54 research outputs found
Agonist-dependent Single Channel Current and Gating in α4β2δ and α1β2γ2S GABAA Receptors
The family of γ-aminobutyric acid type A receptors (GABAARs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABAARs that are mainly comprised of α1, β2, and γ2 subunits, whereas tonic inhibition is mediated by extrasynaptic GABAARs comprised of α4/6, β2, and δ subunits. We investigated the activation properties of recombinant α4β2δ and α1β2γ2S GABAARs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at α4β2δ GABAARs (β ∼1600 s−1) than at α1β2γ2S GABAARs (β ∼ 460 s−1), whereas GABA activated α1β2γ2S GABAARs more rapidly (β ∼1800 s−1) than α4β2δ GABAARs (β < 440 s−1). Single channel recordings of α1β2γ2S and α4β2δ GABAARs showed that both channels open to a main conductance state of ∼25 pS at −70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited ∼36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (PO ∼ 0.3) at α4β2δ GABAARs and at least two “modes” of single channel bursting activity, lasting ∼100 ms at α1β2γ2S GABAARs. The most prevalent bursting mode had a PO of ∼0.7 and was described by a reaction scheme with three open and three shut states, whereas the “high” PO mode (∼0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in α4β2δ and α1β2γ2S GABAARs occurred as a single population of bursts (PO ∼0.4–0.5) of moderate duration (∼33 ms) that could be described by schemes containing two shut and two open states for both GABAARs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance
GABAa receptor α and γ subunits shape synaptic currents via different mechanisms
Synaptic GABAA receptors (GABAARs) mediate most of the inhibitory neurotransmission in the brain. The majority of these receptors are comprised of α1, β2, and γ2 subunits. The amygdala, a structure involved in processing emotional stimuli, expresses α2 and γ1 subunits at high levels. The effect of these subunits on GABA AR-mediated synaptic transmission is not known. Understanding the influence of these subunits on GABAAR-mediated synaptic currents may help in identifying the roles and locations of amygdala synapses that contain these subunits. Here, we describe the biophysical and synaptic properties of pure populations of α1β2γ2, α2β2γ2, α1β2γ1 and α2β2γ1 GABAARs. Their synaptic properties were examined in engineered synapses, whereas their kinetic properties were studied using rapid agonist application, and single channel recordings. All macropatch currents activated rapidly
Single Channel Analysis of Conductance and Rectification in Cation-selective, Mutant Glycine Receptor Channels
Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of α1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2'Δ) GlyR, had mean inward chord conductances (at −60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance–voltage relationship linear (14 pS at both 60 and −60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels
Cation-selective Mutations in the M2 Domain of the Inhibitory Glycine Receptor Channel Reveal Determinants of Ion-Charge Selectivity
Ligand-gated ion channel receptors mediate neuronal inhibition or excitation depending on their ion charge selectivity. An investigation into the determinants of ion charge selectivity of the anion-selective α1 homomeric glycine receptor (α1 glycine receptor [GlyR]) was undertaken using point mutations to residues lining the extra- and intracellular ends of the ion channel. Five mutant GlyRs were studied. A single substitution at the intracellular mouth of the channel (A-1′E GlyR) was sufficient to convert the channels to select cations over anions with PCl/PNa = 0.34. This result delimits the selectivity filter and provides evidence that electrostatic interactions between permeating ions and pore residues are a critical factor in ion charge selectivity. The P-2′Δ mutant GlyR retained its anion selectivity (PCl/PNa = 3.81), but it was much reduced compared with the wild-type (WT) GlyR (PCl/PNa = 27.9). When the A-1′E and the P-2′Δ mutations were combined (selectivity double mutant [SDM] GlyR), the relative cation permeability was enhanced (PCl/PNa = 0.13). The SDM GlyR was also Ca2+ permeable (PCa/PNa = 0.29). Neutralizing the extracellular mouth of the SDM GlyR ion channel (SDM+R19′A GlyR) produced a more Ca2+-permeable channel (PCa/PNa = 0.73), without drastically altering monovalent charge selectivity (PCl/PNa = 0.23). The SDM+R19′E GlyR, which introduces a negatively charged ring at the extracellular mouth of the channel, further enhanced Ca2+ permeability (PCa/PNa = 0.92), with little effect on monovalent selectivity (PCl/PNa = 0.19). Estimates of the minimum pore diameter of the A-1′E, SDM, SDM+R19′A, and SDM+R19′E GlyRs revealed that these pores are larger than the α1 GlyR, with the SDM-based GlyRs being comparable in diameter to the cation-selective nicotinic acetylcholine receptors. This result provides evidence that the diameter of the ion channel is also an important factor in ion charge selectivity
Taurine is a potent activator of extrasynaptic GABAA receptors in the thalamus
Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (>= 100 mu M), and to be a weak agonist at GABA(A) receptors (GABA(A)-Rs), which are usually activated at high concentrations (>= 1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABAA-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10 - 100 mu M) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 mu M taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 mu M Zn2+, consistent with the pharmacological properties of extrasynaptic GABA(A)-Rs (alpha 4 beta 2 delta subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABA(A)-R alpha 4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant alpha(4)beta 2 delta GABA(A)-Rs than at alpha 1 beta 2 gamma 2 GABA(A)-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABA(A)-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus
Inhibitory synapse deficits caused by familial α1 GABAA receptor mutations in epilepsy
Epilepsy is a spectrum of neurological disorders with many causal factors. The GABA type-A receptor (GABA(A)R) is a major genetic target for heritable human epilepsies. Here we examine the functional effects of three epilepsy causing mutations to the alpha 1 subunit (alpha 1(T10T), alpha 1(D192N) and alpha 1(A295D)) on inhibitory postsynaptic currents (IPSCs) mediated by the major synaptic GABA(A)R isoform, alpha 1 eta 2 gamma 2L. We employed a neuron - HEK293 cell heterosynapse preparation to record IPSCs mediated by mutant-containing GABA(A)Rs in isolation from other GABA(A)R isoforms. IPSCs were recorded in the presence of the anticonvulsant drugs, carbamazepine and midazolam, and at elevated temperatures (22, 37 and 40 degrees C) to gain insight into mechanisms of febrile seizures. The mutant subunits were also transfected into cultured cortical neurons to investigate changes in synapse formation and neuronal morphology using fluorescence microscopy. We found that IPSCs mediated by alpha 1(T10T)beta 2 gamma 2L, alpha 1(D192N)beta 2 gamma 2L GABA(A)Rs decayed faster than those mediated by alpha 1 beta 2 gamma 2L receptors. IPSCs mediated by alpha 1(D192N)beta 2 gamma 2L and alpha 1(A295D) beta 2 gamma 2L receptors also exhibited a heightened temperature sensitivity. In addition, the alpha 1(T10T)beta 2 gamma 2L GABA(A)Rs were refractory to modulation by carbamazepine or midazolam. In agreement with previous studies, we found that alpha 1(A295D)beta 2 gamma 2L GABA(A)Rs were retained intracellularly in HEK293 cells and neurons. However, pre-incubation with 100 nM suberanilohydroxamic acid (SAHA) induced alpha 1(A295D)beta 2 gamma 2L GABA(A)Rs to mediate IPSCs that were indistinguishable in magnitude and waveform from those mediated by alpha 1 beta 2 gamma 2L receptors. Finally, mutation specific changes to synaptic bouton size, synapse number and neurite branching were also observed. These results provide new insights into the mechanisms of epileptogenesis of alpha 1 epilepsy mutations and suggest possible leads for improving treatments for patients harbouring these mutations
Identification of molluscan nicotinic acetylcholine receptor (nAChR) subunits involved in formation of cation- and anion-selective nAChRs
Acetylcholine (ACh) is a neurotransmitter commonly found in all animal species. It was shown to mediate fast excitatory and inhibitory neurotransmission in the molluscan CNS. Since early intracellular recordings, it was shown that the receptors mediating these currents belong to the family of neuronal nicotinic acetylcholine receptors and that they can be distinguished on the basis of their pharmacology. We previously identified 12 Lymnaea cDNAs that were predicted to encode ion channel subunits of the family of the neuronal nicotinic acetylcholine receptors. These Lymnaea nAChRs can be subdivided in groups according to the residues supposedly contributing to the selectivity of ion conductance. Functional analysis in Xenopus oocytes revealed that two types of subunits with predicted distinct ion selectivities form homopentameric nicotinic ACh receptor (nAChR) subtypes conducting either cations or anions. Phylogenetic analysis of the nAChR gene sequences suggests that molluscan anionic nAChRs probably evolved from cationic ancestors through amino acid substitutions in the ion channel pore, a mechanism different from acetylcholine-gated channels in other invertebrates
The activation mechanism of α1β2γ2S and α3β3γ2S GABAA receptors
The α1β2γ2 and α3β3γ2 are two isoforms of γ-aminobutyric acid type A (GABAA) receptor that are widely distributed in the brain. Both are found at synapses, for example in the thalamus, where they mediate distinctly different inhibitory postsynaptic current profiles, particularly with respect to decay time. The two isoforms were expressed in HEK293 cells, and single-channel activity was recorded from outside-out patches. The kinetic characteristics of both isoforms were investigated by analyzing single-channel currents over a wide range of GABA concentrations. α1β2γ2 channels exhibited briefer active periods than α3β3γ2 channels over the entire range of agonist concentrations and had lower intraburst open probabilities at subsaturating concentrations. Activation mechanisms were constructed by fitting postulated schemes to data recorded at saturating and subsaturating GABA concentrations simultaneously. Reaction mechanisms were ranked according to log-likelihood values and how accurately they simulated ensemble currents. The highest ranked mechanism for both channels consisted of two sequential binding steps, followed by three conducting and three nonconducting configurations. The equilibrium dissociation constant for GABA at α3β3γ2 channels was ∼2.6 µM compared with ∼19 µM for α1β2γ2 channels, suggesting that GABA binds to the α3β3γ2 channels with higher affinity. A notable feature of the mechanism was that two consecutive doubly liganded shut states preceded all three open configurations. The lifetime of the third shut state was briefer for the α3β3γ2 channels. The longer active periods, higher affinity, and preference for conducting states are consistent with the slower decay of inhibitory currents at synapses that contain α3β3γ2 channels. The reaction mechanism we describe here may also be appropriate for the analysis of other types of GABAA receptors and provides a framework for rational investigation of the kinetic effects of a variety of therapeutic agents that activate or modulate GABAA receptors and hence influence synaptic and extrasynaptic inhibition in the central nervous system
Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (NaV), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on NaV1.1–1.9 using imaging and electrophysiological approaches. Although P-CTX-1 is essentially a non-selective NaV toxin and shifted the voltage-dependence of activation to more hyperpolarising potentials at all NaV subtypes, an increase in the inactivation time constant was observed only at NaV1.8, while the slope factor of the conductance-voltage curves was significantly increased for NaV1.7 and peak current was significantly increased for NaV1.6. Accordingly, P-CTX-1-induced visceral and cutaneous pain behaviours were significantly decreased after pharmacological inhibition of NaV1.8 and the tetrodotoxin-sensitive isoforms NaV1.7 and NaV1.6, respectively. The contribution of these isoforms to excitability of peripheral C- and A-fibre sensory neurons, confirmed using murine skin and visceral single-fibre recordings, reflects the expression pattern of NaV isoforms in peripheral sensory neurons and their contribution to membrane depolarisation, action potential initiation and propagation
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