Gating of cyclic nucleotide-gated (CNGA1) channels by cGMP jumps and depolarizing voltage steps

We expressed rod-type homotetrameric cyclic nucleotide-gated (CNGA1) channels in Xenopus oocytes and studied activation by photolysis-induced jumps of the 3',5'-cyclic guanosine monophosphate (cGMP) concentration and by voltage steps. cGMP jumps to increasing concentrations up to the EC50 value of 46.5 microM decelerate the activation gating, indicative that even at concentrations of cGMP < EC50 binding is not rate limiting. Above the EC50 value, activation by cGMP jumps is again accelerated to the higher concentrations. At the same cGMP concentration, the speed of the activation gating by depolarizing voltage steps is roughly similar to that by cGMP jumps. Permeating ions passing the pore more slowly (Rb+ > K+ > Na+) slow down the activation time course. At the single-channel level, cGMP jumps to high concentrations cause openings directly to the main open level without passing sublevels. From these results it is concluded that at both low and high cGMP the gating of homotetrameric CNGA1 channels is not rate-limited by the cGMP binding but by conformational changes of the channel which are voltage dependent and include movements in the pore region.     

Thermodynamics of activation gating in olfactory-type cyclic nucleotide-gated (CNGA2) channels

Olfactory-type cyclic nucleotide-gated (CNG) ion channels open by the binding of cyclic nucleotides to a binding domain in the C-terminus. Employing the Eyring rate theory, we performed a thermodynamic analysis of the activation gating in homotetrameric CNGA2 channels. Lowering the temperature shifted the concentration-response relationship to lower concentrations, resulting in a decrease of both the enthalpy ΔH and entropy ΔS upon channel opening, suggesting that the order of an open CNGA2 channel plus its environment is higher than that of the closed channel. Activation time courses induced by cGMP concentration jumps were used to study thermodynamics of the transition state. The activation enthalpies ΔH^‡ were positive at all cGMP concentrations. In contrast, the activation entropy ΔS^‡ was positive at low cGMP concentrations and became then negative at increasing cGMP concentrations. The enthalpic and entropic parts of the activation energies approximately balance each other at all cGMP concentrations, leaving the free enthalpy of activation in the range between 19 and 21 kcal/mol. We conclude that channel activation proceeds through different pathways at different cGMP concentrations. Compared to the unliganded channel, low cGMP concentrations generate a transitional state of lower order whereas high cGMP concentrations generate a transitional state of higher order.     

Stoichiometry and arrangement of subunits in rod cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) ion channels mediate the response to light in retinal rods. They are tetramers of two homologous subunits (alpha and beta), each of which is essential for the function of the channels in vivo. We have investigated the stoichiometry and arrangement of these two subunits to determine how they come together within an individual channel complex. We exploited the very specific geometric and spatial requirements for forming a high-affinity Ni2+-binding site to examine the number and relative positions of the subunits. We found that only an order of alpha/alpha/beta/beta could account qualitatively and quantitatively for the observed intersubunit coordination of Ni2+ in wild-type and mutant alpha/beta channels. Furthermore, our results suggest a structural dimerization among like subunits, at least at the level of the Ni2+-binding site.     

Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitivity and pore properties of heteromeric channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits.     

Regulation of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are found in several cell types, and are best studied in photoreceptors and olfactory sensory neurons. There, CNG channels are gated by the second messengers of the visual and olfactory signalling cascades, cGMP and cAMP respectively, and operate as transduction channels generating the stimulus-induced receptor potentials. In visual and olfactory sensory cells CNG channels conduct cationic currents. Calcium can contribute a large fraction of this current, and calcium influx serves a modulatory role in CNG-channel mediated signal transduction. There have been recent developments in our understanding of how the regulation of CNG channels contributes to the physiological properties of photoreceptors and olfactory sensory cells, and in particular on the role of calcium-mediated feedback.     

Loss of CNGB1 protein leads to olfactory dysfunction and subciliary cyclic nucleotide-gated channel trapping

Olfactory receptor neurons (ORNs) employ a cyclic nucleotide-gated (CNG) channel to generate a receptor current in response to an odorant-induced rise in cAMP. This channel contains three types of subunits, the principal CNGA2 subunit and two modulatory subunits (CNGA4 and CNGB1b). Here, we have analyzed the functional relevance of CNGB1 for olfaction by gene targeting in mice. Electro-olfactogram responses of CNGB1-deficient (CNGB1-/-) mice displayed a reduced maximal amplitude and decelerated onset and recovery kinetics compared with wild-type mice. In a behavioral test, CNGB1-/- mice exhibited a profoundly decreased olfactory performance. Electrophysiological recordings revealed that ORNs of CNGB1-/- mice weakly expressed a CNG current with decreased cAMP sensitivity, very rapid flicker-gating behavior and no fast modulation by Ca2+-calmodulin. Co-immunoprecipitation confirmed the presence of a CNGA2/CNGA4 channel in the olfactory epithelium of CNGB1-/- mice. This CNGA2/CNGA4 channel was targeted to the plasma membrane of olfactory knobs, but failed to be trafficked into olfactory cilia. Interestingly, we observed a similar trafficking defect in mice deficient for the CNGA4 subunit. In conclusion, these results demonstrate that CNGB1 has a dual function in vivo. First, it endows the olfactory CNG channel with a variety of biophysical properties tailored to the specific requirements of olfactory transduction. Second, together with the CNGA4 subunit, CNGB1 is needed for ciliary targeting of the olfactory CNG channel.     

Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit

Progressive cone dystrophies are a genetically heterogeneous group of disorders characterized by early deterioration of visual acuity and color vision, together with psychophysical and electrophysiological evidence of abnormal cone function and cone degeneration. Recently, three mutations in the gene encoding the CNGA3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels have been linked to progressive cone dystrophy in humans. To investigate the functional consequences of these mutations, we expressed mutant human CNGA3 subunits in Xenopus oocytes, alone or together with human CNGB3, and studied these channels using patch-clamp recording. Compared with wild-type channels, homomeric and heteromeric channels containing CNGA3-N471S or CNGA3-R563H subunits exhibited an increase in apparent affinity for cGMP and an increase in the relative agonist efficacy of cAMP compared with cGMP. In contrast, R277C subunits did not form functional homomeric or heteromeric channels. Cell surface expression levels, determined using confocal microscopy of green fluorescent protein-tagged subunits and patch-clamp recording, were significantly reduced for both R563H and R277C but unchanged for N471S. Overall, these results suggest that the plasma membrane localization and gating properties of cone CNG channels are altered by progressive cone dystrophy-associated mutations, providing evidence that supports the pathogenicity of these mutations. phosphodiesterase     

Molecular mechanisms of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are highly specialized to carry out their unique role in cell signaling. Significant progress has been made in the last several years determining the molecular mechanisms for these specializations. The activation of the channels begins with the binding of cyclic nucleotide to a domain in the carboxyl terminal region. This binding, in turn, produces an induced fit of the protein that involves a movement of the C-helix portion of the binding domain. The induced fit of the binding domain is coupled to an allosteric conformational change that opens the channel pore. The pore is formed primarily from the sequence between the S5 and S6 segments. A single glutamic acid in the pore represents the binding site for multiple monovalent cations, the blocking site for external divalent cations, and the site for the effect of protons on permeation.     

Functionally important calmodulin-binding sites in both NH2- and COOH-terminal regions of the cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit

Whereas an important aspect of sensory adaptation in rod photoreceptors and olfactory receptor neurons is thought to be the regulation of cyclic nucleotide-gated (CNG) channel activity by calcium-calmodulin (Ca2+-CaM), it is not clear that cone photoreceptor CNG channels are similarly modulated. Cone CNG channels are composed of at least two different subunit types, CNGA3 and CNGB3. We have investigated whether calmodulin modulates the activity of these channels by direct binding to the CNGB3 subunit. Heteromeric channels were formed by co-expression of human CNGB3 with human CNGA3 subunits in Xenopus oocytes; CNGB3 subunits conferred sensitivity to regulation by Ca2+-CaM, whereas CaM regulation of homomeric CNGA3 channels was not detected. To explore the mechanism underlying this regulation, we localized potential CaM-binding sites in both NH2- and COOH-terminal cytoplasmic domains of CNGB3 using gel-overlay and glutathione S-transferase pull-down assays. For both sites, binding of CaM depended on the presence of Ca2+. Individual deletions of either CaM-binding site in CNGB3 generated channels that remained sensitive to regulation by Ca2+-CaM, but deletion of both together resulted in heteromeric channels that were not modulated. Thus, both NH2- and COOH-terminal CaM-binding sites in CNGB3 are functionally important for regulation of recombinant cone CNG channels. These studies suggest a potential role for direct binding and unbinding of Ca2+-CaM to human CNGB3 during cone photoreceptor adaptation and recovery.     

Stoichiometry and assembly of olfactory cyclic nucleotide-gated channels

Native ion channels are precisely tuned to their physiological role in neuronal signaling. This tuning frequently involves the controlled assembly of heteromeric channels comprising multiple types of subunits. Cyclic nucleotide-gated (CNG) channels of olfactory neurons are tetramers and require three types of subunits, CNGA2, CNGA4, and CNGB1b, to exhibit properties necessary for olfactory transduction. Using fluorescently tagged subunits and fluorescence resonance energy transfer (FRET), we find the subunit composition of heteromeric olfactory channels in the surface membrane is fixed, with 2:1:1 CNGA2:CNGA4:CNGB1b. Furthermore, when expressed individually with CNGA2, CNGA4 and CNGB1b subunits were still present in only a single copy and, when expressed alone, did not self-assemble. These results suggest that the precise assembly of heteromeric olfactory channels results from a mechanism where CNGA4 and CNGB1b subunits have a high affinity for CNGA2 but not for self-assembly, precluding more than one CNGA4 or CNGB1b subunit in the channel complex.     

Role of subunit heteromerization and N-linked glycosylation in the formation of functional hyperpolarization-activated cyclic nucleotide-gated channels

The coassembly of homologous subunits to heteromeric complexes serves as an important mechanism in generating ion channel diversity. Here, we have studied heteromerization in the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel family. Using a combination of fluorescence confocal microscopy, coimmunoprecipitation, and electrophysiology we found that upon coexpression in HEK293 cells almost all dimeric combinations of HCN channel subunits give rise to the formation of stable channel complexes in the plasma membrane. We also identified HCN1/HCN2 heteromers in mouse brain indicating that heteromeric channels exist in vivo. Surprisingly, HCN2 and HCN3 did not coassemble to heteromeric channels. This finding indicates that heteromerization requires specific structural determinants that are not present in all HCN channel combinations. Using N-glycosidase F we show that native as well as recombinant HCN channels are glycosylated resulting in a 10-20-kDa shift in the molecular weight. Tunicamycin, an inhibitor of N-linked glycosylation, blocked surface membrane expression of HCN2. Similarly, a mutant HCN2 channel in which the putative N-glycosylation site in the loop between S5 and the pore helix was replaced by glutamine (HCN2N380Q) was not inserted into the plasma membrane and did not yield detectable whole-cell currents. These results indicate that N-linked glycosylation is required for cell surface trafficking of HCN channels. Cotransfection of HCN2N380Q with HCN4, but not with HCN3, rescued cell surface expression of HCN2N380Q. Immunoprecipitation revealed that this rescue was due to the formation of a HCN2N380Q/HCN4 heteromeric channel. Taken together our results indicate that subunit heteromerization and glycosylation are important determinants of the formation of native HCN channels.     

Electrophysiological analysis of cloned cyclic nucleotide-gated ion channels

Electrophysiological studies were conducted on the cloned plant cyclic nucleotide-gated ion channels AtCNGC2 and AtCNGC1 from Arabidopsis, and NtCBP4 from tobacco (Nicotiana tobacum). The nucleotide coding sequences for these proteins were expressed in Xenopus laevis oocytes or HEK 293 cells. Channel characteristics were evaluated using voltage clamp analysis of currents in the presence of cAMP. AtCNGC2 was demonstrated to conduct K(+) and other monovalent cations, but exclude Na(+); this conductivity profile is unique for any ion channel not possessing the amino acid sequence found in the selectivity filter of K(+)-selective ion channels. Application of cAMP evoked currents in membrane patches of oocytes injected with AtCNGC2 cRNA. Direct activation of the channel by cyclic nucleotide, demonstrated by application of cyclic nucleotide to patches of membranes expressing such channels, is a hallmark characteristic of this ion channel family. Voltage clamp studies (two-electrode configuration) demonstrated that AtCNGC1 and NtCBP4 are also cyclic nucleotide-gated channels. Addition of a lipophilic analog of cAMP to the perfusion bath of oocytes injected with NtCBP4 and AtCNGC1 cRNAs induced inward rectified, noninactivating K(+) currents.     

Subunit configuration of heteromeric cone cyclic nucleotide-gated channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to be tetrameric assemblies of CNGB3 (B3) and CNGA3 (A3) subunits. We have used functional and biochemical approaches to investigate the stoichiometry and arrangement of these subunits in recombinant channels. First, tandem dimers of linked subunits were used to constrain the order of CNGB3 and CNGA3 subunits; the properties of channels formed by B3/B3+A3/A3 dimers, or A3/B3+B3/A3 dimers, closely resembled those of channels arising from B3+A3 monomers. Functional markers in B3/B3 (or A3/A3) dimers confirmed that both B3 subunits (and both A3 subunits) gained membership into the pore-forming tetramer and that like subunits were positioned adjacent to each other. Second, chemical crosslinking and co-immunoprecipitation studies using epitope-tagged monomer subunits both demonstrated the presence of two CNGB3 subunits in cone channels. Together, these data support a preferred subunit arrangement for cone CNG channels (B3-B3-A3-A3) that is distinct from the 3A:1B configuration of rod channels.     

All-trans-retinal is a closed-state inhibitor of rod cyclic nucleotide-gated ion channels

Rod vision begins when 11-cis-retinal absorbs a photon and isomerizes to all-trans-retinal (ATR) within the photopigment, rhodopsin. Photoactivated rhodopsin triggers an enzyme cascade that lowers the concentration of cGMP, thereby closing cyclic nucleotide-gated (CNG) ion channels. After isomerization, ATR dissociates from rhodopsin, and after a bright light, this release is expected to produce a large surge of ATR near the CNG channels. Using excised patches from Xenopus oocytes, we recently showed that ATR shuts down cloned rod CNG channels, and that this inhibition occurs in the nanomolar range (aqueous concentration) at near-physiological concentrations of cGMP. Here we further characterize the ATR effect and present mechanistic information. ATR was found to decrease the apparent cGMP affinity, as well as the maximum current at saturating cGMP. When ATR was applied to outside-out patches, inhibition was much slower and less effective than when it was applied to inside-out patches, suggesting that ATR requires access to the intracellular surface of the channel or membrane. The apparent ATR affinity and maximal inhibition of heteromeric (CNGA1/CNGB1) channels was similar to that of homomeric (CNGA1) channels. Single-channel and multichannel data suggest that channel inhibition by ATR is reversible. Inhibition by ATR was not voltage dependent, and the form of its dose-response relation suggested multiple ATR molecules interacting per channel. Modeling of the data obtained with cAMP and cGMP suggests that ATR acts by interfering with the allosteric opening transition of the channel and that it prefers closed, unliganded channels. It remains to be determined whether ATR acts directly on the channel protein or instead alters channel-bilayer interactions.     

Mechanism of allosteric modulation of rod cyclic nucleotide-gated channels

The cyclic nucleotide-gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of alpha subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26-30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C left and right arrow C left and right arrow O scheme, all rate constants were dependent on cyclic nucleotide. For the C left and right arrow O left and right arrow C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 microM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.     

Modification of cyclic nucleotide-gated ion channels by ultraviolet light

We irradiated cyclic nucleotide-gated ion channels in situ with ultraviolet light to probe the role of aromatic residues in ion channel function. UV light reduced the current through excised membrane patches from Xenopus oocytes expressing the alpha subunit of bovine retinal cyclic nucleotide-gated channels irreversibly, a result consistent with permanent covalent modification of channel amino acids by UV light. The magnitude of the current reduction depended only on the total photon dose delivered to the patches, and not on the intensity of the exciting light, indicating that the functionally important photochemical modification(s) occurred from an excited state reached by a one-photon absorption process. The wavelength dependence of the channels' UV light sensitivity (the action spectrum) was quantitatively consistent with the absorption spectrum of tryptophan, with a small component at long wavelengths, possibly due to cystine absorption. This spectral analysis suggests that UV light reduced the currents at most wavelengths studied by modifying one or more "target" tryptophans in the channels. Comparison of the channels' action spectrum to the absorption spectrum of tryptophan in various solvents suggests that the UV light targets are in a water-like chemical environment. Experiments on mutant channels indicated that the UV light sensitivity of wild-type channels was not conferred exclusively by any one of the 10 tryptophan residues in a subunit. The similarity in the dose dependences of channel current reduction and tryptophan photolysis in solution suggests that photochemical modification of a small number of tryptophan targets in the channels is sufficient to decrease the currents.     

Expression of plant cyclic nucleotide-gated cation channels in yeast

The functional properties of inwardly conducting plant cyclic nucleotide-gated cation channels (CNGCs) have not been thoroughly characterized due in part to the recalcitrance of their functional expression in heterologous systems. Here, K+ uptake-deficient mutants of yeast (trk1,2) and Escherichia coli (LB650), as well as the Ca2+-uptake yeast mutant mid1,cch1, were used for functional characterization of Arabidopsis thaliana CNGCs, with the aim of identifying some of the cultural and physiological conditions that impact on plant CNGC function in heterologous systems. Use of the Ca2+-uptake yeast mutant provided the first evidence consistent with Ca2+ conduction by the A. thaliana CNGC AtCNGC1. Expression of AtCNGC1 in LB650 demonstrated that mutants of Escherichia coli (which has no endogenous calmodulin) can also be used to study functional properties of CNGCs. Expression of AtCNGC2 and AtCNGC4 enhanced growth of trk1,2 in the presence of hygromycin; AtCNGC1 has less of an effect. Deletion of the AtCNGC1 calmodulin-binding domain enhanced growth of trk1,2 at low external K+ but not of LB650, suggesting that yeast calmodulin may bind to, and down-regulate this plant channel. In vitro binding studies confirmed this physical interaction. Northern analysis, green fluorescent protein:AtCNGC1 fusion protein expression, as well as an antibody raised against a portion of AtCNGC1, were used to monitor expression of AtCNGC1 and deletion constructs of the channel in the heterologous systems. In the presence of the activating ligand cAMP, expression of the AtCNGC1 channel with the calmodulin-binding domain deleted increased intracellular [K+] of trk1,2. Trk1,2 is hypersensitive to the toxic cations spermine, tetramethylamine, and NH4+. These compounds, as well as amiloride, inhibited trk1,2 growth and thereby improved the efficacy of this yeast mutant as a heterologous expression system for CNGCs. In addition to characterizing mutants of yeast and E. coli as assay systems for plant CNGCs, work presented in this report demonstrates, for the first time, that a plant CNGC can retain ion channel function despite (partial) deletion of its calmodulin-binding domain and that yeast calmodulin can bind to and possibly down-regulate a plant CNGC.