Change In Reversal Potential Research Articles

Distinct effects of heterogeneity and noise on gamma oscillation in a model of neuronal network with different reversal potential

Gamma oscillation is crucial in brain functions such as attentional selection, and is inextricably linked to both heterogeneity and noise (or so-called stochastic fluctuation) in neuronal networks. However, under coexistence of these factors, it has not been clarified how the synaptic reversal potential modulates the entraining of gamma oscillation. Here we show distinct effects of heterogeneity and noise in a population of modified theta neurons randomly coupled via GABAergic synapses. By introducing the Fokker-Planck equation and circular cumulants, we derive a set of two-cumulant macroscopic equations. In bifurcation analyses, we find a stabilizing effect of heterogeneity and a nontrivial effect of noise that results in promoting, diminishing, and shifting the oscillatory region, and is largely dependent on the reversal potential of GABAergic synapses. These findings are verified by numerical simulations of a finite-size neuronal network. Our results reveal that slight changes in reversal potential and magnitude of stochastic fluctuations can lead to immediate control of gamma oscillation, which would results in complex spatio-temporal dynamics for attentional selection and recognition.

Transient receptor potential vanilloid 4 (TRPV4) channel as a target of crotamiton and its bimodal effects.

The sensation of itching can be defined as "an unpleasant cutaneous sensation that provokes a desire to scratch." The perception of itching is not critical for the maintenance of life, but persistent itching can be extremely irritating and decreases the quality of life. Crotamiton (N-ethyl-o-crotonotoluidide) has been used as an anti-itch agent for humans for around 70years. In spite of the long use of crotamiton, its mechanism of action remains unknown. We hypothesized that crotamiton might have effects on transient receptor potential (TRP) channels expressed in the peripheral nervous system and the skin. We first examined the effects of crotamiton on TRP channels by whole-cell patch-clamp recordings. We found that crotamiton strongly inhibited TRPV (vanilloid) 4 channels followed by large currents after crotamiton washout. In mice, crotamiton inhibited itch-related behaviors induced by a TRPV4-selective agonist (GSK1016790A). We biophysically investigated the large TRPV4 currents after crotamiton washout. Comparing single-channel open probabilities and current amplitudes of TRPV4, increases in both parameters were found to contribute to the large washout currents of TRPV4. Because the change in current amplitudes suggested pore dilation of TRPV4, we examined this possibility with cation replacement experiments and by measuring changes in reversal potentials. Greater cation influxes and changes in reversal potentials upon crotamiton washout were observed, suggesting that the TRPV4 pore dilated in its uninhibited state. From these results, we identified the molecular target of crotamiton as TRPV4 and demonstrated pore dilation of TRPV4 upon crotamiton washout.

Acid-Induced Chloride Current in Distal Convoluted Tubule

The distal convoluted tubule (DCT) of the kidney is responsible for the fine-tuning of urinary filtrate before excretion. The DCT is known to facilitate the movement of Na, K, Ca, Mg, and Cl via transporters and ion channels. Recent research has focussed on the functional activity of TRP channels in the DCT, with heavy emphasis on TRPM6/7 (Mg at neutral pH 7.4; Na at acidic pH) and TRPV5/6 (Ca). Several studies have suggested that these channels should give rise to large macroscopic currents in DCT cells. Therefore we undertook this study to determine the ionic species representing the dominant current in mouse DCT (MDCT) cells via whole-cell voltage-clamp electrophysiology. Using ionic permeation studies and analysis of reversal potential changes, our results unequivocally demonstrate that MDCT cells exhibit a significant Cl current under both neutral and acidic extracellular solutions. We further identified that this current is rapidly activating, differentiating it from the endogenous acid-induced anion current discovered in HEK293 cells. We noted that currents elicited under acidic pH were substantially larger than those at neutral pH, and thus hypothesized that extracellular acid induced a different, larger Cl current in MDCT cells. This was supported pharmacologically, where only the acid-induced Cl current was blocked by furosemide, a clinically prescribed loop-diuretic. To further understand the significance of a large acid-induced Cl current in the late nephron, we investigated inner medullary collecting duct (IMCD) cells as they represent the most acidic portion of the nephron. IMCD current was not only acid-induced, but shared an identical I-V relationship, pH sensitivity, and time-course of activation with the acid-induced induced Cl current discovered in MDCT cells. We thus conclude that the late nephron contains a large acid-induced Cl current that may play a significant role in anion reabsorption.

Effect of Diaminopropionic Acid (Dap) on the Biophysical Properties of a Modified Synthetic Channel-Forming Peptide

Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK4-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na+, Cl- or K+, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers.

Re-Introduction of Transmembrane Serine Residues Reduce the Minimum Pore Diameter of Channelrhodopsin-2

Channelrhodopsin-2 (ChR2) is a microbial-type rhodopsin found in the green algae Chlamydomonas reinhardtii. Under physiological conditions, ChR2 is an inwardly rectifying cation channel that permeates a wide range of mono- and divalent cations. Although this protein shares a high sequence homology with other microbial-type rhodopsins, which are ion pumps, ChR2 is an ion channel. A sequence alignment of ChR2 with bacteriorhodopsin, a proton pump, reveals that ChR2 lacks specific motifs and residues, such as serine and threonine, known to contribute to non-covalent interactions within transmembrane domains. We hypothesized that reintroduction of the eight transmembrane serine residues present in bacteriorhodopsin, but not in ChR2, will restrict the conformational flexibility and reduce the pore diameter of ChR2. In this work, eight single serine mutations were created at homologous positions in ChR2. Additionally, an endogenous transmembrane serine was replaced with alanine. We measured kinetics, changes in reversal potential, and permeability ratios in different alkali metal solutions using two-electrode voltage clamp. Applying excluded volume theory, we calculated the minimum pore diameter of ChR2 constructs. An analysis of the results from our experiments show that reintroducing serine residues into the transmembrane domain of ChR2 can restrict the minimum pore diameter through inter- and intrahelical hydrogen bonds while the removal of a transmembrane serine results in a larger pore diameter. Therefore, multiple positions along the intracellular side of the transmembrane domains contribute to the cation permeability of ChR2.

ANO1‐ther brick in the wall – role of Ca2+‐activated Cl− channels of interstitial cells of Cajal in cholinergic motor control of gastrointestinal smooth muscle

ANO1‐ther brick in the wall – role of Ca²⁺‐activated Cl⁻ channels of interstitial cells of Cajal in cholinergic motor control of gastrointestinal smooth muscle

Extracellular potassium accumulation may contribute to long-term potentiation induced with long 100 Hz stimulus trains

Long-term potentiation is often induced by high-frequency stimulation (100 Hz) for 1 second. However, in hippocampal CA1 pyramidal cells we observe that EPSPs quickly become depressed in such trains and it is rare to see action potentials in the pyramidal cell after 200 ms into the train. Because LTP induction depends on postsynaptic depolarization and there appeared to be little depolarization after 200 ms, we predicted that lengthening the train from 200 ms to 1600 ms should produce little additional LTP. However we found that LTP magnitude progressively increased with train length. Further investigation revealed that fiber volley amplitude decreased and latency increased during a long train and that these changes could be reproduced by raising extracellular potassium concentration in the bath. In addition NMDA receptor mediated field EPSPs were increased with elevated extracellular potassium. These experimental findings strongly suggest that decreased afferent excitability and decreased glutamate release during a train are more than compensated by increases in extracellular potassium which enhances the NMDA receptor response and allows long trains to produce additional LTP. We have constructed computational models to test this idea and gain insight into how elevated extracellular potassium might be produced by long high-frequency trains and how this might affect LTP. If extracellular potassium concentration is raised from 3.4 mM to 13 mM, we note that: i) the NMDA reversal potential changes only 1.5 mV, ii) the potassium reversal potential depolarizes by 35 mV, and iii) resting potential depolarizes by 20 mV. The effect of these changes is to double the amount of calcium coming into a dendritic spine through NMDA receptor channels, but these changes also make the neuron fire action potentials even when input is depressed, in contradiction to the experimental results. This suggests that changes in extracellular potassium are likely to be restricted to regions around the afferent axons and the sites of synaptic contact. We are currently investigating this hypothesis and are seeking parameter conditions that reproduce the experimental findings.

Fast activity at seizure onset is mediated by inhibitory circuits in the entorhinal cortex in vitro

Network mechanisms responsible for focal seizure initiation are still largely unknown. One of the prevalent seizure patterns observed during diagnostic intracranial recordings performed in patients with mesial temporal lobe epilepsy is characterized by fast activity at 20 to 30 Hz. We reproduced 20 to 30 Hz oscillations at seizure onset in the temporal lobe of the in vitro isolated guinea pig brain to study cellular and network mechanisms involved in its generation. Seizure-like activity was induced in the isolated brain by 3-minute arterial perfusion of 50 microM bicuculline. Intracellular, extracellular, and ion-selective electrophysiological recordings were performed simultaneously in the entorhinal cortex (EC) during interictal-ictal transition. Principal neurons in deep and superficial layers of the EC did not generate action potentials during fast activity at ictal onset, whereas sustained firing was observed in putative interneurons. Within 5 to 10 seconds from seizure initiation, principal neurons generated a prominent firing that correlated with the appearance of extracellular hypersynchronous bursting discharges. In superficial neurons, fast activity correlated with rhythmic IPSPs that progressively decreased in amplitude during the development of a slow depolarization associated with an increase in extracellular potassium. We conclude that in an acute model of temporal lobe ictogenesis, sustained inhibition without firing of EC principal neurons correlates with the onset of a focal seizure. The progression of the ictal discharge is contributed by a potassium-dependent change in reversal potential of inhibitory postsynaptic potentials. These findings demonstrate a prominent role of inhibitory networks during the transition to seizure in the EC.

Bursting regimes in map-based neuron models coupled through fast threshold modulation

A system consisting of two map-based neurons coupled through reciprocal excitatory or inhibitory chemical synapses is discussed. After a brief explanation of the basic mechanism behind generation and synchronization of bursts, parameter space is explored to determine less obvious but biologically meaningful regimes and effects. Among them, we show how excitatory synapses without any delays may induce antiphase synchronization; that a synapse may change its character from excitatory to inhibitory and vice versa by changing its conductance, without any change in reversal potential; and that small variations in the synaptic threshold may result in drastic changes in the synchronization of spikes within bursts. Finally we show how the synchronization effects found in the two-neuron system carry over to larger networks.

Bestrophin Cl− channels are highly permeable to HCO3−

Bestrophin-1 (Best1) is a Cl(-) channel that is linked to various retinopathies in both humans and dogs. Dysfunction of the Best1 Cl(-) channel has been proposed to cause retinopathy because of altered Cl(-) transport across the retinal pigment epithelium (RPE). In addition to Cl(-), many Cl(-) channels also transport HCO3(-). Because HCO3(-) is physiologically important in pH regulation and in fluid and ion transport across the RPE, we measured the permeability and conductance of bestrophins to HCO3(-) relative to Cl(-). Four human bestrophin homologs (hBest1, hBest2, hBest3, and hBest4) and mouse Best2 (mBest2) were expressed in HEK cells, and the relative HCO3(-) permeability (P HCO3/PCl) and conductance (G HCO3/GCl) were determined. P HCO3/PCl was calculated from the change in reversal potential (Erev) produced by replacing extracellular Cl(-) with HCO3(-). hBest1 was highly permeable to HCO3(-) (P HCO3)/PCl = approximately 0.44). hBest2, hBest4, and mBest2 had an even higher relative HCO3(-) permeability (P HCO3/PCl = 0.6-0.7). All four bestrophins had HCO3(-) conductances that were nearly the same as Cl(-) (G HCO3/GCl = 0.9-1.1). Extracellular Na+ did not affect the permeation of hBest1 to HCO3(-). At physiological HCO3(-) concentration, HCO3(-) was also highly conductive. The hBest1 disease-causing mutations Y85H, R92C, and W93C abolished both Cl(-) and HCO3(-) currents equally. The V78C mutation changed P HCO3/PCl and G HCO3/GCl of mBest2 channels. These results raise the possibility that disease-causing mutations in hBest1 produce disease by altering HCO3(-) homeostasis as well as Cl(-) transport in the retina.

On the Role of the First Transmembrane Domain in Cation Permeability and Flux of the ATP-gated P2X2 Receptor

P2X receptors are a family of seven ligand-gated ion channels (P2X1-P2X7) that open in the presence of ATP. We used alanine-scanning mutagenesis and patch clamp photometry to study the role of the first transmembrane domain of the rat P2X2 receptor in cation permeability and flux. Three alanine-substituted mutants did not respond to ATP, and 19 of the 22 functional receptors resembled the wild-type receptor with regard to the fraction of the total ATP-gated current carried by calcium or the permeability of calcium relative to cesium. The remaining three mutants showed modest changes in calcium dynamics. Two of these occurred at sites (Gly30 and Phe44) that are unlikely to interact with permeating cations in a meaningful way. The third was a conserved tyrosine (Tyr43) that may form an inter-pore binding site for calcium. The data suggest that, with the possible exception of Tyr43, the first transmembrane domain contributes little to the permeation properties of the P2X2 receptor.

Overexpression of CLC-3 in HEK293T cells yields novel currents that are pH dependent

ClC-3 is a member of the ClC family of anion channels/transporters. Recently, the closely related proteins ClC-4 and ClC-5 were shown to be Cl(-)/H(+) antiporters (39, 44). The function of ClC-3 has been controversial. We studied anion currents in HEK293T cells expressing wild-type or mutant ClC-3. The basic biophysical properties of ClC-3 currents were very similar to those of ClC-4 and ClC-5, and distinct from those of the swelling-activated anion channel. ClC-3 expression induced currents with time-dependent activation that rectified sharply in the outward direction. The reversal potential of the current shifted by -48.3 +/- 2.5 mV per 10-fold (decade) change in extracellular Cl(-) concentration, which did not conform to the behavior of an anion-selective channel based upon the Nernst equation, which predicts a -58.4 mV/decade shift at 22 degrees C. Manipulation of extracellular pH (6.35-8.2) altered reversal potential by 10.2 +/- 3.0 mV/decade, suggesting that ClC-3 currents were coupled to proton movement. Mutation of a specific glutamate residue (E224A) changed voltage dependence in a manner similar to that observed in other ClC Cl(-)/H(+) antiporters. Mutant currents exhibited Nernstian changes in reversal potential in response to altered extracellular Cl(-) concentration that averaged -60 +/- 3.4 mV/decade and were pH independent. Thus ClC-3 overexpression induced a pH-sensitive conductance in HEK293T cells that is biophysically similar to ClC-4 and ClC-5.

Calcium‐dependent inactivation of calcium channels in cochlear hair cells of the chicken

Voltage-gated calcium channels support both spontaneous and sound-evoked neurotransmitter release from ribbon synapses of cochlear hair cells. A variety of regulatory mechanisms must cooperate to ensure the appropriate level of activity in the restricted pool of synaptic calcium channels ( approximately 100) available to each synaptic ribbon. One potential feedback mechanism, calcium-dependent inactivation (CDI) of voltage-gated, L-type calcium channels, can be modulated by calmodulin-like calcium-binding proteins. CDI of voltage-gated calcium current was studied in hair cells of the chicken's basilar papilla (analogous to the mammalian cochlea) after blocking the predominant potassium conductances. For inactivating currents produced by 2.5 s steps to the peak of the current-voltage relation (1 mm EGTA internal calcium buffer), single exponential fits yielded an average decay time constant of 1.92 +/- 0.18 s (mean +/- s.e.m., n = 12) at 20-22 degrees C, while recovery occurred with a half-time of approximately 10 s. Inactivation produced no change in reversal potential, arguing that the observed relaxation did not result from alternative processes such as calcium accumulation or activation of residual potassium currents. Substitution of external calcium with barium greatly reduced inactivation, while inhibition of endoplasmic calcium pumps with t-benzohydroquinone (BHQ) or thapsigargin made inactivation occur faster and to a greater extent. Raising external calcium 10-fold (from 2 to 20 mm) increased peak current 3-fold, but did not alter the extent or time course of CDI. However, increasing levels of internal calcium buffer consistently reduced the rate and extent of inactivation. With 1 mm EGTA buffering and in 2 mm external calcium, the available pool of calcium channels was half-inactivated near the resting membrane potential (-50 mV). CDI may be further regulated by calmodulin-like calcium-binding proteins (CaBPs). mRNAs for several CaBPs are expressed in chicken cochlear tissue, and antibodies to CaBP4 label hair cells, but not supporting cells, equivalent to the pattern seen in mammalian cochlea. Thus, molecular mechanisms that underlie CDI appeared to be conserved across vertebrate species, may provide a means to adjust calcium channel open probability, and could serve to maintain the set-point for spontaneous release from the ribbon synapse.

NIPA1(SPG6), the Basis for Autosomal Dominant Form of Hereditary Spastic Paraplegia, Encodes a Functional Mg2+ Transporter

Mutations in the NIPA1(SPG6) gene, named for "nonimprinted in Prader-Willi/Angelman" has been implicated in one form of autosomal dominant hereditary spastic paraplegia (HSP), a neurodegenerative disorder characterized by progressive lower limb spasticity and weakness. However, the function of NIPA1 is unknown. Here, we show that reduced magnesium concentration enhances expression of NIPA1 suggesting a role in cellular magnesium metabolism. Indeed NIPA1 mediates Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with a Michaelis constant of 0.69+/-0.21 mM when expressed in Xenopus oocytes. Subcellular localization with immunofluorescence showed that endogenous NIPA1 protein associates with early endosomes and the cell surface in a variety of neuronal and epithelial cells. As expected of a magnesium-responsive gene, we find that altered magnesium concentration leads to a redistribution between the endosomal compartment and the plasma membrane; high magnesium results in diminished cell surface NIPA1 whereas low magnesium leads to accumulation in early endosomes and recruitment to the plasma membrane. The mouse NIPA1 mutants, T39R and G100R, corresponding to the respective human mutants showed a loss-of-function when expressed in oocytes and altered trafficking in transfected COS7 cells. We conclude that NIPA1 normally encodes a Mg2+ transporter and the loss-of function of NIPA1(SPG6) due to abnormal trafficking of the mutated protein provides the basis of the HSP phenotype.

Linkage between ‘disruption of inactivation’ and ‘reduction of K+ selectivity’ among hERG mutants in the S5‐P linker region

Linkage between ‘disruption of inactivation’ and ‘reduction of K⁺ selectivity’ among hERG mutants in the S5‐P linker region

Reply from Jamie I. Vandenberg, Adam P. Hill, Terence J. Campbell, Catherine E. Clarke

Professor Tseng has objected to the statement in the discussion of our paper: ‘… the majority of the S5P α-helix cysteine [mutants] that had marked changes in reversal potential… form intersubunit disulphide bonds… significantly disrupted outer pore structure’… and its implication that the data interpretation in Liu et al. (2002) was incorrect.

Effects of osmotic shrinkage on voltage-gated Ca2+ channel currents in rat anterior pituitary cells

Increased extracellular osmolarity ([Os]e) suppresses stimulated hormone secretion from anterior pituitary cells. Ca2+ influx may mediate this effect. We show that increase in [Os]e (by 18-125%) differentially suppresses L-type and T-type Ca2+ channel currents (IL and IT, respectively); IL was more sensitive than IT. Hyperosmotic suppression of IL depended on the magnitude of increase in [Os]e and was correlated with the percent decrease in pituitary cell volume, suggesting that pituitary cell shrinkage can modulate L-type currents. The hyperosmotic suppression of IL and IT persisted after incubation of pituitary cells either with the actin-disrupter cytochalasin D or with the actin stabilizer phalloidin, suggesting that the actin cytoskeleton is not involved in this modulation. The hyperosmotic suppression of Ca2+ influx was not correlated with changes in reversal potential, membrane capacitance, and access resistance. Together, these results suggest that the hyperosmotic suppression of Ca2+ influx involves Ca2+ channel proteins. We therefore recorded the activity of L-type Ca2+ channels from cell-attached patches while exposing the cell outside the patch pipette to hyperosmotic media. Increased [Os]e reduced the activity of Ca2+ channels but did not change single-channel conductance. This hyperosmotic suppression of Ca2+ currents may therefore contribute to the previously reported hyperosmotic suppression of hormone secretion.

Single Neurons Can Induce Phase Transitions of Cortical Recurrent Networks with Multiple Internal States

Fluctuations of membrane potential of cortical neurons, referred to here as internal states, are essential for brain function, but little is known about how these internal states emerge and are maintained, or what determines transitions between these states. We performed intracellular recordings from hippocampal CA3 pyramidal cells ex vivo and found that neurons display multiple and hierarchical internal states, which are linked to cholinergic activity and are characterized by several power law structures in membrane potential dynamics. Multiple recordings from adjacent neurons revealed that the internal states were coherent between neurons, indicating that the internal state of any given cell in a local network could represent the network activity state. Repeated stimulation of single neurons led over time to transitions to different internal states in both the stimulated neuron and neighboring neurons. Thus, single-cell activation is sufficient to shift the state of the entire local network. As the states shift to more active levels, theta- and gamma-frequency components developed in the form of subthreshold oscillations. State transitions were associated with changes in membrane conductance but were not accompanied by a change in reversal potential. These data suggest that the recurrent network organizes the internal states of individual neurons into synchronization through network activity with balanced excitation and inhibition, and that this organization is discrete, heterogeneous and dynamic in nature. Thus, neuronal states reflect the 'phase' of an active network, a novel demonstration of the dynamics and flexibility of cortical microcircuitry.

Voltage and concentration dependence of Ca(2+) permeability in recombinant glutamate receptor subtypes.

The channels associated with glutamate receptor (GluR) subtypes, namely N-methyl-D-aspartate receptors (NMDARs), and Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) and kainate receptors (KARs), are to varying degrees permeable to Ca(2+). To compare the mechanism of Ca(2+) influx, we measured Ca(2+) permeability relative to that of Na(+) (P(Ca)/P(Na)) using fractional Ca(2+) currents (P(f)) and reversal potential measurements over a wide voltage and Ca(2+) concentration range in recombinant NMDAR NR1-NR2A, AMPAR GluR-A(Q) and KAR GluR-6(Q) channels. For NR1-NR2A channels, P(Ca)/P(Na) derived from P(f) measurements was voltage independent but showed a weak concentration dependence. A stronger concentration dependence was found when P(Ca)/P(Na) was derived from changes in reversal potentials on going from a Na(+) reference solution to a solution with Ca(2+) as the only permeant ion ('biionic' condition). In contrast, P(Ca)/P(Na) was concentration independent when derived from changes in reversal potentials on going from a Na(+) reference solution to the same solution with added Ca(2+) ('high monovalent' condition). For GluR-A(Q) channels, P(Ca)/P(Na) derived from all three approaches was concentration independent, and for the reversal potential-based approaches were of comparable magnitude. Their most distinctive property was that P(Ca)/P(Na) derived from P(f) measurements was strongly voltage dependent. For GluR-6(Q) channels, P(Ca)/P(Na) derived from P(f) measurements was weakly voltage dependent. On the other hand, P(Ca)/P(Na) derived from all three approaches was the most strongly concentration dependent of any GluR subtype and, except for low Ca(2+) concentrations, the values were of comparable magnitude. Thus, the three Ca(2+)-permeable GluR subtypes showed unique patterns of Ca(2+) permeability, indicating that distinct biophysical and molecular events underlie Ca(2+) influx in each subtype.

Polar Residues of the Second Transmembrane Domain Influence Cation Permeability of the ATP-gated P2X2 Receptor

P2X receptors are simple polypeptide channels that mediate fast purinergic depolarizations in both nerve and muscle. Although the depolarization results mainly from the influx of Na(+), these channels also conduct a significant Ca(2+) current that is large enough to evoke transmitter release from presynaptic neurons. We sought to determine the molecular basis of this Ca(2+) conductance by a mutational analysis of recombinant P2X(2) receptors. Wild type and 31 mutant P2X(2) receptors were expressed in HEK-293 cells and studied under voltage-clamp. We found that the relative Ca(2+) permeability measured from the reversal potentials of ATP-gated currents was unaffected by neutralizing fixed charge (Asp(315), Asp(349)) near the mouths of the channel pore. By contrast, mutations that changed the character or side chain volume of three polar residues (Thr(336), Thr(339), Ser(340)) within the pore led to significant changes in P(Ca)/P(Cs). The largest changes occurred when Thr(339) and Ser(340) were replaced with tyrosine; these mutations almost completely abolished Ca(2+) permeability, reduced P(Li)/P(Cs) by about one-half, and shifted the relative permeability sequence of Cs(+), Rb(+), K(+), and Na(+) to their relative mobility in water. Our results suggest that the permeability sequence of the P2X(2) receptor arises in part from interactions of permeating cations with the polar side chains of three amino acids located in a short stretch of the second transmembrane domain.