Open channel block and alteration of N-methyl-D-aspartic acid receptor gating by an analog of phencyclidine.

We investigated inhibition of the N-methyl-D-aspartic acid (NMDA) receptor-channel complex by N-ethyl-1,4,9, 9alpha-tetrahydro-4alphaR-cis-4alphaH-fluoren-++ +4alpha-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC50 of 0.51 microM at -66 mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate (k+) of 39.9 microM-1 s-1. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel.     

Kv1.3 potassium channel-blocking toxin Ctri9577, novel gating modifier of Kv4.3 potassium channel from the scorpion toxin family

Scorpion toxin Ctri9577, as a potent Kv1.3 channel blocker, is a new member of the α-KTx15 subfamily which are a group of blockers for Kv4.x potassium channels. However, the pharmacological function of Ctri9577 for Kv4.x channels remains unknown. Scorpion toxin Ctri9577 was found to effectively inhibit Kv4.3 channel currents with IC₅₀ value of 1.34 ± 0.03 μM. Different from the mechanism of scorpion toxins as the blocker recognizing channel extracellular pore entryways, Ctri9577 was a novel gating modifier affecting voltage dependence of activation, steady-state inactivation, and the recovery process from the inactivation of Kv4.3 channel. However, Ctri9755, as a potent Kv1.3 channel blocker, was found not to affect voltage dependence of activation of Kv1.3 channel. Interestingly, pharmacological experiments indicated that 1 μM Ctri9755 showed less inhibition on Kv4.1 and Kv4.2 channel currents. Similar to the classical gating modifier of spider toxins, Ctri9577 was shown to interact with the linker between the transmembrane S3 and S4 helical domains through the mutagenesis experiments. To the best of our knowledge, Ctri9577 was the first gating modifier of potassium channels among scorpion toxin family, and the first scorpion toxin as both gating modifier and blocker for different potassium channels. These findings further highlighted the structural and functional diversity of scorpion toxins specific for the potassium channels.     

Quantitative analysis of tetrapentylammonium-induced blockade of open N-methyl-D-aspartate channels

The blockade of open N-methyl-d-aspartate (NMDA) channels by tetrapentylammonium (TPentA) in acutely isolated rat hippocampal neurons was studied using whole-cell patch-clamp techniques. TPentA prevented the closure of the NMDA channel following what is known as the foot-in-the-door mechanism. Hooked tail currents appearing after termination of the agonist (aspartate) and TPentA coapplication were analyzed quantitatively according to the corresponding sequential kinetic model. Studies of the hooked tail current amplitude and the degree of the stationary current inhibition dependence on the blocker concentration led to a new method for estimation of fast foot-in-the-door blocker binding/unbinding rate constants. The application of this method to the NMDA channel blockade by TPentA allowed finding the values of its binding (1.48 microM(-1)s(-1)) and unbinding (14 s(-1)) rate constants. An analysis of the dependence of the electric charge carried during the hooked tail current on the blocker concentration led to a new method for estimation of the maximum NMDA channel open probability, P(0). The value of P(0) found in experiments with TPentA was 0.04.     

Molecular size and hydrophobicity as factors which determine the efficacy of the blocking action of amino-adamantane derivatives on NMDA channels

Neurons isolated from the CA-1 region of rat hippocampal slices by the "vibrodissociation" method were voltage-clamped in the whole cell configuration. The currents through NMDA channels were recorded in response to rapid application (solution exchange time <30 ms) of 100 microM aspartate (ASP) in a Mg2+-free solution in the presence of 3 microM glycine. When added to the ASP solution, amantadine as well as other amino-adamantane derivatives (AAD) produced an open-channel blockade of NMDA channels. Membrane hyperpolarization enhanced the AAD block. The affinity between NMDA channels and AAD was different for various AAD. The analysis of the experimental data led us to conclude that this affinity depended both on the molecular size of the blocker (calculated using HyperChem molecular modeling program) and on the blocker's hydrophobicity (calculated according to Hansch and Leo, 1979). The affinity between NMDA channels and AAD diminished with an increase in molecular size and raised with an increase in blocker's hydrophobicity. We propose an empirical equation which describes the dependence of affinity on the size and hydrophobicity of the blocker. The estimated critical diameter of the NMDA channel pore where the AAD blocking site is located proved to be about 17 A.     

Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.     

Disruption of interdomain interactions in the glutamate binding pocket affects differentially agonist affinity and efficacy of N-methyl-D-aspartate receptor activation

In ionotropic glutamate receptors, agonist binding occurs in a conserved clam shell-like domain composed of the two lobes D1 and D2. Docking of glutamate into the binding cleft promotes rotation in the hinge region of the two lobes, resulting in closure of the binding pocket, which is thought to represent a prerequisite for channel gating. Here, we disrupted D1D2 interlobe interactions in the NR2A subunit of N-methyl-d-aspartate (NMDA) receptors through systematic mutation of individual residues and studied the influence on the activation kinetics of currents from NR1/NR2 NMDA receptors heterologously expressed in HEK cells. We show that the mutations affect differentially glutamate binding and channel gating, depending on their location within the binding domain, mainly by altering k(off) and k(cl), respectively. Whereas impaired stability of glutamate in its binding site is the only effect of mutations on one side of the ligand binding pocket, close to the hinge region, alterations in gating are the predominant consequence of mutations on the opposite side, at the entrance of the binding pocket. A mutation increasing D1D2 interaction at the entrance of the pocket resulted in an NMDA receptor with an increased open probability as demonstrated by single channel and whole cell kinetic analysis. Thus, the results indicate that agonist-induced binding domain closure is itself a complex process, certain aspects of which are coupled either to binding or to gating. Specifically, we propose that late steps of domain closure, in kinetic terms, represent part of channel gating.     

KCNQ4 channels expressed in mammalian cells: functional characteristics and pharmacology

Human cloned KCNQ4 channels were stably expressed in HEK-293 cells and characterized with respect to function and pharmacology. Patch-clamp measurements showed that the KCNQ4 channels conducted slowly activating currents at potentials more positive than -60 mV. From the Boltzmann function fitted to the activation curve, a half-activation potential of -32 mV and an equivalent gating charge of 1.4 elementary charges was determined. The instantaneous current-voltage relationship revealed strong inward rectification. The KCNQ4 channels were blocked in a voltage-independent manner by the memory-enhancing M current blockers XE-991 and linopirdine with IC(50) values of 5.5 and 14 microM, respectively. The antiarrhythmic KCNQ1 channel blocker bepridil inhibited KCNQ4 with an IC(50) value of 9.4 microM, whereas clofilium was without significant effect at 100 microM. The KCNQ4-expressing cells exhibited average resting membrane potentials of -56 mV in contrast to -12 mV recorded in the nontransfected cells. In conclusion, the activation and pharmacology of KCNQ4 channels resemble those of M currents, and it is likely that the function of the KCNQ4 channel is to regulate the subthreshold electrical activity of excitable cells.     

Characterization of the gating conformational changes in the felbamate binding site in NMDA channels

The anticonvulsant effect of felbamate (FBM) is ascribable to inhibition of N-methyl-d-aspartate (NMDA) currents. Using electrophysiological studies in rat hippocampal neurons to examine the kinetics of FBM binding to and unbinding from the NMDA channel, we show that FBM modifies NMDA channel gating via a one-to-one binding stoichiometry and has quantitatively the same enhancement effect on NMDA and glycine binding to the NMDA channel. Moreover, the binding rates of FBM to the closed and the open/desensitized NMDA channels are 187.5 and 4.6 x 10(4) M(-1) s(-1), respectively. The unbinding rates of FBM from the closed and the open/desensitized NMDA channels are approximately 6.2 x 10(-2) and approximately 3.1 s(-1), respectively. From the binding and unbinding rate constants, apparent dissociation constants of approximately 300 and approximately 70 microM could be calculated for FBM binding to the closed and the open/desensitized NMDA channels, respectively. The slight (approximately fourfold) difference in FBM binding affinity to the closed and to the open/desensitized NMDA channels thus is composed of much larger differences in the binding and unbinding kinetics (approximately 250- and approximately 60-fold difference, respectively). These findings suggest that the effects of NMDA and glycine binding coalesce or are interrelated before or at the actual activation gate, and FBM binding seems to modulate NMDA channel gating at or after this coalescing point. Moreover, the entrance zone of the FBM binding site very likely undergoes a much larger conformational change along the gating process than that in the binding region(s) of the binding site. In other words, the FBM binding site becomes much more accessible to FBM with NMDA channel activation, although the spatial configurations of the binding ligand(s) for FBM themselves are not altered so much along the gating process.     

FPL 64176 modification of Ca(V)1.2 L-type calcium channels: dissociation of effects on ionic current and gating current

FPL 64176 (FPL) is a nondihydropyridine compound that dramatically increases macroscopic inward current through L-type calcium channels and slows activation and deactivation. To understand the mechanism by which channel behavior is altered, we compared the effects of the drug on the kinetics and voltage dependence of ionic currents and gating currents. Currents from a homogeneous population of channels were obtained using cloned rabbit Ca(V)1.2 (alpha1C, cardiac L-type) channels stably expressed in baby hamster kidney cells together with beta1a and alpha2delta1 subunits. We found a striking dissociation between effects of FPL on ionic currents, which were modified strongly, and on gating currents, which were not detectably altered. Inward ionic currents were enhanced approximately 5-fold for a voltage step from -90 mV to +10 mV. Kinetics of activation and deactivation were slowed dramatically at most voltages. Curiously, however, at very hyperpolarized voltages (< -250 mV), deactivation was actually faster in FPL than in control. Gating currents were measured using a variety of inorganic ions to block ionic current and also without blockers, by recording gating current at the reversal potential for ionic current (+50 mV). Despite the slowed kinetics of ionic currents, FPL had no discernible effect on the fundamental movements of gating charge that drive channel gating. Instead, FPL somehow affects the coupling of charge movement to opening and closing of the pore. An intriguing possibility is that the drug causes an inactivated state to become conducting without otherwise affecting gating transitions.     

Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers

An alpha-toxin-binding membrane protein, isolated from the head and thoracic ganglia of the locus (Locusta migratoria), was reconstituted into planar lipid bilayers. Cholinergic agonists such as acetylcholine, carbamylcholine, and suberyldicholine induced fluctuations of single channels, which suggests that the protein represents a functional cholinergic receptor channel. The antagonist d-tubocurarine blocked the activation of the channels, whereas hexamethonium had only a weak effect; similar properties have been described for nicotinic insect receptors in situ. The channel was selectively permeable to monovalent cations but was impermeable to anions. The conductance of the channel (75 pS in 100 mM NaCl) was independent of the type of agonist used to activate the receptor. Kinetic analysis of the channel gating revealed that, at high agonist concentrations (50 microM carbamylcholine), more than one closed state exists and that multiple gating events, bursting as well as fast flickering, appeared. At very high agonist concentrations (500 microM carbamylcholine), desensitization was observed. Channel kinetics were dependent on the transmembrane potential. Comparing the conductance, the kinetics, and the pharmacology of nicotinic acetylcholine receptor from insect ganglia and fish electroplax reconstituted into bilayers revealed obvious similarities but also significant differences.     

Different effects of L-, N- and T-type calcium channel blockers on striatal dopamine release measured by microdialysis in freely moving rats.

Using a microdialysis method, we have investigated effects of the voltage-dependent calcium channel blockers, verapamil, nicardipine, omega-conotoxin and flunarizine on the dopamine release and metabolism in the striatum of freely moving rat. Perfusion of verapamil (1-300 microM) and nicardipine (1-100 microM), an L-type calcium channel blocker, into the striatum through the dialysis membrane showed a dose-dependent decrease of dopamine release in the dialysate and slight increase of 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) levels. Treatment of omega-conotoxin (0.1, 1 microM), an N-type channel blocker, decreased about 50% basal dopamine release and slightly decreased DOPAC and HVA levels. Treatment with flunarizine (10 microM), an T-type channel blocker, did not affect the dopamine release and metabolism. From these data, it appears that treatments of the L- and N-type voltage-dependent calcium channel blockers in rat striatum suppress basal dopamine release, but T-type blocker does not suppress it, suggesting that L-, N- and T-type calcium channels regulate in vivo dopamine release in a different mechanism.     

NMDA channel gating is influenced by a tryptophan residue in the M2 domain but calcium permeation is not altered

N-Methyl-D-aspartate (NMDA) receptors are susceptible to open-channel block by dizolcipine (MK-801), ketamine and Mg(2+) and are permeable to Ca(2+). It is thought that a tryptophan residue in the second membrane-associated domain (M2) may form part of the binding site for open-channel blockers and contribute to Ca(2+) permeability. We tested this hypothesis using recombinant wild-type and mutant NMDA receptors expressed in HEK-293 cells. The tryptophan was mutated to a leucine (W-5L) in both the NMDAR1 and NMDAR2A subunits. MK-801 and ketamine progressively inhibited currents evoked by glutamate, and the rate of inhibition was increased by the W-5L mutation. An increase in open channel probability accounted for the acceleration. Fluctuation analysis of the glutamate-evoked current revealed that the NMDAR1 W-5L mutation increased channel mean open time, providing further evidence for an alteration in gating. However, the equilibrium affinities of Mg(2+) and ketamine were largely unaffected by the W-5L mutation, and Ca(2+) permeability was not decreased. Therefore, the M2 tryptophan residue of the NMDA channel is not involved in Ca(2+) permeation or the binding of open-channel blockers, but plays an important role in channel gating.     

Mechanisms of blockade of glutamate receptor ionic channels: Paradox of 9-aminoacridine

9-Aminoacridine and tacrine differ from other channel blockers of NMDA receptors in that their binding prevents the closing of blocked channels and subsequent dissociation of the agonist. Structural determinants of aminoacridine derivatives underlying the blocking mechanism are still unknown. The aim of this study was to elucidate the effects of a dicationic 9-aminoacridine derivative and some other tricyclic compounds on NMDA receptors of rat hippocampal pyramidal neurons. All the compounds under study are voltage-dependent blockers of NMDA channels; their IC₅₀ values recorded at −80 mV vary from 1 to 50 μM. The dicationic derivatives demonstrate the same voltage dependence of the block as the monocationic derivatives. The monoand dicationic tricyclic compounds under study are weak blockers of AMPA receptor channels and differ from adamantane, phenylcyclohexyl and other dicationic derivatives that exhibit greater voltage dependence of the NMDA channel block and are able to induce effective suppression of AMPA channels. We conclude that the mechanisms of action of the tricyclic and dicationic 9-aminoacridine derivatives are different from that of 9-aminoacridine, since these compounds do not prevent closing of the blocked channels. This suggests that the binding site for 9-aminoacridine has specific properties and high selectivity with respect to ligand structure. Original Russian Text ? K.H. Kim, V.E. Gmiro, D.B. Tikhonov, L.G. Magazanik, 2007, published in Biologicheskie Membrany, 2007, Vol. 24, No. 1, pp. 96–104.     

Intracellular Mg2+ is a voltage-dependent pore blocker of HCN channels

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are activated by membrane hyperpolarization that creates time-dependent, inward rectifying currents, gated by the movement of the intrinsic voltage sensor S4. However, inward rectification of the HCN currents is not only observed in the time-dependent HCN currents, but also in the instantaneous HCN tail currents. Inward rectification can also be seen in mutant HCN channels that have mainly time-independent currents (5). In the present study, we show that intracellular Mg(2+) functions as a voltage-dependent blocker of HCN channels, acting to reduce the outward currents. The affinity of HCN channels for Mg(2+) is in the physiological range, with Mg(2+) binding with an IC(50) of 0.53 mM in HCN2 channels and 0.82 mM in HCN1 channels at +50 mV. The effective electrical distance for the Mg(2+) binding site was found to be 0.19 for HCN1 channels, suggesting that the binding site is in the pore. Removing a cysteine in the selectivity filter of HCN1 channels reduced the affinity for Mg(2+), suggesting that this residue forms part of the binding site deep within the pore. Our results suggest that Mg(2+) acts as a voltage-dependent pore blocker and, therefore, reduces outward currents through HCN channels. The pore-blocking action of Mg(2+) may play an important physiological role, especially for the slowly gating HCN2 and HCN4 channels. Mg(2+) could potentially block outward hyperpolarizing HCN currents at the plateau of action potentials, thus preventing a premature termination of the action potential.     

Tolbutamide causes open channel blockade of cystic fibrosis transmembrane conductance regulator Cl- channels.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl- channel that is regulated by protein kinase A and cytosolic nucleotides. Previously, Sheppard and Welsh reported that the sulfonylureas glibenclamide and tolbutamide reduced CFTR whole cell currents. The aim of this study was to quantify the effects of tolbutamide on CFTR gating in excised membrane patches containing multiple channels. We chose tolbutamide because weak (i.e., fast-type) open channel blockers introduce brief events into multichannel recordings that can be readily quantified by current fluctuation analysis. Inspection of current records revealed that the addition of tolbutamide reduced the apparent single-channel current amplitude and increased the open-channel noise, as expected for a fast-type open channel blocker. The apparent decrease in unitary current amplitude provides a measure of open probability within a burst (P0 Burst), and the resulting concentration-response relationship was described by a simple Michaelis-Menten inhibition function. The concentration of tolbutamide causing a 50% reduction of Po Burst (540 +/- 20 microM) was similar to the concentration producing a 50% inhibition of short-circuit current across T84 colonic epithelial cell monolayers (400 +/- 20 microM). Changes in CFTR gating were then quantified by analyzing current fluctuations. Tolbutamide caused a high-frequency Lorentzian (corner frequency, fc > 300 Hz) to appear in the power density spectrum. The fc of this Lorentzian component increased as a linear function of tolbutamide concentration, as expected for a pseudo-first-order open-blocked mechanism and yielded estimates of the on rate (koff = 2.8 +/- 0.3 microM-1 s-1), the off rate (kon = 1210 +/- 225 s-1), and the dissociation constant (KD = 430 +/- 80 microM). Based on these observations, we propose that there is a bimolecular interaction between tolbutamide and CFTR, causing open channel blockade.     

Desensitization of the skeletal muscle ryanodine receptor: evidence for heterogeneity of calcium release channels.

Ca release channels from the junctional sarcoplasmic reticulum (SR) membranes of rabbit skeletal muscle were incorporated into the lipid bilayer membrane, and the inactivation kinetics of the channel were studied at large membrane potentials. The channels conducting Cs currents exhibited a characteristic desensitization that is both ligand and voltage dependent: 1) with a test pulse to -100 mV (myoplasmic minus luminal SR), the channel inactivated with a time constant of 3.9 s; 2) the inactivation had an asymmetric voltage dependence; it was only observed at voltages more negative than -80 mV; and 3) repetitive tests to -100 mV usually led to immobilization of the channel, which could be recovered by a conditioning pulse to positive voltages. The apparent desensitization was seen in approximately 50% of the experiments, with both the native Ca release channel (in the absence of ryanodine) and the ryanodine-activated channel (1 microM ryanodine). The native Ca release channels revealed heterogeneous gating with regard to activation by ATP and binding to ryanodine. Most channels had high affinity to ATP activation (average open probability (po) = 0.55, 2 mM ATP, 100 microM Ca), whereas a small portion of channels had low affinity to ATP activation (po = 0.11, 2 mM ATP, 100 microM Ca), and some channels bound ryanodine faster (< 2 min), whereas others bound much slower (> 20 min). The faster ryanodine-binding channels always desensitized at large negative voltages, whereas those that bound slowly did not show apparent desensitization. The heterogeneity of the reconstituted Ca release channels is likely due to the regulatory roles of other junctional SR membrane proteins on the Ca release channel.     

An inactivation stabilizer of the Na+ channel acts as an opportunistic pore blocker modulated by external Na+

The Na+ channel is the primary target of anticonvulsants carbamazepine, phenytoin, and lamotrigine. These drugs modify Na+ channel gating as they have much higher binding affinity to the inactivated state than to the resting state of the channel. It has been proposed that these drugs bind to the Na+ channel pore with a common diphenyl structural motif. Diclofenac is a widely prescribed anti-inflammatory agent that has a similar diphenyl motif in its structure. In this study, we found that diclofenac modifies Na+ channel gating in a way similar to the foregoing anticonvulsants. The dissociation constants of diclofenac binding to the resting, activated, and inactivated Na+ channels are approximately 880 microM, approximately 88 microM, and approximately 7 microM, respectively. The changing affinity well depicts the gradual shaping of a use-dependent receptor along the gating process. Most interestingly, diclofenac does not show the pore-blocking effect of carbamazepine on the Na+ channel when the external solution contains 150 mM Na+, but is turned into an effective Na+ channel pore blocker if the extracellular solution contains no Na+. In contrast, internal Na+ has only negligible effect on the functional consequences of diclofenac binding. Diclofenac thus acts as an "opportunistic" pore blocker modulated by external but not internal Na+, indicating that the diclofenac binding site is located at the junction of a widened part and an acutely narrowed part of the ion conduction pathway, and faces the extracellular rather than the intracellular solution. The diclofenac binding site thus is most likely located at the external pore mouth, and undergoes delicate conformational changes modulated by external Na+ along the gating process of the Na+ channel.     

A nonequilibrium binary elements-based kinetic model for benzodiazepine regulation of GABAA receptors

Ion channels, like many other proteins, are composed of multiple structural domains. A stimulus that impinges on one domain, such as binding of a ligand to its recognition site, can influence the activity of another domain, such as a transmembrane channel gate, through interdomain interactions. Kinetic schemes that describe the function of interacting domains typically incorporate a minimal number of states and transitions, and do not explicitly model interactions between domains. Here, we develop a kinetic model of the GABA_A receptor, a ligand-gated ion channel modulated by numerous compounds including benzodiazepines, a class of drugs used clinically as sedatives and anxiolytics. Our model explicitly treats both the kinetics of distinct functional domains within the receptor and the interactions between these domains. The model describes not only how benzodiazepines that potentiate GABA_A receptor activity, such as diazepam, affect peak current dose–response relationships in the presence of desensitization, but also their effect on the detailed kinetics of current activation, desensitization, and deactivation in response to various stimulation protocols. Finally, our model explains positive modulation by benzodiazepines of receptor currents elicited by either full or partial agonists, and can resolve conflicting observations arguing for benzodiazepine modulation of agonist binding versus channel gating.     

The relationship between agonist potency and AMPA receptor kinetics

AMPA-type glutamate receptors are tetrameric ion channels that mediate fast excitatory synaptic transmission in the mammalian brain. When agonists occupy the binding domain of individual receptor subunits, this domain closes, triggering rearrangements that couple agonist binding to channel opening. Here we compare the kinetic behavior of GluR2 channels activated by four different ligands, glutamate, AMPA, quisqualate, and 2-Me-Tet-AMPA, full agonists that vary in potency by up to two orders of magnitude. After reduction of desensitization with cyclothiazide, deactivation decays were strongly agonist dependent. The time constants of decay increased with potency, and slow components in the multiexponential decays became more prominent. The desensitization decays of agonist-activated currents also contained multiple exponential components, but they were similar for the four agonists. The time course of recovery from desensitization produced by each agonist was described by two sigmoid components, and the speed of recovery varied substantially. Recovery was fastest for glutamate and slowest for 2-Me-Tet-AMPA, and the amplitude of the slow component of recovery increased with agonist potency. The multiple kinetic components appear to arise from closed-state transitions that precede channel gating. Stargazin increases the slow kinetic components, and they likely contribute to the biexponential decay of excitatory postsynaptic currents.     

Nickel block of three cloned T-type calcium channels: low concentrations selectively block alpha1H

Nickel has been proposed to be a selective blocker of low-voltage-activated, T-type calcium channels. However, studies on cloned high-voltage-activated Ca(2+) channels indicated that some subtypes, such as alpha1E, are also blocked by low micromolar concentrations of NiCl(2). There are considerable differences in the sensitivity to Ni(2+) among native T-type currents, leading to the hypothesis that there may be more than one T-type channel. We confirmed part of this hypothesis by cloning three novel Ca(2+) channels, alpha1G, H, and I, whose currents are nearly identical to the biophysical properties of native T-type channels. In this study we examined the nickel block of these cloned T-type channels expressed in both Xenopus oocytes and HEK-293 cells (10 mM Ba(2+)). Only alpha1H currents were sensitive to low micromolar concentrations (IC(50) = 13 microM). Much higher concentrations were required to half-block alpha1I (216 microM) and alpha1G currents (250 microM). Nickel block varied with the test potential, with less block at potentials above -30 mV. Outward currents through the T channels were blocked even less. We show that depolarizations can unblock the channel and that this can occur in the absence of permeating ions. We conclude that Ni(2+) is only a selective blocker of alpha1H currents and that the concentrations required to block alpha1G and alpha1I will also affect high-voltage-activated calcium currents.