cGMP and cyclic nucleotide-gated channels participate in mouse sperm capacitation

During capacitation of mammalian sperm intracellular [Ca(2+)] and cyclic nucleotides increase, suggesting that CNG channels play a role in the physiology of sperm. Here we study the effect of capacitation, 8Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate) and 8Br-cGMP (8-bromoguanosine 3',5'-cyclic monophosphate) on the macroscopic ionic currents of mouse sperm, finding the existence of different populations of sperm, in terms of the recorded current and its response to cyclic nucleotides. Our results show that capacitation and cyclic nucleotides increase the ionic current, having a differential sensitivity to cGMP (cyclic guanosine monophosphate) and cAMP (cyclic adenosine monophosphate). Using a specific inhibitor we determine the contribution of CNG channels to macroscopic current and capacitation.     

Subunit interactions in the activation of cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) ion channels of retinal photoreceptors and olfactory neurons are multimeric proteins of unknown stoichiometry. To investigate the subunit interactions that occur during CNG channel activation, we have used tandem cDNA constructs of the rod CNG channel to generate heteromultimeric channels composed of wild-type and mutant subunits. We introduced point mutations that affect channel activation: 1) D604M, which alters the relative ability of agonists to promote the allosteric conformational change(s) associated with channel opening, and 2) T560A, which primarily affects the initial binding affinity for cGMP, and to a lesser extent, the allosteric transition. At saturating concentrations of agonist, heteromultimeric channels were intermediate between wild-type and mutant homomultimers in agonist efficacy and apparent affinity for cGMP, cIMP, and cAMP, consistent with a model for the allosteric transition involving a concerted conformational change in all of the channel subunits. Results were also consistent with a model involving independent transitions in two or three, but not one or four, of the channel subunits. The behavior of the heterodimers implies that the channel stoichiometry is some multiple of 2 and is consistent with a tetrameric quaternary structure for the functional channel complex. Steady-state dose-response relations for homomultimeric and heteromultimeric channels were well fit by a Monod, Wyman, and Changeux model with a concerted allosteric opening transition stabilized by binding of agonist.     

In vivo assessment of local phosphodiesterase activity using tailored cyclic nucleotide-gated channels as cAMP sensors.

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.     

C terminus-mediated control of voltage and cAMP gating of hyperpolarization-activated cyclic nucleotide-gated channels

The hyperpolarization-activated cyclic nucleotide-gated (HCN) family of "pacemaker" channels includes 4 isoforms, the kinetics and cAMP-induced modulation of which differ quantitatively. Because HCN isoforms are highly homologous in the central region, but diverge more substantially in the N and C termini, we asked whether these latter regions could contribute to the determination of channel properties. To this aim, we analyzed activation/deactivation kinetics and the response to cAMP of heterologously expressed isoforms mHCN1 and rbHCN4 and verified that mHCN1 has much faster kinetics and lower cAMP sensitivity than rbHCN4. We then constructed rbHCN4 chimeras by replacing either the N or the C terminus, or both, with the analogous domains from mHCN1. We found that: 1) replacement of the N terminus (chimera N1-4) did not substantially modify either the kinetics or cAMP dependence of wild-type channels; 2) replacement of the C terminus, on the contrary, resulted in a chimeric channel (4-C1), the kinetics of which were strongly accelerated compared with rbHCN4, and that was fully insensitive to cAMP; 3) replacement of both N and C termini led to the same results as replacement of the C terminus alone. These results indicate that the C terminus of rbHCN4 contributes to the regulation of voltage- and cAMP-dependent channel gating, possibly through interaction with other intracellular regions not belonging to the N terminus.     

Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). HCN isoforms respond differently to cAMP; binding of cAMP shifts activation of HCN2 and HCN4 by 17 mV but shifts that of HCN1 by only 2-4 mV. To explain the peculiarity of HCN1, we solved the crystal structures and performed a biochemical-biophysical characterization of the C-terminal domain (C-linker plus CNBD) of the three isoforms. Our main finding is that tetramerization of the C-terminal domain of HCN1 occurs at basal cAMP concentrations, whereas those of HCN2 and HCN4 require cAMP saturating levels. Therefore, HCN1 responds less markedly than HCN2 and HCN4 to cAMP increase because its CNBD is already partly tetrameric. This is confirmed by voltage clamp experiments showing that the right-shifted position of V(½) in HCN1 is correlated with its propensity to tetramerize in vitro. These data underscore that ligand-induced CNBD tetramerization removes tonic inhibition from the pore of HCN channels.     

Bimodal agonism in heteromeric cyclic nucleotide-gated channels

Direct binding of cGMP or cAMP to tetrameric cyclic nucleotide-gated (CNG) channels will normally promote the open (conductive) conformation. However, the catfish CNGA2 subtype exhibits bimodal agonism, whereby open probability (P_o) increases with initial cGMP binding events ("pro" action) but decreases with subsequent cGMP binding events ("con" action) that occur at concentrations above 3 mM. We constructed, and heterologously expressed, chimeric CNG channel subunits with sequence substitutions in the binding domain (BD), and tested their activation using patch-clamp of cell-free membranes. A normal subunit with the rat CNGA4 BD (with only pro action) could be converted into a bimodal subunit (both pro and con action) by replacing the N-terminal portion of the BD with catfish CNGA2 sequence. We then fused two bimodal and two normal subunits in tandem tetramers, to form heteromeric CNG channels with bimodal pseudo-subunits either adjacent (cis) or diagonally opposite (trans). The cis tetramer showed con action, with a mean ratio of steady-state conductances g_(30mMcGMP) / g_(3mMcGMP) = 0.87, demonstrating bimodal agonism in a heteromeric CNG channel for the first time. In contrast, trans tetramers showed normal cGMP agonism up to 30 mM cGMP with mean g_(30mMcGMP) / g_(3mMcGMP) = 1.02, although a minority of oocytes (4 of 15) expressed anomalous channel populations with con action. Rearranging subunits in a heteromer thus influences a channel's P_o at high cGMP concentration. The sensitivity of con action to neighbouring subunits implies a cooperative mechanism.     

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.     

Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels

In cyclic nucleotide-gated (CNG) ion channels, binding of cGMP or cAMP drives a conformational change that leads to opening of an ion-conducting pore. One region implicated in the coupling of ligand binding to opening of the pore is the C linker region. Here, we used crosslinking of endogenous cysteines to study interregion proximity. We demonstrate that an individual amino acid--C481--in the C linker region of each of two neighboring subunits can form a disulfide bond. Further, using tandem dimers, we show that a disulfide bond between C35 in the N-terminal region and C481 in the C linker region can form either within a subunit or between subunits. From our data on proximity between individual amino acids and previous studies, a picture emerges of the C linker as a potential dimerization interface.     

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.     

Molecular rearrangements in the ligand-binding domain of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels are activated in response to the direct binding of cyclic nucleotides to an intracellular domain. This domain is thought to contain a beta roll and two alpha helices, designated the B and C helices. To probe the conformational changes occurring in the ligand-binding domain during channel activation, we used the substituted cysteine accessibility method (SCAM). We found that a residue in the beta roll, C505, is more accessible in unliganded channels than in liganded channels, whereas a residue in the C helix, G597C, is more accessible in closed channels than in open channels. These results support a molecular mechanism for channel activation in which the ligand initially binds to the beta roll, followed by an opening allosteric transition involving the relative movement of the C helix toward the beta roll.     

Ca2+ permeation in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels conduct Na+, K+ and Ca2+ currents under the control of cGMP and cAMP. Activation of CNG channels leads to depolarization of the membrane voltage and to a concomitant increase of the cytosolic Ca2+ concentration. Several polypeptides were identified that constitute principal and modulatory subunits of CNG channels in both neurons and non-excitable cells, co-assembling to form a variety of heteromeric proteins with distinct biophysical properties. Since the contribution of each channel type to Ca2+ signaling depends on its specific Ca2+ conductance, it is necessary to analyze Ca2+ permeation for each individual channel type. We have analyzed Ca2+ permeation in all principal subunits of vertebrates and for a principal subunit from Drosophila melanogaster. We measured the fractional Ca2+ current over the physiological range of Ca2+ concentrations and found that Ca2+ permeation is determined by subunit composition and modulated by membrane voltage and extracellular pH. Ca2+ permeation is controlled by the Ca2+-binding affinity of the intrapore cation-binding site, which varies profoundly between members of the CNG channel family, and gives rise to a surprising diversity in the ability to generate Ca2+ signals.     

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.     

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.     

Gating rearrangements in cyclic nucleotide-gated channels revealed by patch-clamp fluorometry

Site-specific fluorescence recordings have shown great promise in understanding conformational changes in signaling proteins. The reported applications on ion channels have been limited to extracellular sites in whole oocyte preparations. We are now able to directly monitor gating movements of the intracellular domains of cyclic nucleotide-gated channels using simultaneous site-specific fluorescence recording and patchclamp current recording from inside-out patches. Fluorescence signals were reliably observed when fluorophore was covalently attached to a site between the cyclic nucleotide-binding domain and the pore. While iodide, an anionic quencher, has a higher quenching efficiency in the channel's closed state, thallium ion, a cationic quencher, has a higher quenching efficiency in the open state. The state and charge dependence of quenching suggests movements of charged or dipolar residues near the fluorophore during CNG channel activation.     

Influence of permeant ions on gating in cyclic nucleotide-gated channels

Cyclic nucleotide-gated channels are key components in the transduction of visual and olfactory signals where their role is to respond to changes in the intracellular concentration of cyclic nucleotides. Although these channels poorly select between physiologically relevant monovalent cations, the gating by cyclic nucleotide is different in the presence of Na(+) or K(+) ions. This property was investigated using rod cyclic nucleotide-gated channels formed by expressing the subunit 1 (or alpha) in HEK293 cells. In the presence of K(+) as the permeant ion, the affinity for cGMP is higher than the affinity measured in the presence of Na(+). At the single channel level, subsaturating concentrations of cGMP show that the main effect of the permeant K(+) ions is to prolong the time channels remain open without major changes in the shut time distribution. In addition, the maximal open probability was higher when K(+) was the permeant ion (0.99 for K(+) vs. 0.95 for Na(+)) due to an increase in the apparent mean open time. Similarly, in the presence of saturating concentrations of cAMP, known to bind but unable to efficiently open the channel, permeant K(+) ions also prolong the time channels visit the open state. Together, these results suggest that permeant ions alter the stability of the open conformation by influencing of the O-->C transition.     

Structural dynamics in the gating ring of cyclic nucleotide-gated ion channels

For ligand-gated ion channels, the binding of a ligand to an intracellular or extracellular domain generates changes in transmembrane pore-forming helices, which alters ion flow. The molecular mechanism for this allostery, however, remains unknown. Here we explore the structure and conformational rearrangements of the C-terminal gating ring of the cyclic nucleotide-gated channel CNGA1 during activation by cyclic nucleotides with patch-clamp fluorometry. By monitoring fluorescent resonance energy transfer (FRET) between membrane-resident quenchers and fluorophores attached to the channel, we detected no movement orthogonal to the membrane during channel activation. By monitoring FRET between fluorophores within the C-terminal region, we determined that the C-terminal end of the C-linker and the end of the C-helix move apart when channels open. We conclude that during channel activation, a portion of the gating ring moves parallel to the plasma membrane, hinging toward the central axis of the channel.     

Multimerization of the ligand binding domains of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels comprise four subunits and are activated by the direct binding of cyclic nucleotide to an intracellular domain on each subunit. This ligand binding domain is thought to contain a beta roll followed by two alpha helices, designated the B and C helices. To examine the quaternary structure of CNG channels and how it changes during ion channel gating, we introduced single cysteines along the C helix of each subunit in an otherwise cysteineless channel. We found that cysteines on the C helices could form intersubunit disulfide bonds, even between diagonal subunits. Disulfide bond formation occurred primarily in closed channels and inhibited channel opening. These data suggest that the C helices from all four channel subunits are in close proximity in the closed state and move apart during channel opening.