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PubMed2000

Coexpression of alpha and beta subunits of the rod cyclic GMP-gated channel restores native sensitivity to cyclic AMP: role of D604/N1201.

Coexpression of the betawt and alphawt subunits of the bovine rod channel restores two characteristics of the native channels: higher sensitivity to cAMP and potentiation of cGMP-induced currents by low cAMP concentrations. To test whether the increased sensitivity to cAMP is due to the uncharged nature of the asparagine residue (N1201) situated in place of aspartate D604 in the beta subunit as previously suggested (, Neuron. 15:619-625), we compared currents from wild-type (alphawt and alphawt/betawt) and from mutated channels (alphaD604N, alphaD604N/betawt, and alphawt/betaN1201D). The results show that the sensitivity to cAMP and cAMP potentiation is partly but not entirely determined by the charge of residue 1201 in the beta subunit. The D604N mutation in the alpha subunit and, to a lesser extent, coexpression of the betawt subunit with the alphawt subunit reduce the open probability for cGMP compared to that of the alphawt channel. Interpretation of the data with the MWC allosteric model (model of Monod, Wyman, Changeux;, J. Mol. Biol. 12:88-118) suggests that the D604N mutation in the alpha subunits and coassembly of alpha and beta subunits alter the free energy of gating by cAMP more than that of cAMP binding.

Amino Acid Substitution↗
PubMed2001

Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.

In cyclic nucleotide-gated channels (CNG), direct binding of cyclic nucleotides in the carboxy-terminal region is allosterically coupled to opening of the pore. A CNG1 channel pore was probed using site-directed cysteine substitution to elucidate conformational changes associated with channel opening. The effects of cysteine modification on permeation suggest a structural homology between CNG and KcsA pores. We found that intersubunit disulfide bonds form spontaneously between S399C residues in the helix bundle when channels are in the closed but not in the open state. While MTSET modification of pore-lining residues was state dependent, Ag(+) modification of V391C, in the inner vestibule, occurred at the same diffusion-limited rate in both open and closed states. Our results suggest that the helix bundle undergoes a conformational change associated with gating but is not the activation gate for CNG channels.

Amino Acid Sequence↗
PubMed2001

Rotational movement during cyclic nucleotide-gated channel opening.

Cyclic nucleotide-gated (CNG) channels are crucial components of visual, olfactory and gustatory signalling pathways. They open in response to direct binding of intracellular cyclic nucleotides and thus contribute to cellular control of both the membrane potential and intracellular Ca2+ levels. Cytosolic Ni2+ potentiates the rod channel (CNG1) response to cyclic nucleotides and inhibits the olfactory channel (CNG2) response. Modulation is due to coordination of Ni2+ by channel-specific histidines in the C-linker, between the S6 transmembrane segment and the cyclic nucleotide-binding domain. Here we report, using a histidine scan of the initial C-linker of the CNG1 channel, stripes of sites producing Ni2+ potentiation or Ni2+ inhibition, separated by 50 degrees on an alpha-helix. These results suggest a model for channel gating where rotation of the post-S6 region around the channel's central axis realigns the Ni2+-coordinating residues of multiple subunits. This rotation probably initiates movement of the S6 and pore opening.

Amino Acid Sequence↗
PubMed1997

Single cyclic nucleotide-gated channels locked in different ligand-bound states.

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.

Animals↗
PubMed2002

Mechanism of calcium/calmodulin inhibition of rod cyclic nucleotide-gated channels.

Rod cyclic nucleotide-gated (CNG) channels are heterotetramers comprised of both CNGA1 and CNGB1 subunits. Calcium/calmodulin (Ca(2+)/CaM) binds to a site in the N-terminal region of CNGB1 subunits and inhibits the opening conformational change in CNGA1/CNGB1 channels. Here, we show that polypeptides derived from an N-terminal region of CNGB1 form a specific interaction with polypeptides derived from a C-terminal region of CNGA1 that is distal to the cyclic nucleotide-binding domain. Deletion of the Ca(2+)/CaM-binding site from the N-terminal region of CNGB1 eliminated both Ca(2+)/CaM modulation of the channel and the intersubunit interaction. Furthermore, the interaction was disrupted by the presence of Ca(2+)/CaM. These results suggest that Ca(2+)/CaM-dependent inhibition of rod channels is caused by the direct binding of Ca(2+)/CaM to a site in the N-terminal region in CNGB1, which disrupts the interaction between this region and a distal C-terminal region of CNGA1. The mechanism underlying Ca(2+)/CaM modulation of rod channels is distinct from that in olfactory (CNGA2) CNG channels.

Animals↗
PubMed1999

Gating by cyclic GMP and voltage in the alpha subunit of the cyclic GMP-gated channel from rod photoreceptors.

Gating by cGMP and voltage of the alpha subunit of the cGMP-gated channel from rod photoreceptor was examined with a patch-clamp technique. The channels were expressed in Xenopus oocytes. At low [cGMP] (<20 microM), the current displayed strong outward rectification. At low and high (700 microM) [cGMP], the channel activity was dominated by only one conductance level. Therefore, the outward rectification at low [cGMP] results solely from an increase in the open probability, P(o). Kinetic analysis of single-channel openings revealed two exponential distributions. At low [cGMP], the larger P(o) at positive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, depolarizing voltage steps, starting from -100 mV, generated a time-dependent current that increased with the step size (activation). At low [cGMP] (20 microM), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective changes were small and fast. The dose-response relation at -100 mV was shifted to the right and saturated at significantly lower P(o) values with respect to that at +100 mV (0.77 vs. 0.96). P(o) was determined as function of the [cGMP] (at +100 and -100 mV) and voltage (at 20, 70, and 700 microM, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation time course was monoexponential, which allowed us to determine the individual rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is concluded that an allosteric model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric reaction, describes the cGMP- and voltage-dependent gating of cGMP-gated channels adequately.

Animals↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1996

Predicted ligand interactions of 3'5'-cyclic nucleotide-gated channel binding sites: comparison of retina and olfactory binding site models.

Cyclic nucleotide-gated channels (CNGC) open in response to the binding of 3'5'-cyclic nucleotides. Members of the CNGC family vary as much as 100-fold in their ability to respond to cAMP and cGMP. Molecular models of the nucleotide binding domains of the bovine retina and catfish and rat olfactory CNGCs were built from the crystal structure of cAMP bound to catabolite gene activator protein (CAP) with AMMP, a program for molecular mechanics and dynamics. The nucleotide conformation can be predicted from the number of strong and weak interactions between the purine ring and the binding site. The amino acids predicted to be important for determining the nucleotide affinity and specificity are residues 61, 83 (mediated through a water molecule), 119 and 127 (CAP sequence numbers) which interact with the purine ring. These residues also dictate the conformation of the ligand in the binding pocket. cGMP is preferentially bound in the syn conformation in bovine retina, bovine olfactory and rat olfactory CNGCs due to Thr83, while either conformation can bind in catfish olfactory CNGC. cAMP is predicted to bind either in syn or anti conformation, depending on the interaction with residue 119: the anti conformation is preferentially bound in olfactory CNGCs.

Animals↗
PubMed1999

A gustatory cyclic nucleotide-gated channels CNGgust, is expressed in the retina.

Cyclic nucleotide-gated (CNG) channels are essential proteins that contribute to the intracellular signal transduction of the senses of sight and smell. Recently, we found a novel CNG channel (CNGgust) in rat taste buds, and demonstrated its possible involvement in taste signal transduction. In the present study, we used RT-PCR and immunostaining to prove that this gustatory CNG channel is expressed in the outer segments of rat cone photoreceptor cells. The study strongly suggests that the senses of taste and sight share, at least in part, a common signal transduction pathway.

Animals↗
PubMed2006

Locking CNGA1 channels in the open and closed state.

With the aim of understanding the relation between structure and gating of CNGA1 channels from bovine rod, an extensive cysteine scanning mutagenesis was performed. Each residue from Phe-375 to Val-424 was mutated into a cysteine one at a time and the modification caused by various sulfhydryl reagents was analyzed. The addition of the mild oxidizing agent copper phenanthroline (CuP) in the open (presence of 1 mM cGMP) or closed state locked the channel in the respective states. A subsequent treatment with the reducing agent DTT restored normal gating fully in the open state and partially in the closed state. This action of CuP was not observed when F380 was mutated into a cysteine in the cysteine-free CNGA1 channel and in the double mutant C314S&F380C. These observations suggest that these effects are mediated by the formation of a disulfide bond (S-S) between F380C and the endogenous Cys-314 in the S5 segment. It can be rationalized by supposing that during gating the S6 segment rotates anticlockwise-when viewed from the extracellular side-by approximately 30 degrees .

Animals↗
PubMed2005

Activation of olfactory-type cyclic nucleotide-gated channels is highly cooperative.

Cyclic nucleotide-gated (CNG) ion channels play a key role in the sensory transduction of vision and olfaction. The channels are opened by the binding of cyclic nucleotides. Native olfactory CNG channels are heterotetramers of CNGA2, CNGA4, and CNGB1b subunits. Upon heterologous expression, only CNGA2 subunits can form functional homotetrameric channels. It is presently not known how the binding of the ligands to the four subunits is translated to channel opening. We studied activation of olfactory CNG channels by photolysis-induced jumps of cGMP or cAMP, two cyclic nucleotides with markedly different apparent affinity. It is shown that at equal degree of activation, the activation time course of homotetrameric channels is similar with cGMP and cAMP and it is also similar in homo- and heterotetrameric channels with the same cyclic nucleotide. Kinetic models were globally fitted to activation time courses of homotetrameric channels. While all models containing equivalent binding sites failed, a model containing three binding sites with a ligand affinity high-low-high described the data adequately. Only the second binding step switches from a very low to a very high open probability. We propose a unique gating mechanism for homotetrameric and heterotetrameric channels that involves only three highly cooperative binding steps.

Animals↗
PubMed1995

Voltage-dependent gating of an asymmetric gramicidin channel.

In an effort to understand the molecular mechanisms of voltage activation of ion channels, we have chosen a system of known structure and examined the properties of heterodimeric channels formed between [Val1]gramicidin A ([Val1]gA) and [F6Val1]gramicidin A ([F6Val1]gA). Gramicidin channels are usually not voltage-dependent; but the introduction of a single symmetry-breaking dipolar F6Val1 residue into a ([Val1]gA)2 dimer to form the [F6Val1]gA/[Val1]gA heterodimer induces voltage-dependent transitions between two conducting states: a high-conductance state and a zero conductance (closed) state. The distribution between these states varies as a function of the applied potential but is not dependent on the nature of the permeant ion (H+ or Cs+). The permeating ions do not seem to contribute to the apparent gating charge.

Calcium Channels↗
PubMed2002

Dominant-negative suppression of HCN1- and HCN2-encoded pacemaker currents by an engineered HCN1 construct: insights into structure-function relationships and multimerization.

I(f), a diastolic depolarizing current activated by hyperpolarization, is a key player in cardiac pacing. Despite the fact that I(f) has been known for over 20 years, the encoding genes, namely HCN1 to 4, have only recently been identified. Functional data imply that different HCN isoforms may coassemble to form heteromeric channel complexes, but little direct evidence is available. Subunit stoichiometry is also unknown. Although the pore region of HCN channels contains the glycine-tyrosine-glycine (GYG) signature motif found in K+-selective channels, they permeate both Na+ and K+. In the present study, we probed the functional importance of the GYG selectivity motif in pacemaker channels by replacing this triplet in HCN1 with alanines (GYG(349-351)AAA or HCN1-AAA). HCN1-AAA did not yield functional currents; coexpression of HCN1-AAA with wild-type (WT) HCN1 suppressed normal channel activity in a dominant-negative manner (55.2+/-3.2%, 68.3+/-4.3%, 78.7+/-1.6%, 91.7+/-0.8%, and 97.9+/-0.2% current reduction at -140 mV for WT:AAA cRNA ratios of 4:1, 3:1, 2:1, 1:1, and 1:2, respectively) without affecting gating (steady-state activation, activation and deactivation kinetics) or permeation (reversal potential) properties. HCN1-AAA coexpression, however, did not alter the expressed current amplitudes of Kv1.4 and Kv2.1 channels, indicating that its suppressive effect was channel-specific. Statistical analysis reveals that a single HCN channel is composed of 4 monomeric subunits. Interestingly, HCN1-AAA also inhibited HCN2 in a dominant-negative manner with the same efficacy. We conclude that the GYG motif is a critical determinant of ion permeation for HCN channels, and that HCN1 and HCN2 readily coassemble to form heterotetrameric complexes.

Amino Acid Motifs↗
PubMed2004

Molecular structure of P2X receptors.

P2X receptors are ligand-gated ion channels that transduce many of the physiological effects of extracellular ATP. There has been a dramatic increase in awareness of these receptors over the past 5 or so years, in great part due to their molecular cloning and characterization. The availability of cDNA clones for the various subunits has led to rapid progress in identifying their tissue-specific expression, resulting in new ideas concerning the functional roles these receptors might play in physiological and pathophysiological processes. In addition, molecular approaches have yielded much information regarding the structure and function of the receptor proteins themselves. In this review we seek to review recent findings concerning the molecular determinants of receptor-channel function, with particular focus on ligand binding and gating, ion selectivity, and subunit assembly.

Animals↗
PubMed1993

Symmetrical segregation of potassium channels at cytokinesis.

To determine how voltage-gated ion channels segregate between sibling cells at cytokinesis, we used a whole-cell patch clamp to measure the electrophysiological phenotypes of siblings within 45 min of division. Recently born siblings in an immortalized line of embryonic retinal cells were identified as pairs of spherical cells adhering to one another. All siblings were electrically coupled when cells were simultaneously voltage clamped, whereas nonsiblings were not coupled. Twelve pairs of siblings were electrically isolated by mechanical separation so that their phenotypes could be measured independently. Cells expressed two principal membrane conductances, delayed rectifier-like (IK) and inward rectifier (IK(IR)) potassium currents. Despite qualitative and quantitative variability in IK and IK(IR) expression within the population, each cell of a given pair expressed similar steady-state current densities between -110 and +50 mV. We estimated IK(IR) slope conductance by blocking the current specifically with 5 mM Cs and calculated IK(IR) ratios in siblings and nonsiblings. Three pairs of siblings expressed IK(IR) ratios of approximately 1.2, while ratios in three pairs of adhered nonsiblings varied between 1.6 and 5.4. When currents were sampled continuously through cytokinesis by using the perforated-patch recording mode, current amplitude showed no net change within 30 min of division. Because channel number did not appear to change in siblings during this interval, parental channels were inherited by each daughter in proportion to the area of membrane received. Heterogeneity therefore arises after siblings reenter interphase and is not due to the asymmetrical segregation of channels at cytokinesis.

Animals↗
PubMed1996

Depolarization as a regulatory signal at voltage-gated calcium channels.

Changes in membrane potential are a regulatory signal for voltage-gated ion channels including the family of Ca2+ channels. This regulatory role includes the voltage-dependent opening, closing, and inactivation of ion channels and the control of drug access and affinity for discrete channels states. Membrane potential is both a short- and long-term regulatory signal controlling the number and function of these voltage-gated channels. Depolarization of neuronal and neurosecretory cells produces down-regulation of L-type voltage-gated Ca2+ channels. These processes are described and their relevance to physiologic and pathologic processes of neuronal development and neuroprotection are indicated.

Animals↗