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PubMed1995

InsP3-gated ion channels in rat olfactory cilia membrane.

InsP3-gated channels present in isolated rat olfactory cilia membranes were studied by recording current fluctuations in cilia membranes fused onto phospholipid bilayers formed at the tip of a patch pipette. We found that InsP3 (1.25 to 30 microM) induced current fluctuations in 18 of 157 otherwise silent bilayers. The ion channels opened by InsP3 could be classified into two groups that differed in terms of conductance and kinetics. One channel, which had a conductance of 37 pS, displayed two current levels, and the larger single channel currents were associated with longer open dwell times. The other channel displayed a larger conductance (103-184 pS). Both types of channels displayed reversal potentials near zero millivolts, consistent with nonspecific cation channels. These experiments suggest that mammalian olfactory neurons possess two types of ciliary InsP3-gated channels.

Animals↗
PubMed2003

Therapeutic approaches targeting prostate cancer progression using novel voltage-gated ion channel blockers.

The early detection and treatment of prostate cancer have increased survival and improved clinical outcomes. The nature of the disease and pathologic understaging result in a high proportion of patients developing locally recurrent disease or distant metastases. The development of prostate cancer the time from tumor initiation and progression to invasive carcinoma often begins in men in the fourth or fifth decades of life and extends across decades. This prolonged window highlights the tremendous clinical impact that early intervention with therapeutic agents that selectively target the invasive and metastatic potential of the prostate cancer cell could have on patient survival and quality of life. Our research is currently focused on the development and testing of novel voltage-gated ion channel blockers. The expression of voltage-gated sodium channels (VGSCs) was recently associated with the metastatic behavior of prostate cancer cells. In these studies, VGSC blockers altered prostate cancer cell morphology and arrested prostate cancer cell migration. Clinically, one of the most widely used sodium channel blockers is phenytoin. We have used rational drug design based on the phenytoin binding site in a VGSC to make novel sodium channel blockers with enhanced activity and minimal acute toxicity. Our initial studies in vitro demonstrate enhanced binding of the compounds to the sodium channel and increased inhibition of prostate cancer cell growth in culture and in soft agarose compared with phenytoin. These derivatives are currently being tested for their antitumor activity in human prostate cancer xenografts.

Adenocarcinoma↗
PubMed1991

Structure-activity relationship of philanthotoxins--II. Effects on the glutamate gated ion channels of the locust muscle fibre membrane.

1. PTX-4.3.3, the synthetic product of the wasp toxin, delta-philanthotoxin, blocks the open, glutamate gated, ion channels of the locust muscle. 2. This block is accompanied by the appearance of double exponential distribution of the closed times frequency, and a decrease of the inverse relationship between the open and closed states. 3. The analogues with the shortened polyamine chain, PTX-4.3 and PTX-3.3, are less potent, trifluoromethyl-PTX-4.3.3 is a more potent analogue. 4. Yet, they still behave like PTX-4.3.3 as open ion-channel blockers. 5. Another analogue, without the aromatic nucleus, dephenol-PTX-4.3.3, proved to act in a different way. 6. This less potent analogue, doesn't block the open ion channels. 7. Most likely, the not-activation induced, non-competitive antagonism, caused by dephenol-PTX-4.3.3, has a modulatory effect on glutamate receptors. 8. Methyl-PTX-4.3.3 is also an open ion-channel blocker. However, at relatively low concentrations it has an agonistic-like effect, probably of a modulatory origin as well. 9. A long-term inhibition of PTX-4.3.3 applied at low concentrations also indicates that beside an open ion-channel block, also other inhibitory processes are involved.

Animals↗
PubMed2002

A site in the fourth membrane-associated domain of the N-methyl-D-aspartate receptor regulates desensitization and ion channel gating.

The N-methyl-d-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2). The M1-M3 domains have been demonstrated to influence the function of the ion channel, but a similar role for the M4 domain has not been reported. We have identified a methionine residue (Met(823)) in the M4 domain of the NR2A subunit that regulates desensitization and ion channel gating. A tryptophan substitution at this site did not alter the EC(50) for glycine or the peak NMDA EC(50) but decreased the steady-state NMDA EC(50) and markedly increased apparent desensitization, mean open time, and peak current density. Results of rapid solution exchange experiments revealed that changes in microscopic desensitization rates and closing rates could account for the changes in macroscopic desensitization, steady-state NMDA EC(50), and current density. Other amino acid substitutions at this site could increase or decrease the rate of desensitization and mean open time of the ion channel. Both mean open time and desensitization were dependent primarily upon the hydrophobic character of the amino acid at the position. These results demonstrate an important role for hydrophobic interactions at Met(823) in regulation of NMDA receptor function.

Amino Acid Sequence↗
PubMed2004

An amino-acid substitution in the influenza-B NB protein affects ion-channel gating.

The effects of site-directed mutations in NB, a protein encoded by the influenza B virus that has been shown to form cation-selective ion channels at pH 6.0, were studied on ion channel characteristics in artificial lipid bilayers. It was thought that the residues in the hydrophobic region of NB we selected for mutation might be involved in the transport of cations across the channel and that changes in these residues might affect channel properties such as gating and ion-selectivity. Serine residues at positions 20 and 28, threonine at position 24 and cysteine at position 26 were replaced by alanine. We found that the mutation S20A gave channels that did not gate and that remained open most of the time. Proton permeability of NB channels, as detected by fluorescence quenching, was also altered by the mutation S20A: channels were no longer proton-permeable. The other mutations, S28A, T24A and C26A, did not have any detectable effect on the activity or proton permeability of channels formed by NB. The results indicate that serine 20 may have an important role in normal function of NB channels.

Amino Acid Substitution↗
PubMed2001

Phosphorylation of voltage-gated ion channels in rat olfactory receptor neurons.

In olfactory receptor neurons (ORNs), ligand-odorant receptor interactions cause G protein-mediated activation of adenylate cyclase and a subsequent increase in concentration of the intracellular messenger cAMP. Odorant-evoked elevation in cAMP is thought to directly activate a cation-selective cyclic nucleotide-gated channel, which causes external Ca2+ influx, leading to membrane depolarization and the generation of action potentials. Our data show that in freshly dissociated rat ORNs, odorant-induced elevation in cAMP also activates cAMP-dependent protein kinase (PKA), which is then able to phosphorylate various protein targets in the olfactory signal transduction pathway, specifically voltage-gated sodium and calcium channels. The presence of PKI (PKA inhibitor peptide) blocked the modulatory action of cAMP on voltage-gated ion channels. By modulating the input/output properties of the sensory neurons, this mechanism could take part in the complex adaptation process in odorant perception. In addition, we found modulation of voltage-gated sodium and calcium channel currents by 5-hydroxytryptamine and the dopamine D1 receptor agonist SKF 38393. These findings suggest that in situ ORNs might also be a target for efferent modulation.

Animals↗
PubMed2000

Intravenous anesthetics differentially modulate ligand-gated ion channels.

BACKGROUND: Heteromeric neuronal nicotinic acetylcholine receptors (nAChRs) are potently inhibited by volatile anesthetics, but it is not known whether they are affected by intravenous anesthetics. Ketamine potentiates gamma-aminobutyric acid type A (GABAA) receptors at high concentrations, but it is unknown whether there is potentiation at clinically relevant concentrations. Information about the effects of intravenous anesthetics with different behavioral profiles on specific ligand-gated ion channels may lead to hypotheses as to which ion channel effect produces a specific anesthetic behavior. METHODS: A heteromeric nAChR composed of alpha4 and beta4 subunits was expressed heterologously in Xenopus laevis oocytes. Using the two-electrode voltage clamp technique, peak ACh-gated current was measured before and during application of ketamine, etomidate, or thiopental. The response to GABA of alpha1beta2gamma2s GABAA receptors expressed in human embryonic kidney cells and Xenopus oocytes was compared with and without coapplication of ketamine from 1 microm to 10 mm. RESULTS: Ketamine caused potent, concentration-dependent inhibition of the alpha4beta4 nAChR current with an IC50 of 0.24 microm. The inhibition by ketamine was use-dependent; the antagonist was more effective when the channel had been opened by agonist. Ketamine did not modulate the alpha1beta2gamma2s GABAA receptor response in the clinically relevant concentration range. Thiopental caused 27% inhibition of ACh response at its clinical EC50. Etomidate did not modulate the alpha4beta4 nAChR response in the clinically relevant concentration range, although there was inhibition at very high concentrations. CONCLUSIONS: The alpha4beta4 nAChR, which is predominantly found in the central nervous system (CNS), is differentially affected by clinically relevant concentrations of intravenous anesthetics. Ketamine, commonly known to be an inhibitor at the N-methyl-D-aspartate receptor, is also a potent inhibitor at a central nAChR. It has little effect on a common CNS GABAA receptor in a clinically relevant concentration range. Interaction between ketamine and specific subtypes of nAChRs in the CNS may result in anesthetic behaviors such as inattention to surgical stimulus and in analgesia. Thiopental causes minor inhibition at the alpha4beta4 nAChR. Modulation of the alpha4beta4 nAChR by etomidate is unlikely to be important in anesthesia practice based on the insensitivity of this receptor to clinically used concentrations.

Anesthetics, Dissociative↗
PubMed1999

Membrane potential and time requirements for detection of weak signals by voltage-gated ion channels.

The question of minimum detection limits for biological processes sensitive to membrane potential perturbations has arisen in various contexts. Of special interest are the prediction of theoretical limits for sensory perception processes and for possible biological effects of environmental or therapeutic electric and magnetic fields. A new method is presented here, addressing the particular case in which perturbations of membrane potential affect the gating rate probability of voltage-sensitive ion channels. Using a two-state model for channel gating, the influence of the perturbing potential on the mean fraction of open channels is approximated by a Boltzmann distribution, and integrated over time to obtain a quantity proportional to the net change in expected charge transfer through the membrane. This change in net charge transfer (the signal, S) is compared to the expected root mean variance in charge transfer (the noise, N) due to random channel gating. Using a nominal criterion of S/N = 1, a model is developed for predicting the minimum time and number of ion channels necessary to detect a given membrane potential. Example calculations, carried out for a gating charge of 6, indicate that a 1 microV induced membrane potential can be detected after 10 ms by an ensemble of less than 10(8) ion channels.

Algorithms↗
PubMed1992

A new look at the mechanism of activation and inactivation of voltage-gated ion channels.

Studies on the kinetics of activation and inactivation of the sodium channels of the squid giant axon, on the sodium gating current, and on the properties of the non-inactivating steady-state current, are briefly reviewed. Taken in conjunction with recent evidence on the structure of voltage-gated ion channels, they have led to the development of a series-parallel model of the sodium channel that can be regarded as a modernized version of the Hodgkin-Huxley model, with some novel features. It is suggested that activation results from conformational changes brought about by the four S4 voltage sensors operating in parallel, each of which makes two discrete steps to reach the fully activated state of the channel. There follows a voltage-independent hydration step, and the channel is ready to open. Inactivation is a potential-dependent process involving a third transition of voltage sensor S4d alone, which, rather than bringing a ball and chain blocking group into position to close the channels, serves to switch the system so that it passes from an initial activated mode, in which there is a high probability of arriving at an open state with a brief latency, to a second steady-state mode, in which the probability of opening is very much lower.

Animals↗
PubMed2000

Irreversible activation of cyclic nucleotide-gated ion channels by sulfhydryl-reactive derivatives of cyclic GMP.

First discovered in the sensory epithelium of the visual and olfactory systems, cyclic nucleotide-gated (CNG) ion channels have now been found in tissues throughout the body. Native rod CNG channels are tetramers composed of homologous, but distinct, alpha- and beta-subunits. The goal of this study was to develop a novel method for targeting covalent attachment of cGMP to individual subunit types. Toward this goal, we have found that treatment of membrane patches expressing rod alpha-subunit channels with sulfhydryl-reactive derivatives of cGMP resulted in irreversible activation. The persistent currents were sensitive to block by both Mg(2+) and tetracaine. Pretreatment of the patch with the sulfhydryl-blocking reagents N-ethylmaleimide (NEM) and bis-dithionitrobenzoic acid (DTNB) prevented covalent activation; the effect of DTNB was reversed by reduction with DTT. Furthermore, the process of covalent activation was dramatically slowed by the presence of an excess of 8-Br-cGMP. These results suggested that covalent activation resulted from the tethering of cGMP near the channel's ligand-binding sites by reaction with an endogenous cysteine. The alpha-subunit of the rod channel contains seven cysteine residues, and we set out to determine the site of attachment by site-directed mutagenesis. Surprisingly, irreversible activation was not abolished by elimination of all seven cysteine residues. This result suggests that the site of attachment is on a tightly associated protein, rather than on the channel protein itself. To further investigate these results, we treated patches containing irreversibly activated channels with 100 microg/mL trypsin and discovered two modes of covalent activation. One type developed rapidly and was removed by trypsin treatment, and the second developed slowly and was resistant to trypsin treatment. Both types of covalent activation were present in all mutants tested and were also present when CNG channels were expressed in HEK-293 cells. These results suggest that CNG channel subunits may associate with endogenous proteins when they are expressed in heterologous systems.

Affinity Labels↗
PubMed1991

Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.

The neurotransmitter serotonin (5HT) activates a variety of second messenger signaling systems and through them indirectly regulates the function of ion channels. Serotonin also activates ion channels directly, suggesting that it may also mediate rapid, excitatory responses. A complementary DNA clone containing the coding sequence of one of these rapidly responding channels, a 5HT3 subtype of the serotonin receptor, has been isolated by screening a neuroblastoma expression library for functional expression of serotonin-gated currents in Xenopus oocytes. The predicted protein product has many of the features shared by other members of the ligand-gated ion channel family. The pharmacological and electrophysiological characteristics of the cloned receptor are largely consistent with the properties of native 5HT3 receptors. Messenger RNA encoding this receptor is found in the brain, spinal cord, and heart. This receptor defines a new class of excitatory ligand-gated channels.

Amino Acid Sequence↗
PubMed2002

Activation of NMDA receptors linked to modulation of voltage-gated ion channels and functional implications.

Catfish (Ictalurus punctatus) cone horizontal cells contain N-methyl-d-aspartate (NMDA) receptors, the function of which has yet to be determined. In the present study, we have examined the effect of NMDA receptor activation on voltage-gated ion channel activity. NMDA receptor activation produced a long-term downregulation of voltage-gated sodium and calcium currents but had no effect on the delayed rectifying potassium current. NMDA's effect was eliminated in the presence of AP-7. To determine whether NMDA receptor activation had functional implications, isolated catfish cone horizontal cells were current clamped to mimic the cell's physiological response. When horizontal cells were depolarized, they elicited a single depolarizing overshoot and maintained a depolarized steady state membrane potential. NMDA reduced the amplitude of the depolarizing overshoot and increased the depolarized steady-state membrane potential. Both effects of NMDA were eliminated in the presence of AP-7. These results support the hypothesis that activation of NMDA receptors in catfish horizontal cells may affect the type of visual information conveyed through the distal retina.

Animals↗
PubMed2006

Phosphodiesterase 3 and 5 and cyclic nucleotide-gated ion channel expression in rat trigeminovascular system.

Activation of the trigeminovascular pain signalling system appears involved in migraine pathophysiology. However, the molecular mechanisms are only partially known. Stimulation of cAMP and cGMP production as well as inhibition of their breakdown induce migraine-like headache. Additionally, migraine may be associated with mutations in ion channels. The aim of the present study was to describe the expression of phosphodiesterase 3 (PDE3) and 5 (PDE5) and cyclic nucleotide-gated ion channels (CNG) in cerebral arteries, meninges, and the trigeminal ganglion. mRNA for PDE and CNG was determined in the rat middle cerebral artery, basilar artery, trigeminal ganglion, and dura mater using real-time PCR. PDE and CNG proteins were identified using Western blot. For comparison, rat aorta and mesenteric artery were analysed. PDE3A, PDE3B, and PDE5A mRNA were detected in all tissues examined except for PDE3A mRNA in dura mater and the trigeminal ganglion. PDE5A and PDE3A protein expression was present in both cerebral and peripheral arteries, whereas PDE3B protein was present only in the cerebral arteries. The CNGA4 and B1 subunit mRNAs were detected in cerebral arteries and CNGA2 also in the mesenteric artery. CNGA2 and A3 proteins were found in cerebral arteries and dura and CNGA1, CNGA2 and CNGA3 in the trigeminal ganglion. In conclusion, PDE3A, PDE3B, PDE5A, and five CNG subunits were expressed in several components of the trigeminovascular system of the rat. This suggests that modulation of cAMP and cGMP levels by PDE and activation of CNG may play a role in trigeminovascular pain signalling leading to migraine headache.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Pseudechetoxin: a peptide blocker of cyclic nucleotide-gated ion channels.

Ion channels activated by the binding of cyclic nucleotides first were discovered in retinal rods where they generate the cell's response to light. In other systems, however, it has been difficult to unambiguously determine whether cyclic nucleotide-dependent processes are mediated by protein kinases, their classical effector enzymes, or cyclic nucleotide-gated (CNG) ion channels. Part of this difficulty has been caused by the lack of specific pharmacological tools. Here we report the purification from the venom of the Australian King Brown snake of a peptide toxin that inhibits current through CNG channels. This toxin, which we have named Pseudechetoxin (PsTx), was purified by cation exchange and RP-HPLC and has a molecular mass of about 24 kDa. When applied to the extracellular face of membrane patches containing the alpha-subunit of the rat olfactory CNG channel, PsTx blocked the cGMP-dependent current with a Ki of 5 nM. Block was independent of voltage and required only a single molecule of toxin. PsTx also blocked CNG channels containing the bovine rod alpha-subunit with high affinity (100 nM), but it was less effective on the heteromeric version of the rod channel (Ki approximately 3 microM). We have obtained N-terminal and partial internal sequence data and the amino acid composition of PsTx. These data indicate that PsTx is a basic protein that exhibits some homology with helothermine, a toxin isolated from the venom of the Mexican beaded lizard. PsTx promises to be a valuable pharmacological tool for studies on the structure and physiology of CNG channels.

Amino Acids↗
PubMed1990

Reptation theory of ion channel gating.

Reptation theory is a highly successful approach for describing polymer dynamics in entangled systems. In turn, this molecular process is the basis of viscoelasticity. We apply a modified version of reptation dynamics to develop an actual physical model of ion channel gating. We show that at times longer than microseconds these dynamics predict an alpha-helix-screw motion for the amphipathic protein segment that partially lines the channel pore. Such motion has been implicated in several molecular mechanics studies of both voltage-gated and transmitter-gated channels. The experimental probability density function (pdf) for this process follows t-3/2 which has been observed in several experimental systems. Reptation theory predicts that channel gating will occur on the millisecond time scale and this is consistent with experimental results from single-channel recording. We examine the consequences of reptation over random barriers and we show that, to first order, the pdf remains unchanged. In the case of a charged helix undergoing reptation in the presence of a transmembrane potential we show that the tail of the pdf will be exponential. We provide a list of practical experimental predictions to test the validity of this physical theory.

Elasticity↗
PubMed1995

P2X receptors bring new structure to ligand-gated ion channels.

P2X receptors are cation-selective ion channels that open on binding to extracellular ATP; they play a role in fast synaptic transmission between neurones, and from autonomic nerves to smooth muscles. Isolation of cDNAs that encode P2X receptors in the smooth muscle of vas deferens and in phaeochromocytoma cells indicates that the receptors are not related to other ligand-gated ion channels. Their overall structure resembles more closely that of epithelial Na+ channels and the proteins that are thought to form mechanosensitive channels in Caenorhabditis elegans. The type of P2X RNA that is found in vas deferens is expressed preferentially by apoptotic thymocytes, and the type of P2X RNA that is found in PC12 cells is abundant in the pituitary gland, suggesting hitherto unsuspected roles for ATP-gated channels in endocrine and immune function.

Adenosine Triphosphate↗
PubMed2003

Acetylcholine binding protein (AChBP): a secreted glial protein that provides a high-resolution model for the extracellular domain of pentameric ligand-gated ion channels.

Acetylcholine binding protein (AChBP) has recently been identified from molluskan glial cells. Glial cells secrete it into cholinergic synapses, where it plays a role in modulating synaptic transmission. This novel mechanism resembles glia-dependent modulation of glutamate synapses, with several key differences. AChBP is a homolog of the ligand binding domain of the pentameric ligand-gated ion-channels. The crystal structure of AChBP provides the first high-resolution structure for this family of Cys-loop receptors. Nicotinic acetylcholine receptors and related ion-channels such as GABAA, serotonin 5HT3, and glycine can be interpreted in the light of the 2.7 A AChBP structure. The structural template provides critical details of the binding site and helps create models for toxin binding, mutational effects, and molecular gating.

Amino Acid Sequence↗
PubMed2006

Statistical evaluation of ion-channel gating models based on distributions of log-likelihood ratios.

The distributions of log-likelihood ratios (DeltaLL) obtained from fitting ion-channel dwell-time distributions with nested pairs of gating models (Xi, full model; Xi(R), submodel) were studied both theoretically and using simulated data. When Xi is true, DeltaLL is asymptotically normally distributed with predictable mean and variance that increase linearly with data length (n). When Xi(R) is true and corresponds to a distinct point in full parameter space, DeltaLL is Gamma-distributed (2DeltaLL is chi-square). However, when data generated by an l-component multiexponential distribution are fitted by l+1 components, Xi(R) corresponds to an infinite set of points in parameter space. The distribution of DeltaLL is a mixture of two components, one identically zero, the other approximated by a Gamma-distribution. This empirical distribution of DeltaLL, assuming Xi(R), allows construction of a valid log-likelihood ratio test. The log-likelihood ratio test, the Akaike information criterion, and the Schwarz criterion all produce asymmetrical Type I and II errors and inefficiently recognize Xi, when true, from short datasets. A new decision strategy, which considers both the parameter estimates and DeltaLL, yields more symmetrical errors and a larger discrimination power for small n. These observations are explained by the distributions of DeltaLL when Xi or Xi(R) is true.

Computer Simulation↗