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PubMed1997

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.

Amino Acid Sequence↗
PubMed2000

Mutation of a single residue in the S2-S3 loop of CNG channels alters the gating properties and sensitivity to inhibitors.

We previously found that native cyclic nucleotide-gated (CNG) cation channels from amphibian rod cells are directly and reversibly inhibited by analogues of diacylglycerol (DAG), but little is known about the mechanism of this inhibition. We recently determined that, at saturating cGMP concentrations, DAG completely inhibits cloned bovine rod (Brod) CNG channels while only partially inhibiting cloned rat olfactory (Rolf) channels (Crary, J.I., D.M. Dean, W. Nguitragool, P.T. Kurshan, and A.L. Zimmerman. 2000. J. Gen. Phys. 116:755-768; in this issue). Here, we report that a point mutation at position 204 in the S2-S3 loop of Rolf and a mouse CNG channel (Molf) found in olfactory epithelium and heart, increased DAG sensitivity to that of the Brod channel. Mutation of this residue from the wild-type glycine to a glutamate (Molf G204E) or aspartate (Molf G204D) gave dramatic increases in DAG sensitivity without changing the apparent cGMP or cAMP affinities or efficacies. However, unlike the wild-type olfactory channels, these mutants demonstrated voltage-dependent gating with obvious activation and deactivation kinetics. Interestingly, the mutants were also more sensitive to inhibition by the local anesthetic, tetracaine. Replacement of the position 204 glycine with a tryptophan residue (Rolf G204W) not only gave voltage-dependent gating and an increased sensitivity to DAG and tetracaine, but also showed reduced apparent agonist affinity and cAMP efficacy. Sequence comparisons show that the glycine at position 204 in the S2-S3 loop is highly conserved, and our findings indicate that its alteration can have critical consequences for channel gating and inhibition.

Amino Acid Sequence↗
PubMed1999

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.

Animals↗
PubMed1997

Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel.

We used Northern blot analysis, ribonuclease protection assay (RPA), reverse transcriptase-polymerase chain reaction, and in situ hybridization to investigate the hypothesis that the CNG1 isoform of the cyclic nucleotide-gated nonselective cation channel may be widely distributed in tissues of the rat. A cDNA encoding the CNG1 isoform was isolated from rat eye and human retina, and partial sequences were isolated from rat pineal gland and human kidney. Northern blot analysis revealed a 3.1-kilobase (kb) CNG1 transcript in rat eye, pineal gland, pituitary, adrenal gland, and spleen, and a larger transcript of 3.5 kb was found in testis. RPA confirmed the identity of CNG1 mRNA in rat eye, lung, spleen, and brain. Polymerase chain reaction-based detection of the mRNA for CNG1 indicates that the channel is expressed in lower abundance in many other tissues, including thymus, skeletal muscle, heart, and parathyroid gland. The cellular distribution of CNG1 was further studied by in situ hybridization, which demonstrated expression of mRNA in lung, thymus, pineal gland, hippocampus, cerebellum, and cerebral cortex but not in heart or kidney.

Amino Acid Sequence↗
PubMed1999

The native rat olfactory cyclic nucleotide-gated channel is composed of three distinct subunits.

Cyclic nucleotide-gated (CNG) channels play central roles in visual and olfactory signal transduction. In the retina, rod photoreceptors express the subunits CNCalpha1 and CNCbeta1a. In cone photoreceptors, only CNCalpha2 expression has been demonstrated so far. Rat olfactory sensory neurons (OSNs) express two homologous subunits, here designated CNCalpha3 and CNCalpha4. This paper describes the characterization of CNCbeta1b, a third subunit expressed in OSNs and establishes it as a component of the native channel. CNCbeta1b is an alternate splice form of the rod photoreceptor CNCbeta1a subunit. Analysis of mRNA and protein expression together suggest co-expression of all three subunits in sensory cilia of OSNs. From single-channel analyses of native rat olfactory channels and of channels expressed heterologously from all possible combinations of the CNCalpha3, -alpha4, and -beta1b subunits, we conclude that the native CNG channel in OSNs is composed of all three subunits. Thus, CNG channels in both rod photoreceptors and olfactory sensory neurons result from coassembly of specific alpha subunits with various forms of an alternatively spliced beta subunit.

Alternative Splicing↗
PubMed1999

Dual pharmacological properties of a cyclic AMP-sensitive potassium channel.

Bovine adrenal zona fasciculata cells express a novel K+ current (IAC) that sets the resting potential while it couples adrenocorticotropin and angiotensin II receptors to membrane depolarization and cortisol secretion. IAC is distinctive among K+ channels both in its activation by ATP and its inhibition by cyclic AMP. Whole-cell and single-channel patch-clamp recording was used to establish a pharmacological profile of IAC K+ channels. IAC was blocked by antagonists of cyclic nucleotide-gated channels, including the diphenylbutylpiperidine (DPBP) antipsychotic pimozide and l-cis-diltiazem. Other DPBPs, including penfluridol and fluspirilene, also potently inhibited this channel. The inhibition of IAC by DPBPs was selective because 200-fold higher concentrations of penfluridol were required to inhibit voltage-gated IA K+ channels in adrenal zona fasciculata cells. Standard K+ channel antagonists blocked IAC at concentrations 100- to 100,000-fold higher than the DPBPs. IAC channels were also inhibited by the sulfonylureas glyburide and tolbutamide but at concentrations higher than those that typically block ATP-sensitive inward rectifier K+ channels. Overall, the relative order of potency and associated IC50 values for IAC antagonists were as follows: penfluridol (0.187 microM) > fluspirilene (0.232 microM) > pimozide (0.354 microM) >> l-cis-diltiazem (24.9 microM) approximately quinidine (24.1 microM) > bupivacaine (113.2 microM) > tolbutamide (784.4 microM) > BaCl2 (1027 microM) > 4-aminopyridine (2750 microM) > tetraethylammonium (24,270 microM). IAC channels are unique in combining the pharmacological properties of K+-selective channels with those of cyclic nucleotide-gated cation channels. The potent block of IAC channels identifies DPBPs as a new class of K+ channel antagonists and suggests additional targets for these neuroleptics in the central nervous system.

Animals↗
PubMed2003

Disruption of an intersubunit interaction underlies Ca2+-calmodulin modulation of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated channels are key molecular elements for olfactory transduction. Olfactory adaptation caused by repeated exposure to an odorant has been proposed to be mediated by the binding of Ca2+-calmodulin to the NH2-terminal domain of the channel, breaking its interaction with the COOH-terminal domain and downregulating the channel. We used a fluorescence resonance energy transfer (FRET) approach to study the structural aspects of this domain-domain interaction under physiological conditions in real time. Fluorescent proteins enhanced cyan fluorescent protein and enhanced yellow fluorescent protein were genetically attached at sites adjacent to the NH2- and COOH-terminal interacting domains, respectively, allowing direct observation of molecular rearrangements in intact channels. FRET signals caused by the specific interdomain interaction were observed in both intact cells and excised patches. Comparison of the effective FRET efficiencies demonstrated that the interaction occurs specifically between subunits but not within the same subunit. Binding of Ca2+-calmodulin caused a reversible decrease in FRET with the same time course as channel downregulation. These results suggest that a separation or reorientation of the interacting domains between subunits by Ca2+-calmodulin leads to channel downregulation. The quaternary arrangement presents a structural framework for understanding the molecular mechanism of olfactory adaptation.

Adaptation, Physiological↗
PubMed2005

General anesthetic-induced channel gating enhancement of 5-hydroxytryptamine type 3 receptors depends on receptor subunit composition.

5-Hydroxytryptamine (serotonin) (5-HT) type 3 (5-HT(3)) receptors are members of an anesthetic-sensitive superfamily of Cys-loop ligand-gated ion channels that can be formed as homomeric 5-HT(3A) or heteromeric 5-HT(3AB) receptors. When the efficacious agonist 5-HT is used, the inhaled anesthetics halothane and chloroform (at clinically relevant concentrations) significantly reduce the agonist EC(50) for 5-HT(3A) receptors but not for 5-HT(3AB) receptors. In the present study, we used dopamine (DA), a highly inefficacious agonist for 5-HT(3) receptors, to determine whether the difference in sensitivity between 5-HT(3A) and 5-HT(3AB) receptors to the potentiating effects of halothane and chloroform is due to differential modulation of agonist affinity, channel gating, or both. Using the two-electrode voltage-clamp technique with 5-HT(3A) and 5-HT(3AB) receptors expressed in Xenopus oocytes, we found that chloroform and halothane enhanced currents evoked by receptor-saturating concentrations of DA for both receptor subtypes in a concentration-dependent manner but that the magnitude of enhancement was substantially greater for 5-HT(3A) receptors than for 5-HT(3AB) receptors. Isoflurane induced only a small enhancement of currents evoked by receptor-saturating concentrations of DA for 5-HT(3A) receptors and no enhancement for 5-HT(3AB) receptors. For both receptor subtypes, none of the three test anesthetics significantly altered the agonist EC(50) for DA, implying that these anesthetics do not affect agonist binding affinity. Our results show that chloroform, halothane, and (to a much lesser degree) isoflurane enhance channel gating for 5-HT(3A) receptors and that the incorporation of 5-HT(3B) subunits to produce heteromeric 5-HT(3AB) receptors markedly attenuates the ability of these anesthetics to enhance channel gating.

Anesthetics, General↗
PubMed1985

Phasic ion channel blockade. A kinetic model and parameter estimation procedure.

For excitable membranes, use and frequency dependence represent a progressive incorporation of drug into gated ion channels with repetitive stimulation. In contrast to receptors where access to ligand is continuous in time, we define guarded receptors, such as gated ion channels, as receptors whose access to the ligand pool is transient and controlled by the channel-gating process. During repetitive stimulation, the fraction of ligand-bound channels (ion channel blockade) follows an exponential time course, determined by the interstimulus interval, channel-gating processes, drug concentration, and the forward and reverse rate coefficients characteristic of the binding process. Based on a first order model of ligand-receptor binding, we derive a characterization of ion channel blockade via a single diffusion path under conditions of repetitive phasic stimulation. Extension to multiple diffusion paths and multiple drugs leads to a more complex scheme, but these generalizations are straightforward. For the case of one diffusion path, we derive the steady state level of channel blockade for guarded receptors as a function of stimulus rate and develop a data analysis strategy suitable for characterizing ion channel-blocking agents such as local anesthetics and antiarrhythmic drugs. We show that as receptor access time increases, the transient and steady state properties of guarded receptors become equivalent to those derived from the standard continuous access ligand-receptor model. The analysis tools presented simplify the quantitative description of the functional properties of many ion channel blockers and appear to have general applicability to characterization of periodically accessible receptors.

Animals↗
PubMed1999

Mutations in the S4 region isolate the final voltage-dependent cooperative step in potassium channel activation.

Charged residues in the S4 transmembrane segment play a key role in determining the sensitivity of voltage-gated ion channels to changes in voltage across the cell membrane. However, cooperative interactions between subunits also affect the voltage dependence of channel opening, and these interactions can be altered by making substitutions at uncharged residues in the S4 region. We have studied the activation of two mutant Shaker channels that have different S4 amino acid sequences, ILT (V369I, I372L, and S376T) and Shaw S4 (the S4 of Drosophila Shaw substituted into Shaker), and yet have very similar ionic current properties. Both mutations affect cooperativity, making a cooperative transition in the activation pathway rate limiting and shifting it to very positive voltages, but analysis of gating and ionic current recordings reveals that the ILT and Shaw S4 mutant channels have different activation pathways. Analysis of gating currents suggests that the dominant effect of the ILT mutation is to make the final cooperative transition to the open state of the channel rate limiting in an activation pathway that otherwise resembles that of Shaker. The charge movement associated with the final gating transition in ILT activation can be measured as an isolated component of charge movement in the voltage range of channel opening and accounts for 13% ( approximately 1.8 e0) of the total charge moved in the ILT activation pathway. The remainder of the ILT gating charge (87%) moves at negative voltages, where channels do not open, and confirms the presence of Shaker-like conformational changes between closed states in the activation pathway. In contrast to ILT, the activation pathway of Shaw S4 seems to involve a single cooperative charge-moving step between a closed and an open state. We cannot detect any voltage-dependent transitions between closed states for Shaw S4. Restoring basic residues that are missing in Shaw S4 (R1, R2, and K7) rescues charge movement between closed states in the activation pathway, but does not alter the voltage dependence of the rate-limiting transition in activation.

Animals↗
PubMed2004

Voltage-dependent inhibition of rat skeletal muscle sodium channels by aminoglycoside antibiotics.

Aminoglycoside (AG) antibiotics interact with numerous biological molecules, including some voltage-gated ion channels. The present study demonstrates that 4,5-disubstituted (neomycin class) and 4,6-disubstituted (kanamycin class) AGs inhibit whole-cell currents through cloned rat skeletal muscle sodium channels (mu1, Na(V)4.1). Increases in the amplitude of the step command reduced inhibition by extracellular AGs but increased inhibition by intracellularly applied AGs, indicating that the block was voltage dependent. Furthermore, intracellular neamine or sisomycin hastened the rate of macroscopic current decay at positive voltages. Extracellular solution containing sodium ions slowed the rate of current decay in the presence of intracellular sisomycin and decreased the apparent affinity of sisomycin from the intracellular side twofold. Current inhibition by extracellularly or intracellularly applied AGs was well fitted by the Woodhull model of pore block. The model indicated that most extracellularly applied AGs interact at a site that is an electrical distance of approximately 10-15% from the outside, whereas intracellularly applied neamine or sisomycin bind to sites that are approximately 49% and approximately 24%, respectively, into the electric field from the inside. Our data suggested that AG antibiotics induce a low-affinity, voltage-dependent block of mu1 channels.

Algorithms↗
PubMed1993

Chimaeric nicotinic-serotonergic receptor combines distinct ligand binding and channel specificities.

The neuronal nicotinic alpha 7 (nAChR) and 5-hydroxytryptamine (5HT3) receptors are ligand-gated ion channels with a homologous topological organization and have activation and desensitization reactions in common. Yet these homo-oligomeric receptors differ in the pharmacology of their binding sites for agonists and competitive antagonists, and in their sensitivity to Ca2+ ions. The alpha 7 channel is highly permeable to Ca2+ ions and external Ca2+ ions potentiate, in an allosteric manner, the permeability response to acetylcholine, as shown for other neuronal nAChRs. The 5HT3 channel, in contrast, is not permeable to Ca2+ ions, but blocked by them. To assign these properties to delimited domains of the primary structure, we constructed several recombinant chimaeric alpha 7-5HT3 receptors. We report here that one of the constructs expresses a functional receptor that contains the serotonergic channel still blocked by Ca2+ ions, but is activated by nicotinic ligands and potentiated by external Ca2+ ions.

Acetylcholine↗
PubMed1998

Fluorescence imaging of Na+ influx via P2X receptors in cochlear hair cells.

The adenosine 5'-triphosphate (ATP)-activated membrane conductance, mediated by P2X receptors, was examined in isolated guinea-pig cochlear inner and outer hair cells. Photo-activated release of caged-ATP elicted a 30-ms latency inwardly rectifying non-selective cation conductance, blocked by the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS; 10-100 microM), consistent with the direct activation of ATP-gated ion channels. A K(Ca) conductance in the inner hair cells (IHC), activated by the entry of Ca2+ through the ATP-gated ion channels, was blocked by including 10 mM 1,2-his(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) in the internal solution. Real-time confocal slit-scanning fluorescence imaging of Na+ influx through the ATP-gated ion channels was performed using the dye Sodium Green with simultaneous whole-cell recording of membrane currents. The Na+ entry was localized to the endolymphatic surface, with the increase in [Na+]i detected within approximately 200 ms of the onset of the inward current response. Within 600 ms Na+ had diffused throughout the cell cytoplasm with the exception of the subnuclear region of the outer hair cells. Correlation of voltage-clamp measurements of Na+ entry with regional increases in Na+-induced fluorescence demonstrated ATP-induced increases in intracellular Na+ in excess of 45 mM within 4 s. These data provide direct evidence for the Na+ permeability of the ATP-gated ion channels as well as independent evidence for the localization of P2X receptors at the endolymphatic surface of the sensory hair cells. The localization of the ATP-gated ion channels to the apical surface of the hair cells supports an ATP-mediated modulation of 'silent' K+ current across the cochlear partition which could regulate hearing sensitivity by controlling the transcellular driving force for both mechanoelectrical and electromechanical transduction in hair cells.

Adenosine Triphosphate↗
PubMed1991

Sodium and potassium channels in epithelial cells from thymus glands and thymomas of myasthenia gravis patients.

In both normal and neoplastic epithelial cells from human thymus glands and thymomas, respectively, we found voltage-gated sodium and potassium channels that resemble the adult-type Na channel and the delayed outward rectifier K channel, respectively, of human skeletal muscle and mammalian nervous system. These voltage-gated ion channels might be part of a communication system between epithelial cells and other components of the microenvironment of the thymus.

Electric Conductivity↗
PubMed2005

HCN subunit-specific and cAMP-modulated effects of anesthetics on neuronal pacemaker currents.

General anesthetics have been a mainstay of surgical practice for more than 150 years, but the mechanisms by which they mediate their important clinical actions remain unclear. Ion channels represent important anesthetic targets, and, although GABA(A) receptors have emerged as major contributors to sedative, immobilizing, and hypnotic effects of intravenous anesthetics, a role for those receptors is less certain in the case of inhalational anesthetics. The neuronal hyperpolarization-activated pacemaker current (Ih) is essential for oscillatory and integrative properties in numerous cell types. Here, we show that clinically relevant concentrations of inhalational anesthetics modulate neuronal Ih and the corresponding HCN channels in a subunit-specific and cAMP-dependent manner. Anesthetic inhibition of Ih involves a hyperpolarizing shift in voltage dependence of activation and a decrease in maximal current amplitude; these effects can be ascribed to HCN1 and HCN2 subunits, respectively, and both actions are recapitulated in heteromeric HCN1-HCN2 channels. Mutagenesis and simulations suggest that apparently distinct actions of anesthetics on V(1/2) and amplitude represent different manifestations of a single underlying mechanism (i.e., stabilization of channel closed state), with the predominant action determined by basal inhibition imposed by individual subunit C-terminal domains and relieved by cAMP. These data reveal a molecular basis for multiple actions of anesthetics on neuronal HCN channels, highlight the importance of proximal C terminus in modulation of HCN channel gating by diverse agents, and advance neuronal pacemaker channels as potentially relevant targets for clinical actions of inhaled anesthetics.

Anesthetics↗
PubMed1990

Cyclic AMP-dependent protein kinase decreases GABAA receptor current in mouse spinal neurons.

GABA, the major inhibitory neurotransmitter in the mammalian brain, binds to GABAA receptors, which form chloride ion channels. The predicted structure of the GABAA receptor places a consensus phosphorylation site for cAMP-dependent protein kinase (PKA) on an intracellular domain of the channel. Phosphorylation by various protein kinases has been shown to alter the activity of certain ligand- and voltage-gated ion channels. We have examined the role of phosphorylation by the catalytic subunit of PKA in the regulation of GABAA receptor channel function using whole-cell and excised outside-out patch-clamp techniques. Inclusion of the catalytic subunit of PKA in the recording pipettes significantly reduced GABA-evoked whole-cell and single-channel chloride currents. Both heat inactivation of PKA and addition of the specific protein kinase inhibitor peptide prevented the reduction of GABA-evoked currents by PKA. Neither mean channel open time nor channel conductance was affected by PKA. The reduction in GABA receptor current by PKA was primarily due to a reduction in channel opening frequency.

Animals↗