M2 pore mutations convert the glycine receptor channel from being anion- to cation-selective.

Three mutations in the M2 transmembrane domains of the chloride-conducting alpha1 homomeric glycine receptor (P250Delta, A251E, and T265V), which normally mediate fast inhibitory neurotransmission, produced a cation-selective channel with P(Cl)/P(Na), = 0.27 (wild-type P(Cl)/P(Na) = 25), a permeability sequence P(Cs) > P(K) > P(Na) > P(Li), an impermeability to Ca(2+), and a reduced glycine sensitivity. Outside-out patch measurements indicated reversed and accentuated rectification with extremely low mean single channel conductances of 3 pS (inward current) and 11 pS (outward current). The three inverse mutations, to those analyzed in this study, have previously been shown to make the alpha7 acetylcholine receptor channel anion-selective, indicating a common location for determinants of charge selectivity of inhibitory and excitatory ligand-gated ion channels.

Mutation of an arginine residue in the human glycine receptor transforms beta-alanine and taurine from agonists into competitive antagonists.

Agonist binding to the inhibitory glycine receptor (GlyR) initiates the opening of a chloride-selective channel that modulates the neuronal membrane potential. Point mutations of the GlyR, substituting Arg-271 with either Leu or Gln, have been shown to underlie the inherited neurological disorder startle disease (hyperekplexia). We show that these substitutions result in the redistribution of GlyR single-channel conductances to lower conductance levels. Additionally, the binding of the glycinergic agonists beta-alanine and taurine to mutated GlyRs does not initiate a chloride current, but instead competitively antagonizes currents activated by glycine. These findings are consistent with mutations of Arg-271 resulting in the uncoupling of the agonist binding process from the channel activation mechanism of the receptor.

Startle disease mutations reduce the agonist sensitivity of the human inhibitory glycine receptor.

The receptor for the inhibitory neurotransmitter glycine is a member of the ligand-gated ion channel receptor superfamily. Point mutations in the gene encoding the alpha 1 subunit of the glycine receptor-channel complex (GlyR) have recently been identified in pedigrees with the autosomal dominant neurological disorder, startle disease (hyperekplexia). These mutations result in the substitution of leucine or glutamine for arginine 271. This charged residue is located near the ion channel region and is predicted to affect chloride permeation through the GlyR. We found little evidence for this role from the anion/cation selectivity and lack of pronounced rectification of currents flowing through recombinant human alpha 1 subunit GlyRs containing the startle disease mutations. We reveal, however, that the startle disease mutations profoundly disrupt GlyR function by causing 230-410-fold decreases in the sensitivity of receptor currents activated by the agonist glycine. Additionally, we report corresponding 56- and 120-fold reductions in the apparent binding affinity (Ki) of glycine to the mutant GlyRs, but no change in the binding affinity of the competitive antagonist, strychnine. Thus, startle disease reduces the efficacy of glycinergic inhibitory neurotransmission by producing GlyRs with diminished agonist responsiveness. Our results show that startle disease mutations define a novel receptor activation site.

The extracellular disulfide loop motif of the inhibitory glycine receptor does not form the agonist binding site.

Inhibitory (glycine and gamma-aminobutyric acid type A) and excitatory (nicotinic acetylcholine and serotonin 5-hydroxytryptamine type 3) receptors of the ligand-gated ion channel superfamily are related by both structural similarities and primary sequence identity. One invariant feature of all members of this receptor superfamily is the presence of an extracellular disulfide loop motif. This structural motif has been modeled, and Cockcroft et al. [Proteins 8:386-397 (1990)] have suggested that it forms the agonist binding site of the ligand-gated ion channel receptors. Using site-directed mutagenesis of the inhibitory glycine receptor (GlyR), we have specifically tested this hypothesis. The lysine residue at position 143 is proposed to form the binding site for the negatively charged carboxyl group of the agonist glycine. Differing residues at this position in other ligand-gated receptors are proposed to confer agonist specificity. The aspartic acid residue at position 148 is an invariant residue in every known subunit of the ligand-gated ion channel receptor superfamily. This residue has been proposed as the binding site for the positively charged amino group of the various agonists. Mutation of the lysine at position 143 to alanine resulted in essentially unaltered GlyRs, showing only modest decreases in strychnine affinity (Kd, 8.1 +/- 1.4 nM versus 13.4 +/- 1.3 nM), glycine displacement of strychnine binding (Ki, 25 +/- 5 microM versus 49 +/- 9 microM), and glycine activation of chloride currents (EC50, 27 +/- 6 microM versus 114 +/- 14 microM). Thus, we conclude that Lys-143 does not play a major role in either agonist or antagonist binding or agonist activation of the GlyR. Mutation of Asp-148 to either alanine or asparagine disrupted the expression and/or assembly of the receptor, and no binding sites or ion channels were expressed on the cell surface. The conservative mutation of the aspartic acid at position 148 to glutamic acid (D148E) allowed the expression of receptors, although with reduced efficiency. The D148E GlyRs showed a 1 order of magnitude decrease in strychnine affinity (Kd, 8.1 +/- 1.4 nM versus 82 +/- 21 nM), without any change in the glycine displacement of strychnine binding (Ki, 25 +/- 5 microM versus 29 +/- 8 microM) or glycine activation of chloride currents (EC50, 27 +/- 6 microM versus 20 +/- 1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor alpha subunit form the strychnine-binding site.

The inhibitory glycine receptor (GlyR) is a member of the ligand-gated ion channel receptor superfamily. Glycine activation of the receptor is antagonized by the convulsant alkaloid strychnine. Using in vitro mutagenesis and functional analysis of the cDNA encoding the alpha 1 subunit of the human GlyR, we have identified several amino acid residues that form the strychnine-binding site. These residues were identified by transient expression of mutated cDNAs in mammalian (293) cells and examination of resultant [3H]strychnine binding, glycine displacement of [3H]strychnine, and electrophysiological responses to the application of glycine and strychnine. This mutational analysis revealed that residues from two separate domains within the alpha 1 subunit form the binding site for the antagonist strychnine. The first domain includes the amino acid residues Gly-160 and Tyr-161, and the second domain includes the residues Lys-200 and Tyr-202. These results, combined with analyses of other ligand-gated ion channel receptors, suggest a conserved tertiary structure and a common mechanism for antagonism in this receptor superfamily.

A voltage-dependent persistent sodium current in mammalian hippocampal neurons.

Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltage-clamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carried by sodium ions because it was blocked by TTX, decreased in amplitude when extracellular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at approximately -40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 +/- 0.05 nA (mean +/- 1 SEM) and -0.21 +/- 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = gmax/(1 + exp[(V' - V)/k)]). The average gmax was 7.8 +/- 1.1 nS in the 21 neurons in slices and 4.4 +/- 1.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at greater than 50% of its normal amplitude when the transient current was completely inactivated. Because the persistent sodium current activates at potentials close to the resting membrane potential and is very resistant to inactivation, it probably plays an important role in the repetitive firing of action potentials caused by prolonged depolarizations such as those that occur during barrages of synaptic inputs into these cells.

Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices.

Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of -60 to -70 mV elicited a slow inward calcium (Ca2+) current and two outward potassium (K+) currents: an A current and a slower, Ca2+-dependent K+ current. Noradrenaline (NA) (20 microM) depressed the amplitude of the K+ currents without affecting the Ca2+ current. The effect of NA could be blocked with propranolol and was mimicked by isoprenaline, suggesting that NA depresses the K+ currents by binding to beta-receptors.

A threshold sodium current in pyramidal cells in rat hippocampus.

Maintained, inward currents were activated by small depolarizations from the resting membrane potential (-50 to -60 mV) in voltage-clamped, pyramidal neurons in rat hippocampal slices. The currents were apparently Na currents as they were blocked by tetrodotoxin or removal of extracellular Na and were not affected by Cd. They showed little decrease in amplitude during prolonged depolarizations. The increase in Na conductance with depolarization was sigmoidal, with half-maximum conductance at about -50 mV, and saturated at -20 to -30 mV. This 'threshold' Na current may be involved in setting patterns of repetitive firing of action potentials.

The inhibitory role of the visually responsive region of the thalamic reticular nucleus in the rat.

Two-shock inhibition, a feature of 98 of 100 P cells recorded in the dorsal lateral geniculate nucleus of the normal rat, was not observed in 91 of 140 geniculate cells after an electrolytic lesion had been made in the adjacent visually responsive thalamic reticular nucleus. Nine geniculate cells recorded both before and after a reticular lesion had their initial inhibition abolished or substantially reduced after the lesion. The reticular lesion eliminated the bursts of spikes which normally terminate periods of inhibition following electrical or photic stimulation but caused no other changes in receptive field organization of geniculate cells. We conclude that the visually responsive region of the thalamic reticular nucleus in the rat is responsible for the profound two-shock inhibition and for the post-inhibitory bursts which are normal properties of relay cells of the dorsal lateral geniculate nucleus.