Cyclic Nucleotide-activated Channels Research Articles

Mechanisms and physiological implications of cooperative gating of clustered ion channels.

Ion channels play a central role in the regulation of nearly every cellular process. Dating back to the classic 1952 Hodgkin-Huxley model of the generation of the action potential, ion channels have always been thought of as independent agents. A myriad of recent experimental findings exploiting advances in electrophysiology, structural biology, and imaging techniques, however, have posed a serious challenge to this long-held axiom, as several classes of ion channels appear to open and close in a coordinated, cooperative manner. Ion channel cooperativity ranges from variable-sized oligomeric cooperative gating in voltage-gated, dihydropyridine-sensitive CaV1.2 and CaV1.3 channels to obligatory dimeric assembly and gating of voltage-gated NaV1.5 channels. Potassium channels, transient receptor potential channels, hyperpolarization cyclic nucleotide-activated channels, ryanodine receptors (RyRs), and inositol trisphosphate receptors (IP3Rs) have also been shown to gate cooperatively. The implications of cooperative gating of these ion channels range from fine-tuning excitation-contraction coupling in muscle cells to regulating cardiac function and vascular tone, to modulation of action potential and conduction velocity in neurons and cardiac cells, and to control of pacemaking activity in the heart. In this review, we discuss the mechanisms leading to cooperative gating of ion channels, their physiological consequences, and how alterations in cooperative gating of ion channels may induce a range of clinically significant pathologies.

Biophysical analysis of an HCN1 epilepsy variant suggests a critical role for S5 helix Met-305 in voltage sensor to pore domain coupling

Hyperpolarization-gated, cyclic nucleotide-activated (HCN1-4) channels are inwardly rectifying cation channels that display voltage dependent activation and de-activation. Pathogenic variants in HCN1 are associated with severe developmental and epileptic encephalopathies including the de novo HCN1 M305L variant. M305 is located in the S5 domain that is implicated in coupling voltage sensor domain movement to pore opening. This variant lacks voltage-dependent activation and de-activation and displays normal cation selectivity. To elucidate the impact of the mutation on the channel structure-function relations, molecular dynamics simulations of the wild type and mutant homotetramers were compared and identified a sulphur-aromatic interaction between M305 and F389 that contributes to the coupling of the voltage-sensing domain to the pore domain. To mimic the heterozygous condition as a heterotetrameric channel assembly, Xenopus oocytes were co-injected with various ratios of wild-type and mutant subunit cRNAs and the biophysical properties of channels with different subunit stoichiometries were determined. The results showed that a single mutated subunit was sufficient to significantly disrupt the voltage dependence of activation. The functional data were qualitatively consistent with predictions of a model that assumes independent activation of the voltage sensing domains allosterically controlling the closed to open transition of the pore. Overall, the M305L mutation results in an HCN1 channel that lacks voltage dependence and facilitates excitatory cation flow at membrane potentials that would normally close the channel. Our findings provide molecular insights into HCN1 channels and reveal the structural and biophysical basis of the severe epilepsy phenotype associated with the M305L mutation.

Role of Interactions between Transmembrane and C-Terminal Regions in Voltage-Dependent Activation of HCN4 Channels

The HCN4 subtype of Hyperpolarization- and Cyclic Nucleotide-activated (HCN) channels is noted for slower kinetics and a hyperpolarized activation midpoint voltage (V1/2) compared to the HCN2 subtype. To test whether the HCN4 transmembrane (TM) region requires interactions with the C-terminal region to determine V1/2, we substituted the HCN4 TM region into previously characterized HCN2 channels with various truncations of the C-terminal region. Substituting the HCN4 TM region into HCN2 channels - either full-length or truncated after the cyclic nucleotide-binding (CNB) fold - resulted in a significant (more than 8 mV) negative shift of V1/2. Strikingly, in HCN2 channels lacking the CNB fold, or channels where the majority of the CNB fold was present except for the final C-helix, the HCN4 TM substitution failed to shift V1/2 significantly (less than 2 mV difference). This suggests that the hyperpolarized V1/2 of HCN4 channels relative to other HCN subtypes relies on a functional interaction of the TM and C-terminal regions, and that the specific conformation supporting this inhibitory interaction requires an intact CNB fold. To test whether this inhibitory interaction coincides with any inhibition caused by eliminating CNB fold-mediated cAMP potentiation, we repeated the HCN4 TM substitution in a full length HCN2 channel whose CNB fold is mutated (R591E) so it cannot bind cAMP. HCN2 R591E exhibits V1/2 20 mV more negative than cAMP-potentiated HCN2 channels, yet the HCN4 TM substitution in HCN2 R591E produced a further 10 mV negative V1/2 shift, matching that produced by HCN4 TM substitution in cAMP-potentiated channels. This suggests a new role for the CNB fold in supporting an inhibitory interaction between the C-terminal region and the HCN4 TM region, independent of and additive with the well-known autoinhibition caused by eliminating cAMP potentiation.

A Charged Residue in the HCN Channel C-Linker Stabilizes the Open State

Hyperpolarization- and cyclic nucleotide-activated (HCN) channels are regulated by a voltage sensing domain (VSD) as well as a cyclic nucleotide binding domain (CNBD) coupled to the pore by a C-linker. We previously found a mutation in the VSD that slows deactivation kinetics by creating a highly stable open state (Open State Trapping, OST) that is dependent on cAMP binding; deletion of the CNBD and C-linker (ΔC) completely removed OST. I introduced point mutations in the C-linker in an OST mutant, and tested their effects on the stable open conformation. Deactivation kinetics of the mutants, and the ΔC construct, were studied in Xenopus laevis oocytes using two electrode voltage clamp, and fitted to an exponential decay equation. Arginine substitution of a conserved glutamate significantly decreased the deactivation time constant (τ) at +20 mV by ∼3.5 fold when compared to wild type. The same mutation shifted the isochronal deactivation midpoint voltage (Vdeact) by −41 mV. For ΔC, ΔVdeact was −55 mV relative to wild type and τ was ∼8 fold faster than wild type. Therefore, the charge reversal mutation removed a substantial portion of the OST effect on τ and the majority of the OST effect on Vdeact. In comparison, mutating the same residue to glutamine or cysteine partially removed the OST effects, with τ decreased by ∼1.5 fold and 3 fold respectively, and with ΔVdeact of −9 mV and −19 mV respectively. We conclude the native glutamate stabilizes the open state during OST, possibly due to C-linker interactions with other channel regions. Future research will explore how and when this stabilization occurs, for example if it can still occur in the absence of cAMP binding or of the OST mechanism.

HCN Channel C-Terminal Region Speeds Activation Rates Independently of Autoinhibition.

Hyperpolarization- and cyclic nucleotide-activated (HCN) channels contribute to rhythmic oscillations in excitable cells. They possess an intrinsic autoinhibition with a hyperpolarized V 1/2, which can be relieved by cAMP binding to the cyclic nucleotide binding (CNB) fold in the C-terminal region or by deletion of the CNB fold. We questioned whether V 1/2 shifts caused by altering the autoinhibitory CNB fold would be accompanied by parallel changes in activation rates. We used two-electrode voltage clamp on Xenopus oocytes to compare wildtype (WT) HCN2, a constitutively autoinhibited point mutant incapable of cAMP binding (HCN2 R591E), and derivatives with various C-terminal truncations. Activation V 1/2 and deactivation t 1/2 measurements confirmed that a truncated channel lacking the helix αC of the CNB fold (ΔαC) had autoinhibition comparable to HCN2 R591E; however, ΔαC activated approximately two-fold slower than HCN2 R591E over a 60-mV range of hyperpolarizations. A channel with a more drastic truncation deleting the entire CNB fold (ΔCNB) had similar V 1/2 values to HCN2 WT with endogenous cAMP bound, confirming autoinhibition relief, yet it surprisingly activated slower than the autoinhibited HCN2 R591E. Whereas CNB fold truncation slowed down voltage-dependent reaction steps, the voltage-independent closed-open equilibrium subject to autoinhibition in HCN2 was not rate-limiting. Chemically inhibiting formation of the endogenous lipid PIP2 hyperpolarized the V 1/2 of HCN2 WT but did not slow down activation to match ΔCNB rates. Our findings suggest a "quickening conformation" mechanism, requiring a full-length CNB that ensures fast rates for voltage-dependent steps during activation regardless of potentiation by cAMP or PIP2.

Will the Real Single HCN Channel Please Stand Up?

Will the Real Single HCN Channel Please Stand Up?

Synaptic Plasticity: Spatio-Temporal Analysis of Actin Dynamics

Synaptic Plasticity: Spatio-Temporal Analysis of Actin Dynamics Olga Kochan 1 * and Robert S. Zucker** *Molecular & Cell Biology and Psychology Major, College of Letters and Science, **Department of Molecular & Cell Biology, University of California, Berkeley Key words: Synaptic plasticity, synaptic transmission, cAMP dependent enhancement, actin, HCN channels ABSTRACT: Glutamatergic synapses are highly modifiable, making them key targets in processes such as learning and memory. In crayfish glutamatergic neuromuscular junctions, hyperpolarization and cyclic nucleotide-activated (HCN) channels and actin cytoskeleton dynamics are critical intermediate factors in hormonal modulation of glutamatergic synapses which lead to cAMP (3’-5’-cyclic adenosine monophosphate)-dependent enhancement of synaptic transmission. Although models have been proposed, there has been a lack of experimental evidence on the relationship between HCN channels and the integrity of the actin cytoskeleton during cAMP-dependent enhancement. The specific goal of this study is to test the sequence of activation of the aforementioned mediators in synaptic enhancement via precisely controlled pharmacological experiments. At glutamatergic neuromuscular junctions of crayfish limb muscles, HCN channel activator, lamotrigine (50 µM), enhanced synaptic transmission about 20%. This enhancement was completely blocked with actin depolymerizer, latrunculin B (3 µM). These results support previous models of the temporal arrangement of events leading to synaptic enhancement, specifically that changes in actin cytoskeleton follow HCN channel activation. Concurrently, we are also using a spatio-temporal marker called phalloidin, a toxin which binds actin filaments, to further test the hypothesis that activation of HCNCs precedes actin cytoskeleton polymerization. This allows for manipulation of HCN channels and visualization of actin that could propose the associated molecular mechanisms. Preliminary evidence suggests actin reorganization. INTRODUCTION Synaptic plasticity underlies adaptability of the nervous system to changing circumstances and is involved in learning and memory. One such modifiability includes cAMP-dependent enhancement of synaptic transmission. At crayfish neuromuscular junctions (NMJ), the number of glutamate-containing synaptic vesicles available for release by action potentials at the exciter is increased by the circulating neurohormone serotonin (1) acting in part by production of the presynaptic second messenger cAMP (2). This cAMP-dependent enhancement of transmission, which can also be produced directly by stimulating adenylyl cyclase with forskolin, involves the activation of presynaptic hyperpolarization and cyclic nucleotide-activated channels (HCNCs) by cAMP as well as the integrity of the actin cytoskeleton (3). In past studies, it was noted that any of three HCNC blockers (ZD 7288, DK-AH 269, or Cs+) or depolymerization of actin by cytochalasin D, latrunculin B, or swinholide A, greatly reduce the forskolin-induced enhancement of transmission (4). A schematic model of the steps involved in induction of serotonergic (cAMP- dependent) enhancement of transmission has been proposed (4). From the model, it is evident that both HCN channels and actin cytoskeleton dynamics are required to mediate synaptic enhancement; however, the relationship between HCNCs and actin still remains elusive and merely hypothesized (Figure 1). Therefore, in this study, we set out to determine the sequential relationship between the actin cytoskeleton and HCN channels in supporting synaptic plasticity and transmission. We used the crayfish NMJ to study the implicated mediators of synaptic enhancement. This model offers many advantages such as availability, suitability for electrophysiological recording and molecular manipulations, as well as physiological and biochemical properties nearly identical to those of mammalian brain synapse. We investigated the mechanism by which changes in HCN channel activation and actin cytoskeleton dynamics result in synaptic enhancement via two main experimental protocols. The first experimental technique manipulates HCN channels 8-CPT Jasplakinolide Lat B Figure 1: This previously published figure (4) depicts a schematic of the steps involved in induction of serotonergic (cAMP-dependent) enhancement of transmission, tetanic activation of LTF (long term facilitation), and synaptic tagging, which are all mechanisms of synaptic plasticity. cAMP enhances transmission by activating Epac and HCN channels, the latter acting via an actin dependent step. The temporal relationship between HCN channels and actin was still indefinite and needed to be further investigated, leading to the experiments in the present study. Corresponding author: University of California, Department of Molecular and Cell Biology, 111 LSA, Berkeley, Ca 94720. Phone number: (925) 286-8491. E-mail: olga54@berkeley.edu.

Synaptic Plasticity: Spatio-Temporal Analysis Of Actin Dynamics

Glutamatergic synapses are highly modifiable, making them key targets in processes such as learning and memory. In crayfish glutamatergic neuromuscular junctions, hyperpolarization and cyclic nucleotide-activated (HCN) channels and actin cytoskeleton dynamics are critical intermediate factors in hormonal modulation of glutamatergic synapses which lead to cAMP (3’-5’-cyclic adenosine monophosphate)-dependent enhancement of synaptic transmission. Although models have been proposed, there has been a lack of experimental evidence on the relationship between HCN channels and the integrity of the actin cytoskeleton during cAMP-dependent enhancement. The specific goal of this study is to test the sequence of activation of the aforementioned mediators in synaptic enhancement via precisely controlled pharmacological experiments. At glutamatergic neuromuscular junctions of crayfish limb muscles, HCN channel activator, lamotrigine (50 μM), enhanced synaptic transmission about 20%. This enhancement was completely blocked with actin depolymerizer, latrunculin B (3 μM). These results support previous models of the temporal arrangement of events leading to synaptic enhancement, specifically that changes in actin cytoskeleton follow HCN channel activation. Concurrently, we are also using a spatio-temporal marker called phalloidin, a toxin which binds actin filaments, to further test the hypothesis that activation of HCNCs precedes actin cytoskeleton polymerization. This allows for manipulation of HCN channels and visualization of actin that could propose the associated molecular mechanisms. Preliminary evidence suggests actin reorganization.

Presynaptic Mechanism Underlying cAMP-Induced Synaptic Potentiation in Medial Prefrontal Cortex Pyramidal Neurons

cAMP, a classic second messenger, has been proposed recently to participate in regulating prefrontal cortical cognitive functions, yet little is known about how it does so. In this study, we used forskolin, an adenylyl cyclase activator, to examine the effects of cAMP on excitatory synaptic transmission in the medial prefrontal cortex (mPFC) using whole-cell patch-clamp recordings from visually identified layer II-III or V pyramidal cells in vitro. We found that bath application of forskolin significantly increased the amplitude of excitatory postsynaptic currents (EPSCs) in a concentration- and age-dependent manner. This enhancement was completely abolished by coapplication of cAMP-dependent protein kinase (PKA) inhibitor and p42/p44 mitogen-activated protein kinase (MAPK) kinase inhibitor, but not application of either drug alone. The membrane-permeable cAMP analog adenosine 3',5'-cyclic monophosphorothioate, Sp-isomer, triethylammonium salt, or activation of beta-adrenergic receptor by isoproterenol mimicked the effect of forskolin to potentiate EPSCs. However, neither exchange protein activated by cAMP (Epac) inhibitor brefeldin A nor hyperpolarization and cyclic nucleotide-activated channel blocker 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288) affected forskolin response. The augmentation of EPSCs by forskolin was accompanied by a reduction of the synaptic failure rate, coefficient of variation and paired-pulse ratio of EPSCs, and an increase in release probability and number of releasable synaptic vesicles. Forskolin also significantly increased the frequency of miniature EPSCs without altering their amplitude distribution. These results indicate that cAMP acts presynaptically to elicit a synaptic potentiation on the layer V pyramidal neurons of mPFC through converging activation of PKA and p42/p44 MAPK signaling pathways.

Roles of Ca2+, hyperpolarization and cyclic nucleotide-activated channel activation, and actin in temporal synaptic tagging.

At crayfish neuromuscular junctions, cAMP increases transmitter released by action potentials by activating two effectors, hyperpolarization and cyclic nucleotide-activated channels (HCNCs) and a separate target that has been tentatively identified as exchange protein activated by cAMP (Epac). Intense electrical activity in the motor neuron induces a long-term facilitation (LTF) of transmitter release in which hyperpolarization from an electrogenic Na+-K+ exchanger activates HCNCs. The coupling of HCNCs to transmission involves actin. After LTF induction, cAMP further increases transmission in an HCNC-independent manner, activating the second target. This relaxation of the requirement for HCNC activation to enhance release is called temporal synaptic tagging. Tagging lasts at least 1 d but develops only in the 10 min period after electrical activity. The HCNCs are activated by the post-tetanic hyperpolarization occurring during this time. Both synaptic tagging and LTF induction depend on presynaptic Ca2+ accumulation during activity; both are blocked by EGTA-AM, and LTF is also prevented by stimulation in a low-[Ca2+] medium. Actin depolymerizers prevent induction of LTF and tagging, with little effect on HCNCs, whose sensitivity to cAMP and HCNC blockers is unaffected by tagging. Enhancement of actin polymerization can rescue tagging from HCNC block, suggesting that actin acts at a step after HCNC activation. These and other recent results suggest a model in which HCNC activation, followed by a process involving actin polymerization, acts cooperatively with [Ca2+] to induce tagging, after which only Epac activation is required for cAMP to further enhance transmission.

Mutating Three Residues in the Bovine Rod Cyclic Nucleotide-Activated Channel Can Switch a Nucleotide from Inactive to Active

Cyclic nucleotide-gated (CNG) channels, which were initially studied in retina and olfactory neurons, are activated by cytoplasmic cGMP or cAMP. Detailed comparisons of nucleotide-activated currents using nucleotide analogs and mutagenesis revealed channel-specific residues in the nucleotide-binding domain that regulate the binding and channel-activation properties. Of particular interest are N 1-oxide cAMP, which does not activate bovine rod channels, and Rp-cGMPS, which activates bovine rod, but not catfish, olfactory channels. Previously, we showed that four residues coordinate the purine interactions in the binding domain and that three of these residues vary in the α subunits of the bovine rod, catfish, and rat olfactory channels. Here we show that both N 1-oxide cAMP and Rp-cGMPS activate rat olfactory channels. A mutant of the bovine rod α subunit, substituted with residues from the rat olfactory channel at the three variable positions, was weakly activated by N 1-oxide cAMP, and a catfish olfactory-like bovine rod mutant lost activation by Rp-cGMPS. These experiments underscore the functional importance of purine contacts with three residues in the cyclic nucleotide-binding domain. Molecular models of nucleotide analogs in the binding domains, constructed with AMMP, showed differences in the purine contacts among the channels that might account for activation differences.

Molecular determinants of the modulation of cyclic nucleotide-activated channels by calmodulin.

The action of calmodulin (CaM) on target proteins is important for a variety of cellular functions. We demonstrate here, however, that the presence of a CaM-binding site on a protein does not necessarily imply a functional effect. The alpha-subunit of the cGMP-gated cation channel of human retinal cones has a CaM-binding site on its cytoplasmic N-terminal region, but the homomeric channel that it forms is not functionally modulated by CaM. Mutational analysis based on comparison to the highly homologous olfactory cyclic nucleotide-gated channel alpha-subunit, which does form a CaM-modulated channel, indicates that residues downstream of the CaM-binding domain on these channels are also important for CaM to have an effect. These findings suggest that a CaM-binding site and complementary structural features in a protein probably evolve independently, and an effect caused by CaM occurs only in the presence of both elements. More generally, the same may be true for other recognized binding sites on proteins for modulators or activators, so that a demonstrated physical interaction does not necessarily imply functional consequence.

Taste Buds Have a Cyclic Nucleotide-activated Channel, CNGgust

Cyclic nucleotide-gated (CNG) channels have been characterized as important factors involved in physiological processes including sensory reception for vision and olfaction. The possibility thus exists that a certain CNG channel functions in gustation as well. In the present study, we carried out reverse transcription-polymerase chain reaction and genomic DNA cloning and characterized a CNG channel (CNGgust) as a cyclic nucleotide-activated species expressed in rat tongue epithelial tissues where taste reception takes place. Several types of 5'-rapid amplification of cDNA ends clones of CNGgust cDNA were obtained with various 5'-terminal sequences. As the CNGgust gene was a single copy, the formation of such CNGgust variants should result from alternative splicing. The encoded protein was homologous to known vertebrate CNG channels with 50-80% similarities in amino acid sequence, and particularly homologous to bovine testis CNG channel and human cone CNG channel with 82% similarities. CNGgust was functional when expressed in human embryonic kidney cells, where it opened upon the addition of cGMP or cAMP. Immunohistochemical analysis using an antibody raised against a CNGgust peptide demonstrated the channel to be localized on the pore side of each taste bud in the circumvallate papillae, with no signal observed for degenerated taste buds after denervation of the glossopharyngeal nerve. All these results, together with the indication that cyclic nucleotides play a role gustatory signaling pathway(s), strongly suggest the involvement of CNGgust in taste signal transduction.

Cyclic nucleotide-gated ion channels: an extended family with diverse functions.

An ion channel directly activated by cGMP was first discovered about ten years ago. Since then, a number of ion channels with the same property (cyclic nucleotide-activated channels) have been reported that are involved in a variety of cell functions. In addition, other channels have been found that are not primarily controlled by cyclic nucleotides but are modulated by them (cyclic nucleotide-modulated channels). These channels likewise have diverse functions and tissue distributions. Both channel classes are reviewed here. Coverage includes the cyclic-nucleotide binding site on these channels, ion permeation, pharmacological blockers, channel gating and modulation, and physiological functions of the channels.

Localization of regions affecting an allosteric transition in cyclic nucleotide-activated channels

Sensory transduction in olfactory receptors and photoreceptors is mediated by cyclic nucleotide-activated ion channels. We have studied the gating mechanism in olfactory and rod channels expressed in Xenopus oocytes. We report that the differences in cyclic nucleotide affinity and efficacy between these channels resulted from sequence differences outside the cyclic nucleotide-binding domain, especially in the amino-terminal domain, influencing the free energy of the closed to open allosteric conformational change. In addition, Ni 2+ inhibited activation of the olfactory channel, decreasing both the maximum current and the apparent affinity for cyclic nucleotides. Ni 2+ exerted its effect by binding preferentially to the closed configuration of the channel, thereby destabilizing the opening conformational change. We have localized this inhibition to a single histidine (H396) following the last transmembrane segment, suggesting a role for this region in channel gating.

A histidine residue associated with the gate of the cyclic nucleotide-activated channels in rod photoreceptors.

Ion channels directly activated by the binding of cGMP mediate the electrical response to light in rod photoreceptors. Here, we identify a region of the channel associated with the activation gate using potentiation by intracellular Ni2+. Low concentrations of Ni2+ caused a dramatic increase in the response of rod channels expressed in Xenopus oocytes to both cGMP and cAMP. Whereas saturating cAMP normally activated less than 1% of the channels, Ni2+ increased the cAMP potency nearly 50-fold. Ni2+ did not produce potentiation in the related channel from the olfactory epithelium. We localized the Ni(2+)-binding site to a histidine residue in the putative intracellular mouth of the rod channel (H420). We propose a mechanism for potentiation in which Ni2+ binds to H420 primarily when the channel is open, stabilizing the open conformation. These experiments suggest that H420 is associated with the activation gate.

Direct modulation by Ca2+–calmodulin of cyclic nucleotide-activated channel of rat olfactory receptor neurons

Olfactory receptor neurons depolarize in response to odorant stimulation of their sensory cilia. One transduction mechanism involves a G-protein-mediated increase in adenylate cyclase activity, raising the internal cyclic AMP concentration to open a cyclic nucleotide-activated cation channel on the plasma membrane. An influx of Ca2+ through this channel, which is permeable to both monovalent and divalent cations, triggers olfactory adaptation. Previous work has indicated that at least part of this Ca(2+)-mediated adaptation resides in the channel itself, but the mechanism remains unclear and controversial. Here we use the cloned channel from rat expressed in a cell line and the native channel from rat olfactory receptor cells to show that Ca2+ reduces the apparent affinity of the channel for cAMP by up to 20-fold in the presence of calmodulin, an abundant protein in olfactory cilia. This decrease in apparent affinity appears to involve a direct interaction between Ca(2+)-calmodulin and the channel, and it can reduce the activation of the channel by cAMP by up to a few hundred-fold, suggesting that it may be a key component of the Ca(2+)-triggered olfactory adaptation.

Cyclic nucleotide-activated channels in carp olfactory receptor cells

When applied from the cytoplasmic side, cyclic 3′,5′-adenosine and guanosine monophosphates reversibly increased the ion permeability of inside-out patches of carp olfactory neuron plasma membrane. The cAMP (cGMP)-induced permeability via cAMP (cGMP) concentration was fitted by Hill's equation with the exponents of 1.07 ± 0.15 (1.12 ± 0.05) and EC 50 = 1.3 ± 0.6 μ M (0.9 ± 0.3 μ M) . Substitution of NaCl in the bathing solution by chlorides of other alkali metals resulted in a slight shift of reversal potential of the cyclic nucleotide-dependent (CN) current, which indicates a weak selectivity of the channels. Permeability coefficients calculated by Goldman-Hodgkin-Katz's equation corresponded to the following relation: P Na /P K /P Li /P Rb /P Cs = 1:0.98:0.94:0.70:0.61 . Ca 2+ and Mg 2+ in physiological concentrations blocked the channels activated by cyclic nucleotides (CN-channels). In the absence of divalent cations the conductance of single CN-channels was equal to 51 ± 9 pS in 100 mM NaCl solution. Channel density did not exceed 1 μm −2. The maximal open state probability of the channel ( P o ) tended towards 1.0 at a high concentration of cAMP or cGMP. Dichlorobenzamil decreased P o without changing the single CN-channel conductance. CN-channels exhibited burst activity. Mean open and closed times as well as the burst duration depended on agonist concentration. A kinetic model with four states (an inactivated, a closed and two open ones) is suggested to explain the regularities of CN-channel gating and dose—response relations.

Primary structure and functional expression of a cyclic nucleotide-activated channel from olfactory neurons

Odorant signal transduction occurs in the specialized cilia of the olfactory sensory neurons. Considerable biochemical evidence now indicates that this process could be mediated by a G protein-coupled cascade using cyclic AMP as an intracellular second messenger. A stimulatory G protein alpha subunit is expressed at high levels in olfactory neurons and is specifically enriched in the cilia, as is a novel form of adenylyl cyclase. This implies that the olfactory transduction cascade might involve unique molecular components. Electrophysiological studies have identified a cyclic nucleotide-activated ion channel in olfactory cilia. These observations provide evidence for a model in which odorants increase intracellular cAMP concentration, which in turn activates this channel and depolarizes the sensory neuron. An analogous cascade regulating a cGMP-gated channel mediates visual transduction in photoreceptor cells. The formal similarities between olfactory and visual transduction suggest that the two systems might use homologous channels. Here we report the molecular cloning, functional expression and characterization of a channel that is likely to mediate olfactory transduction.

Cyclic nucleotide-activated channels in the frog olfactory receptor plasma membrane

Patch clamp technique was used to record cyclic nucleotide-dependent current of the frog olfactory receptor cell plasma membrane. Data obtained indicate that the channels passing this current are permeable to Ca 2+ or Mg 2+ and moderately selective for monovalent cations according to the sequence Li +, Na +, K + > Rb + > Cs + and are effectively blocked by 1- cis-diltiazem and 3′,4′-dichlorobenzamil. The conductance of single cyclic nucleotide-gated channels in solutions with low Ca 2+ and Mg 2+ content is about 19 pS. The results demonstrate that cyclic nucleotide-activated channels of olfactory receptor cells are virtually identical to photoreceptor ones.