNGA3, suggesting that the integrity of the membrane domains is critical for the channel cellular processing and plasma membrane localization. This study demonstrates the association of photoreceptor CNG channels with membrane domains enriched in raft lipids and indicates, for the first time, that raft lipids modulate the plasma membrane localization and functional activity of photoreceptor CNG channels.     

Natriuretic peptides and nitric oxide stimulate cGMP synthesis in different cellular compartments

Cyclic nucleotide-gated (CNG) channels are a family of ion channels activated by the binding of cyclic nucleotides. Endogenous channels have been used to measure cyclic nucleotide signals in photoreceptor outer segments and olfactory cilia for decades. Here we have investigated the subcellular localization of cGMP signals by monitoring CNG channel activity in response to agonists that activate either particulate or soluble guanylyl cyclase. CNG channels were heterologously expressed in either human embryonic kidney (HEK)-293 cells that stably overexpress a particulate guanylyl cyclase (HEK-NPRA cells), or cultured vascular smooth muscle cells (VSMCs). Atrial natriuretic peptide (ANP) was used to activate the particulate guanylyl cyclase and the nitric oxide donor S-nitroso-n-acetylpenicillamine (SNAP) was used to activate the soluble guanylyl cyclase. CNG channel activity was monitored by measuring Ca2+ or Mn2+ influx through the channels using the fluorescent dye, fura-2. We found that in HEK-NPRA cells, ANP-induced increases in cGMP levels activated CNG channels in a dose-dependent manner (0.05-10 nM), whereas SNAP (0.01-100 microM) induced increases in cGMP levels triggered little or no activation of CNG channels (P < 0.01). After pretreatment with 100 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, ANP-induced Mn2+ influx through CNG channels was significantly enhanced, while SNAP-induced Mn2+ influx remained small. In contrast, we found that in the presence of IBMX, both 1 nM ANP and 100 microM SNAP triggered similar increases in total cGMP levels. We next sought to determine if cGMP signals are compartmentalized in VSMCs, which endogenously express particulate and soluble guanylyl cyclase. We found that 10 nM ANP induced activation of CNG channels more readily than 100 muM SNAP; whereas 100 microM SNAP triggered higher levels of total cellular cGMP accumulation. These results suggest that cGMP signals are spatially segregated within cells, and that the functional compartmentalization of cGMP signals may underlie the unique actions of ANP and nitric oxide.     

Three amino acids in the C-linker are major determinants of gating in cyclic nucleotide-gated channels.

The activation of cyclic nucleotide-gated (CNG) channels is a complex process comprising the initial ligand binding and a consecutive allosteric transition from a closed to an open configuration. The cone and olfactory CNG channels differ considerably in cyclic nucleotide affinity and efficacy. In each channel, the cyclic nucleotide-binding site is connected to the last transmembrane segment of the channel by a linker peptide (C-linker) of approximately 90 amino acids. Here we report that replacement of three amino acids in the cone C-linker by the corresponding amino acids of the olfactory channel (I439V, D481A and D494S) profoundly enhanced the cAMP efficacy and increased the affinities for cAMP and cGMP. Unlike the wild-type cone channel, the mutated channel exhibited similar single-channel kinetics for both cGMP and cAMP, explaining the increase in cAMP efficacy. We thus conclude that the identified amino acids are major determinants of channel gating.     

The cellular and molecular basis of odor adaptation

An important recent advance in the understanding of odor adaptation has come from the discovery that complex mechanisms of odor adaptation already take place at the earliest stage of the olfactory system, in the olfactory cilia. At least two rapid forms and one persistent form of odor adaptation coexist in vertebrate olfactory receptor neurons. These three different adaptation phenomena can be dissected on the basis of their different onset and recovery time courses and their pharmacological properties, indicating that they are controlled, at least in part, by separate molecular mechanisms. Evidence is provided for the involvement of distinct molecular steps in these forms of odor adaptation, including Ca(2+) entry through cyclic nucleotide-gated (CNG) channels, Ca(2+)-dependent CNG channel modulation, Ca(2+)/calmodulin kinase II-dependent attenuation of adenylyl cyclase, and the activity of the carbon monoxide/cyclic GMP second messenger system. Identification of these molecular steps may help to elucidate how the olfactory system extracts temporal and intensity information and to which extent odor perception is influenced by the different mechanisms underlying adaptation.     

Odorant inhibition of the olfactory cyclic nucleotide-gated channel with a native molecular assembly

Human olfaction comprises the opposing actions of excitation and inhibition triggered by odorant molecules. In olfactory receptor neurons, odorant molecules not only trigger a G-protein-coupled signaling cascade but also generate various mechanisms to fine tune the odorant-induced current, including a low-selective odorant inhibition of the olfactory signal. This wide-range olfactory inhibition has been suggested to be at the level of ion channels, but definitive evidence is not available. Here, we report that the cyclic nucleotide-gated (CNG) cation channel, which is a key element that converts odorant stimuli into electrical signals, is inhibited by structurally unrelated odorants, consistent with the expression of wide-range olfactory inhibition. Interestingly, the inhibitory effect was small in the homo-oligomeric CNG channel composed only of the principal channel subunit, CNGA2, but became larger in channels consisting of multiple types of subunits. However, even in the channel containing all native subunits, the potency of the suppression on the cloned CNG channel appeared to be smaller than that previously shown in native olfactory neurons. Nonetheless, our results further showed that odorant suppressions are small in native neurons if the subsequent molecular steps mediated by Ca(2+) are removed. Thus, the present work also suggests that CNG channels switch on and off the olfactory signaling pathway, and that the on and off signals may both be amplified by the subsequent olfactory signaling steps.     

Cyclic GMP-gated CNG channels function in Sema3A-induced growth cone repulsion

Cyclic nucleotide-gated channels (CNGCs) transduce external signals required for sensory processes, e.g., photoreception, olfaction, and taste. Nerve growth cone guidance by diffusible attractive and repulsive molecules is regulated by differential growth cone Ca2+ signaling. However, the Ca2+-conducting ion channels that transduce guidance molecule signals are largely unknown. We show that rod-type CNGC-like channels function in the repulsion of cultured Xenopus spinal neuron growth cones by Sema3A, which triggers the production of the cGMP that activates the Xenopus CNGA1 (xCNGA1) subunit-containing channels in interneurons. Downregulation of xCNGA1 or overexpression of a mutant xCNGA1 incapable of binding cGMP abolished CNG currents and converted growth cone repulsion to attraction in response to Sema3A. We also show that Ca2+ entry through xCNGCs is required to mediate the repulsive Sema3A signal. These studies extend our knowledge of the function of CNGCs by demonstrating their requirement for signal transduction in growth cone guidance.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Structure of the SthK Carboxy-Terminal Region Reveals a Gating Mechanism for Cyclic Nucleotide-Modulated Ion Channels

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the K_v1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.     

Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns

The selectivity of Ca2+ over Na+ is approximately 3.3-fold larger in cGMP-gated channels of cone photoreceptors than in those of rods when measured under saturating cGMP concentrations, where the probability of channel opening is 85-90%. Under physiological conditions, however, the probability of opening of the cGMP-gated channels ranges from its largest value in darkness of 1-5% to essentially zero under continuous, bright illumination. We investigated the ion selectivity of cGMP-gated channels as a function of cyclic nucleotide concentration in membrane patches detached from the outer segments of rod and cone photoreceptors and have found that ion selectivity is linked to gating. We determined ion selectivity relative to Na+ (PX/PNa) from the value of reversal potentials measured under ion concentration gradients. The selectivity for Ca2+ over Na+ increases continuously as the probability of channel opening rises. The dependence of PCa/PNa on cGMP concentration, in both rods and cones, is well described by the same Hill function that describes the cGMP dependence of current amplitude. At the cytoplasmic cGMP concentrations expected in dark-adapted intact photoreceptors, PCa/PNa in cone channels is approximately 7.4-fold greater than that in rods. The linkage between selectivity and gating is specific for divalent cations. The selectivity of Ca2+ and Sr2+ changes with cGMP concentration, but the selectivity of inorganic monovalent cations, Cs+ and NH4+, and organic cations, methylammonium+ and dimethylammonium+, is invariant with cGMP. Cyclic nucleotide-gated channels in rod photoreceptors are heteromeric assemblies of alpha and beta subunits. The maximal PCa/PNa of channels formed from alpha subunits of bovine rod channels is less than that of heteromeric channels formed from alpha and beta subunits. In addition, Ca2+ is a more effective blocker of channels formed by alpha subunits than of channels formed by alpha and beta subunits. The cGMP-dependent shift in divalent cation selectivity is a property of alphabeta channels and not of channels formed from alpha subunits alone.     

In retinal cones, membrane depolarization in darkness activates the cGMP-dependent conductance. A model of Ca homeostasis and the regulation of guanylate cyclase

We measured outer segment currents under voltage clamp in solitary, single cone photoreceptors isolated from the retina of striped bass. In darkness, changes in membrane voltage to values more positive than 10 mV activate a time- and voltage-dependent outward current in the outer segment. This dark, voltage-activated current (DVAC) increases in amplitude with a sigmoidal time course up to a steady-state value, reached in 0.75-1.5 s. DVAC is entirely suppressed by light, and its current-voltage characteristics and reversal potential are the same as those of the light-sensitive currents. DVAC, therefore, arises from the activation by voltage in the dark of the light-sensitive, cGMP-gated channels of the cone outer segment. Since these channels are not directly gated by voltage, we explain DVAC as arising from a voltage- dependent decrease in cytoplasmic Ca concentration that, in turn, activates only guanylate cyclase and results in net synthesis of cGMP. This explanation is supported by the finding that the Ca buffer BAPTA, loaded into the cytoplasm of the cone outer segment, blocks DVAC. To link a decrease in cytoplasmic Ca concentration to the synthesis of cGMP and the characteristics of DVAC, we develop a quantitative model that assumes cytoplasmic Ca concentration can be continuously calculated from the balance between passive Ca influx via the cGMP- gated channel and its active efflux via a Na/Ca,K exchanger, and that further assumes that guanylate cyclase is activated by decreasing cytoplasmic Ca concentration with characteristics identical to those described for the enzyme in rods. The model successfully simulates experimental data by adjusting the Ca conductance of the cGMP-gated channels as a function of voltage and the Ca buffering power of the cytoplasm. This success suggests that the activity of guanylate cyclase in cone outer segments is indistinguishable from that in rods.