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PubMed2004

Identification of the Ca2+ blocking site of acid-sensing ion channel (ASIC) 1: implications for channel gating.

Acid-sensing ion channels ASIC1a and ASIC1b are ligand-gated ion channels that are activated by H+ in the physiological range of pH. The apparent affinity for H+ of ASIC1a and 1b is modulated by extracellular Ca2+ through a competition between Ca2+ and H+. Here we show that, in addition to modulating the apparent H+ affinity, Ca2+ blocks ASIC1a in the open state (IC50 approximately 3.9 mM at pH 5.5), whereas ASIC1b is blocked with reduced affinity (IC50 > 10 mM at pH 4.7). Moreover, we report the identification of the site that mediates this open channel block by Ca2+. ASICs have two transmembrane domains. The second transmembrane domain M2 has been shown to form the ion pore of the related epithelial Na+ channel. Conserved topology and high homology in M2 suggests that M2 forms the ion pore also of ASICs. Combined substitution of an aspartate and a glutamate residue at the beginning of M2 completely abolished block by Ca2+ of ASIC1a, showing that these two amino acids (E425 and D432) are crucial for Ca2+ block. It has previously been suggested that relief of Ca2+ block opens ASIC3 channels. However, substitutions of E425 or D432 individually or in combination did not open channels constitutively and did not abolish gating by H+ and modulation of H+ affinity by Ca2+. These results show that channel block by Ca2+ and H+ gating are not intrinsically linked.

Acid Sensing Ion Channels↗

PubMed2005

Alterations in glutamatergic and gabaergic ion channel activity in hippocampal neurons following exposure to the abused inhalant toluene.

Toluene, a representative member of the large class of abused inhalants, decreases neuronal activity and depresses behavior in both animals and humans. The sites of action of toluene are not completely known but recent studies suggest that ion channels that regulate neuronal excitability may be particularly sensitive. Previous studies with recombinant receptors showed that toluene decreases currents carried by N-methyl-D-aspartate (NMDA)-glutamate receptors without affecting those gated by non-NMDA receptors. In addition, toluene increases currents generated by GABA and glycine receptors. In the present study, primary cultures of rat hippocampal neurons were used to investigate the effects of acute and chronic toluene exposure on native excitatory and inhibitory ligand-gated ion channels. Toluene dose-dependently inhibited NMDA-mediated currents (IC50 1.5 mM) but had no effect on responses evoked by the non-NMDA agonist kainic acid. Prolonged treatment of neurons with toluene (1 mM; 4 days) increased whole-cell responses to exogenously applied NMDA, reduced those evoked by GABA but did not alter responses generated by kainic acid. Immunoblot analysis revealed that prolonged toluene exposure increased levels of NR2A and NR2B NMDA receptor subunits with no change in NR1. Immunohistochemical analysis with confocal imaging showed that toluene-treated neurons had significant increases in the density of NR1 subunits as compared with control neurons. Toluene exposure increased the amplitude of synaptic NMDA currents and decreased those activated by GABA. The results from this study suggest that toluene induces compensatory responses in the functional expression of ion channels that regulate neuronal excitability.

Amino Acid Transport System X-AG↗

PubMed1995

Molecular mechanism for ligand discrimination of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated ion channels of retinal photoreceptors and olfactory neurons are differentially activated by ligands that vary only in their purine ring structure. The nucleotide selectivity of the bovine rod cyclic nucleotide-gated channel (cGMP > cIMP >> cAMP) was significantly altered by neutralization of a single aspartic acid residue in the binding domain (cGMP > or = cAMP > cIMP). Substitution by a nonpolar residue at this position inverted agonist selectivity (cAMP >> cIMP > or = cGMP). These effects resulted from an alteration in the relative ability of the agonists to promote the allosteric conformational change associated with channel activation, not from a modification in their initial binding affinity. We propose a general mechanism for guanine nucleotide discrimination, in common with that observed in high affinity GTP-binding proteins, involving the formation of a pair of hydrogen bonds between the aspartic acid side chain and N1 and N2 of the guanine ring.

Allosteric Site↗

PubMed2006

Regulation of the desensitization and ion selectivity of ATP-gated P2X2 channels by phosphoinositides.

Phosphoinositides (PIP(n)s) are known to regulate the activity of some ion channels. Here we determined that ATP-gated P2X(2) channels also are regulated by PIP(n)s, and investigated the structural background and the unique features of this regulation. We initially used two-electrode voltage clamp to analyse the electrophysiological properties of P2X(2) channels expressed in Xenopus oocytes, and observed that preincubation with wortmannin or LY294002, two PI3K inhibitors, accelerated channel desensitization. K365Q or K369Q mutation of the conserved, positively charged, amino acid residues in the proximal region of the cytoplasmic C-terminal domain also accelerated desensitization, whereas a K365R or K369R mutation did not. We observed that the permeability of the channel to N-methyl-d-glucamine (NMDG) transiently increased and then decreased after ATP application, and that the speed of the decrease was accelerated by K365Q or K369Q mutation or PI3K inhibition. Using GST-tagged recombinant proteins spanning the proximal C-terminal region, we then analysed their binding of the P2X(2) cytoplasmic domain to anionic lipids using PIP(n)s-coated nitrocellulose membranes. We found that the recombinant proteins that included the positively charged region bound to PIPs and PIP(2)s, and that this binding was eliminated by the K365Q and K369Q mutations. We also used a fluorescence assay to confirm that fusion proteins comprising the proximal C-terminal region of P2X(2) with EGFP expressed in COS-7 cells closely associated with the membrane. Taken together, these results show that membrane-bound PIP(n)s play a key role in maintaining channel activity and regulating pore dilation through electrostatic interaction with the proximal region of the P2X(2) cytoplasmic C-terminal domain.

Adenosine Triphosphate↗

PubMed1989

Acetylcholine-gated and chloride conductance channel expression in rat muscle membrane.

1. During the differentiation of skeletal muscle, there is a synchronized expression of a number of muscle-specific proteins including the acetylcholine-gated ion channel (AChR). Another muscle-specific ion channel, responsible for chloride conductance, was shown to be expressed in an anticoordinate fashion to AChR. An organ culture system for rat lumbrical muscles was developed to manipulate the expression of these two ion channels. 2. Denervation induced a change in expression of both channels that was mimicked in culture and reversed by direct electrical stimulation. 3. The time course of the disappearance of both channels was similar and started immediately after denervation (chloride conductance) or stimulation (AChR). The time course of the appearance of AChR was delayed several days after denervation and culture but chloride conductance increased immediately upon stimulation. 4. The loss of chloride conductance in muscle cultured in cycloheximide exhibited first-order kinetics, providing an estimate of the half-life (2.3 days) for the chloride conductance channel. This resembled the disappearance of chloride conductance in normal medium, suggesting that synthesis of this channel ceases following denervation. The decrease in chloride conductance characteristic of denervated muscle was not halted by cycloheximide. 5. Changes in chloride conductance presumably alter the intracellular concentration of chloride. The possibility that chloride might regulate the expression of AChRs in skeletal muscle was tested by altering the intracellular concentration of chloride in muscles maintained in organ culture. 6. Denervated muscles, whose intracellular concentration of chloride is elevated, were cultured in medium containing 9 mM-chloride (low-Cl- medium). AChR expression was reduced by either low-Cl- medium or electrical stimulation. Together, low-Cl- medium and electrical stimulation reduced expression more than either treatment alone. 7. The loss of AChRs in low-Cl- medium was blocked when muscle fibrillation was halted by TTX. 8. When chloride conductance was blocked by 9AC (9-anthracene carboxylic acid) intracellular chloride was elevated to the levels seen in denervated muscle. The elevated levels of chloride did not prevent the reduction in AChR expression induced by electrical stimulation. 9. The uncoupling of AChR expression and the intracellular concentration of chloride showed that they were not rigidly linked. Chloride affects the expression of AChR indirectly, by altering the activity of muscle cells.

Acetylcholine↗

PubMed2002

Antagonistic pathways in neurons exposed to body fluid regulate social feeding in Caenorhabditis elegans.

Wild isolates of Caenorhabditis elegans can feed either alone or in groups. This natural variation in behaviour is associated with a single residue difference in NPR-1, a predicted G-protein-coupled neuropeptide receptor related to Neuropeptide Y receptors. Here we show that the NPR-1 isoform associated with solitary feeding acts in neurons exposed to the body fluid to inhibit social feeding. Furthermore, suppressing the activity of these neurons, called AQR, PQR and URX, using an activated K(+) channel, inhibits social feeding. NPR-1 activity in AQR, PQR and URX neurons seems to suppress social feeding by antagonizing signalling through a cyclic GMP-gated ion channel encoded by tax-2 and tax-4. We show that mutations in tax-2 or tax-4 disrupt social feeding, and that tax-4 is required in several neurons for social feeding, including one or more of AQR, PQR and URX. The AQR, PQR and URX neurons are unusual in C. elegans because they are directly exposed to the pseudocoelomic body fluid. Our data suggest a model in which these neurons integrate antagonistic signals to control the choice between social and solitary feeding behaviour.

Animals↗

PubMed2003

Structure-activity and interaction effects of 14 different pyrethroids on voltage-gated chloride ion channels.

We have proposed that since the type II pyrethroids deltamethrin and cypermethrin, but not the type I pyrethroid cismethrin act on chloride channels, this could contribute to the bimodal nature of pyrethroid poisoning syndromes. We now examine a wider range of pyrethroid structures on the activity of these calcium-independent voltage-gated maxi-chloride channels. Excised inside-out membrane patches from differentiated mouse neuroblastoma cells were used, and mean channel open probabilities calculated. For single dosing at 10 microM, bioallethrin, beta-cyfluthrin, cypermethrin, deltamethrin, and fenpropathrin were all found to significantly decrease open channel probability (p < 0.05). Bifenthrin, bioresmethrin, cispermethrin, cisresmethrin, cyfluthrin isomers 2 and 4, lambda-cyhalothrin, esfenvalerate, and tefluthrin, did not significantly alter open channel probability (p > 0.05). Since the type II pyrethroids, esfenvalerate, and lambda-cyhalothrin were ineffective, we must conclude that actions at the chloride ion channel target cannot in themselves account for the differences between the two types of poisoning syndrome. Sequential dosing with type II pyrethroids caused no further chloride ion channel closure. The type I pyrethroid cisresmethrin did however prevent a subsequent effect by the mixed type pyrethroid fenpropathrin. In contrast, the type I pyrethroid cispermethrin did not prevent a subsequent effect due to the type II pyrethroid deltamethrin. The difference in effect may be the result of differences in potency, as deltamethrin had a greater effect than fenpropathrin. It therefore appears clear that in some combinations the type I and type II pyrethroids can compete and may bind to the same chloride channel target site.

Animals↗

PubMed2003

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

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

Cell Membrane Permeability↗

PubMed1993

Pursuing the voltage sensor of a voltage-gated mammalian potassium channel.

Ion channels are generally classified as voltage- or ligand-gated channels based upon their ability to respond to a change in the transmembrane voltage or to a specific ligand interaction. The voltage sensor of voltage-gated ion channels is thought to be contained within the fourth putative transmembrane segment, S4, and is characterized by basic residues typically spaced apart. Thus far, a full understanding of all charges contributing to the voltage sensor of voltage-gated potassium channels has been lacking since several neutralization mutants in S4 could not be functionally expressed. By construction of multimeric cDNAs encoding a mammalian RCK1 (Kv1.1) potassium channel, we now report functional expression of all charge neutralizations in the S4 segment, providing a more comprehensive insight into the functioning of the voltage sensor.

Animals↗

PubMed2005

Voltage-dependent synchronization of gating of syringomycin E ion channels.

Antifungal lipodepsipeptide syringomycin E (SRE) forms two major conductive states in lipid bilayers: "small" and "large". Large SRE channels are cluster of several small ones, demonstrating synchronous opening and closure. To get insight into the mechanism of such synchronization we investigated how transmembrane potential, membrane surface charge, and ionic strength affect the number of small SRE channels synchronously functioning in the cluster. Here, we report that the large SRE channels can be presented as 3-8 simultaneously gating small channels. The increase in the absolute value of the transmembrane potential (from 50 to 200 mV) decreases the number of synchronously gated channels in the clusters. Voltage-dependence of channel synchronization was influenced by the ionic strength of the bathing solution, but not by membrane surface charge. We propose a mechanism for the voltage-dependent cluster behavior that involves a voltage-induced reorientation of lipid dipoles associated with the channel pores.

Ion Channel Gating↗

PubMed2001

Protein mobility and GABA-induced conformational changes in GABA(A) receptor pore-lining M2 segment.

Protein movements underlying ligand-gated ion channel activation are poorly understood. Here we used disulfide bond trapping to examine the proximity and mobility of cysteines substituted for aligned GABAA receptor alpha1 and beta1 M2 segment channel-lining residues in resting and activated receptors. With or without GABA, disulfide bonds formed at alpha1N275C/beta1E270C (20') and alpha1S272C/beta1H267C (17'), near the extracellular end, suggesting that this end is more mobile and/or flexible than the rest of the segment. Near the middle of M2, at alpha1T261C/beta1T256C (6'), a disulfide bond formed only in the presence of GABA and locked the channels open. Channel activation must involve an asymmetric rotation of two adjacent subunits toward each other. This would move aligned engineered cysteines on different subunits into proximity and allow disulfide bond formation without blocking conduction. Asymmetric rotation of M2 segments is probably a common gating mechanism in other ligand-gated ion channels.

Animals↗

PubMed2000

Capsaicin, acid and heat-evoked currents in rat trigeminal ganglion neurons: relationship to functional VR1 receptors.

Activation of primary trigeminal (TG) neurons by protons, capsaicin, or heat can evoke a variety of sensations, including tingling, stinging, warmth, and burning. Capsaicin and acid are trigeminal stimulants that are important in gustatory physiology. These stimuli can activate H(+)-gated ion channels and heterologously expressed VR1 receptors (vanilloid receptor 1). We have obtained evidence by using electrophysiological and pharmacological measurements on TG neurons that these three stimuli can activate many receptors, and we have determined the extent they behave similarly to VR1 receptors and H(+)-gated channels from the DEGenerin/ENaC superfamily. Whole-cell recordings from rat TG neurons revealed that protons evoked transient (Tp), sustained (Sp), and biphasic (TSp) currents. Tp currents had reversal potentials (Vr) of 24-45 mV, a pH(0.5) range from 5.5 to 6.5, and were inhibited by amiloride, suggesting the presence of functional H(+)-gated channels. Sp currents were inhibited by the VR1 antagonist capsazepine, had Vr's approximately 0 mV, and had pH(0.5) = 6.4. Capsaicin also activated transient (Tc), sustained (Sc), and biphasic (TSc) currents. At pH 5.9, the sensitivity of the Sc currents increased by about a factor of 10, which may partially account for the synergistic responses of acid in foods containing capsaicin. Heating TG neurons evoked a thermally active, capsazepine-inhibitable current with threshold temperature of 43 degrees C and Vr = 5 mV that is also present in neurons activated by and protons (Sp) and capsaicin (Sc). These data suggest that TG neurons have functional receptors that behave similarly to VR1. Activation of such receptors should result in a burning sensation, whereas activation of the transient and biphasic currents should result in other taste descriptors.

Acids↗

PubMed2001

A novel extracellular calcium sensing mechanism in voltage-gated potassium ion channels.

Potassium (K(+)) channels influence neurotransmitter release, burst firing rate activity, pacing, and critical dampening of neuronal circuits. Internal and external factors that further modify K(+) channel function permit fine-tuning of neuronal circuits. Human ether-à-go-go-related gene (HERG) K(+) channels are unusually sensitive to external calcium concentration ([Ca(2+)](o)). Small changes in [Ca(2+)](o) shift the voltage dependence of channel activation to more positive membrane potentials, an effect that cannot be explained by nonspecific surface charge screening or channel pore block. The HERG-calcium concentration-response relationship spans the physiological range for [Ca(2+)](o). The modulatory actions of calcium are attributable to differences in the Ca(2+) affinity between rested and activated channels. Adjacent extracellular, negatively charged amino acids (E518 and E519) near the S4 voltage sensor influence both channel gating and Ca(2+) dependence. Neutralization of these charges had distinct effects on channel gating and calcium sensitivity. A change in the degree of energetic coupling between these amino acids on transition from closed to activated channel states reveals movement in this region during channel gating and defines a molecular mechanism for protein state-dependent ligand interactions. The results suggest a novel extracellular [Ca(2+)](o) sensing mechanism coupled to allosteric changes in channel gating and a mechanism for fine-tuning cell repolarization.

Allosteric Regulation↗

PubMed1998

Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons.

Development of ionic currents underlying changes in action potential waveforms in rat spinal motoneurons. J. Neurophysiol. 80: 3047-3061, 1998. Differentiation of the ionic mechanism underlying changes in action potential properties was investigated in spinal motoneurons of embryonic and postnatal rats using whole cell voltage- and current-clamp recordings. Relatively slow-rising, prolonged, largely Na+-dependent action potentials were recorded in embryonic motoneurons, and afterdepolarizing potentials were elicited in response to prolonged intracellular injections of depolarizing currents. Action potential amplitude, as well as its rates of rise and repolarization significantly increased, and an afterhyperpolarizing potential (AHP) became apparent immediately after birth. Concurrently, repetitive action potential firing was elicited in response to a prolonged current injection. To determine the ionic mechanism underlying these changes, the properties of voltage-gated macroscopic Na+, Ca2+, and K+ currents were examined. Fast-rising Na+ currents (INa) and slow-rising Ca2+ currents (ICa) were expressed early in embryonic development, but only INa was necessary and sufficient to trigger an action potential. INa and ICa densities significantly increased while the time to peak INa and ICa decreased after birth. The postnatal increase in INa resulted in overshooting action potential with significantly faster rate of rise than that recorded before birth. Properties of three types of outward K+ currents were examined: transient type-A current (IA), noninactivating delayed rectifier-type current (IK), and Ca2+-dependent K+ current (IK(Ca)). The twofold postnatal increase in IK and IK(Ca) densities resulted in shorter duration action potential and the generation of AHP. Relatively large IA was expressed early in neuronal development, but unlike IK and IK(Ca) its density did not increase after birth. The three types of K+ channels had opposite modulatory actions on action potential firing behavior: IK and IA increased the firing rate, whereas IK(Ca) decreased it. Our findings demonstrated that the developmental changes in action potential waveforms and the onset of repetitive firing were correlated with large increases in the densities of existing voltage-gated ion channels rather than the expression of new channel types.

Action Potentials↗

PubMed1993

Quantal variability of excitatory transmission in the hippocampus: implications for the opening probability of fast glutamate-gated channels.

The quantal description of synaptic transmission, first formulated at the neuromuscular junction, has recently been extended to the Schaffer collateral-CA1 synapse of the mammalian hippocampus. In a subset of connections, an upper limit can be placed on the coefficient of variation of the quantal amplitude, on occasion lower than 0.2. In the same connections, the average quantal amplitude implies that fewer than 20 fast glutamate-gated ion channels open to carry the quantal current. With so few channels opening, their stochastic behaviour imposes a minimal trial-to-trial variability on the quantal current. These observations can be reconciled by postulating that most of the available receptors in the synaptic cleft are bound by released transmitter and that ionophores open with a high probability.

Animals↗

PubMed2005

Voltage-gated sodium and calcium channels in nerve, muscle, and heart.

Voltage-gated ion channels are membrane proteins which underlie rapid electrical signals among neurons and the spread of excitation in skeletal muscle and heart. We outline some recent advances in the study of voltage-sensitive sodium and calcium channels. Investigations are providing insight into the changes in molecular conformation associated with open-closed gating of the channels, the mechanisms by which they allow only specific ion species to pass through and carry an electric current, and the pathological consequences of small perturbations in channel structure which result from genetic mutations. Determination of three-dimensional structures, coupled with molecular manipulations by site-directed mutagenesis, and parallel electrophysiological analyses of currents through the ion channels, are providing an understanding of the roles and function of these channels at an unprecedented level of molecular detail. Crucial to these advances are studies of bacterial homologues of ion channels from man and other eukaryotes, and the use of naturally occurring peptide toxins which target different ion channel types with exquisite specificity.

Animals↗

PubMed1997

Interdomain interactions underlying activation of cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) ion channels are multimeric proteins that activate in response to the binding of cyclic nucleotide to intracellular domains. Here, an intramolecular protein-protein interaction between the amino-terminal domain and the carboxyl-terminal ligand-binding domain of the rat olfactory CNG channel was shown to exert an autoexcitatory effect on channel activation. Calcium-calmodulin, which modulates CNG channel activity during odorant adaptation, blocked this interaction. A specific deletion within the amino-terminal domain disrupted the interdomain interaction in vitro and altered the gating properties and calmodulin sensitivity of expressed channels. Thus, the amino-terminal domain may promote channel opening by directly interacting with the carboxyl-terminal gating machinery; calmodulin regulates channel activity by targeting this interaction.

Animals↗

PubMed1988

Inhibition of the GABA receptor-gated chloride ion channel in brain by noncompetitive inhibitors of the nicotinic receptor-gated cation channel.

The ability of various noncompetitive inhibitors (NCI) of the nicotinic acetylcholine receptor to inhibit gamma-aminobutyric acidA (GABA) receptor activity in brain was investigated. Micromolar concentrations of NCI such as tetraphenylphosphonium, mepacrine, chlorpromazine and phencyclidine inhibited muscimol-induced 36chloride (36Cl-) uptake in rat cerebral cortical synaptoneurosomes in a noncompetitive manner. D-Tubocurarine behaved as a competitive inhibitor. In experiments measuring the time course of muscimol-induced 36Cl- uptake, a decline in the apparent transport rate constant (k') (reflection of desensitization) occurred over the first 3 sec. At lowered temperature (22 degrees C) the k' did not decline during the first 4 sec. Under these conditions preincubation of the vesicles with tetraphenylphosphonium caused a marked decline in the apparent k' over the first 5 sec (transition T1/2 of 1.04 sec) suggesting that tetraphenylphosphonium promotes desensitization of the GABA receptor. The inhibition of muscimol-induced 36Cl- uptake by NCI was reduced in the absence of Ca++. In addition, Ca++ decreased the potency of muscimol to stimulate 36Cl- uptake with an IC50 = 64 microM. The interaction of NCI with GABA agonist and convulsant sites associated with the GABA receptor-gated Cl- channel also was investigated. NCI antagonized noncompetitively [3H]t-butylbicycloorthobenzoate binding to convulsant sites whereas only mepacrine and chlorpromazine antagonized binding of [3H]muscimol to agonist sites. These findings suggest that 1) inhibition of the GABA receptor-gated Cl- channel by NCI may explain the convulsant properties of several of these compounds and 2) there may be structural domains common to the GABA receptor and nicotinic receptor-gated ion channels that selectively permit interactions with various NCI.

Animals↗