Trevor M. Lewis

Recombinant nicotinic receptors, expressed in Xenopus oocytes, do not resemble native rat sympathetic ganglion receptors in single‐channel behaviour.

1. In order to establish the subunit composition of neuronal nicotinic receptors in rat superior cervical ganglia (SCG), their single‐channel properties were compared with those of recombinant receptors expressed in Xenopus oocytes, using outside‐out excised patch recording. 2. The mean main conductance of SCG channels from adult and 1‐day‐old rats was 34.8 and 36.6 pS, respectively. Less frequent openings to lower conductances occurred both as isolated bursts and as events connected to the main level by direct transitions. There was considerable interpatch variability in the values of the lower conductances. 3. Nicotinic receptors from oocytes expressing alpha3beta4 and alpha4beta4 subunits had chord conductances lower than that of SCG neurones (22 pS for alpha3beta4 and 29 pS for alpha4beta4). 4. Prolonged recording from both native and recombinant channels was precluded by ‘run‐down’, i.e. channel activity could be elicited for only a few minutes after excision. Nevertheless, SCG channel openings were clearly seen to occur as short bursts (slowest component, 38 ms), whereas recombinant channels opened in very prolonged bursts of activity, the major component being the slowest (480 ms). 5. Addition of the alpha5 subunit to the alpha3beta4 pair produced channels with a higher conductance than those observed after injection of the pair alone (24.9 vs. 22 pS), suggesting incorporation of alpha5 into the channel. Addition of the beta2 subunit did not change alpha3beta4 single‐channel properties. In one out of fourteen alpha3alpha5beta4 patches, both ganglion‐like, high conductance, short burst openings and recombinant‐type, low conductance, slow burst openings were observed. 6. Channels produced by expression in Xenopus oocytes of neuronal nicotinic subunits present in rat SCG as a rule differ from native ganglion receptors in single‐channel conductance and gross kinetics. While it is possible that an essential nicotinic subunit remains to be cloned, it is perhaps more likely that oocytes either cannot assemble neuronal nicotinic subunits efficiently into channels with the correct composition and stoichiometry, or that they produce post‐translational channel modifications which differ from those of mammalian neurones.

THE ION CHANNEL PROPERTIES OF A RAT RECOMBINANT NEURONAL NICOTINIC RECEPTOR ARE DEPENDENT ON THE HOST CELL TYPE

1 A stable mammalian cell line (L‐α3β4) has been established which expresses the cloned rat neuronal nicotinic acetylcholine receptor (nAChR) subunits α3 and β4, which are the most abundant in autonomic ganglia. Ion channel properties of nAChRs expressed in L‐α3β4 cells were investigated by single‐channel and whole‐cell recording techniques, and compared with both rat α3β4 nAChRs expressed in Xenopus oocytes, and endogenous nicotinic receptors in rat superior cervical ganglion (SCO) neurones, using identical solutions for all cell types. 2 Acetylcholine (ACh) caused activation of single ion channel currents with a range of amplitudes. Some channels had high conductances (30–40 pS), and relatively brief lifetimes; these resembled the predominant native channel from SCG. Other channels had low conductances (20–26 pS) and long bursts of openings which were quite unlike native channels, but which were similar to channels formed by α3β4 in oocytes. Both types often occurred in the same patch. 3 Cytisine was about 3 times more potent than ACh (low‐concentration potency ratio) in L‐α3β4 cells, which is not dissimilar to the 5‐fold potency ratio found in both SCG and oocytes, whereas 1,1‐dimethyl‐4‐phenylpiperazinium (DMPP) was less potent than ACh in some cells (as in the oocyte), but more potent in others (as in SCG). 4 While the channels expressed in L‐α3β4 cells do not mimic exactly those expressed in rat SCG, they differ considerably from the same subunit combination expressed in oocytes. Larger conductance, SCG‐like channels were detected frequently in L‐α3β4, but were rarely, if ever, seen in oocytes injected with α3 and β4 mRNA. Our results indicate that ion channel properties such as single‐channel conductance can be influenced by the choice of heterologous expression system.

Properties of human glycine receptors containing the hyperekplexia mutation α1(K276E), expressed in Xenopus oocytes

1 Inherited defects in human glycine receptors give rise to hyperekplexia (startle disease). We expressed human glycine receptors in Xenopus oocytes, in order to examine the pharmacological and single‐channel properties of receptors that contain a mutation, α1(K276E), associated with an atypical form of hyperekplexia. 2 Equilibrium concentration‐response curves showed that recombinant human α1(K276E)β receptors had a 29‐fold lower glycine sensitivity than wild‐type α1β receptors, and a greatly reduced Hill coefficient. The maximum response to glycine also appeared much reduced, whereas the equilibrium constant for the glycine receptor antagonist strychnine was unchanged. 3 Both wild‐type and mutant channels opened to multiple conductance levels with similar main conductance levels (33 pS) and weighted mean conductances (41.5 versus 49.8 pS, respectively). 4 Channel openings were shorter for the α1(K276E)β mutant than for the wild‐type α1β, with mean overall apparent open times of 0.82 and 6.85 ms, respectively. 5 The main effect of the α1(K276E) mutation is to impair the opening of the channel rather than the binding of glycine. This is shown by the results of fitting glycine dose‐response curves with particular postulated mechanisms, the shorter open times of mutant channels, the properties of single‐channel bursts, and the lack of an effect of the mutation on the strychnine‐binding site.

Compound heterozygosity and nonsense mutations in the alpha(1)-subunit of the inhibitory glycine receptor in hyperekplexia

The α1-inhibitory glycine receptor is a ligand-gated chloride channel composed of three ligand-binding α1-subunits and two structural β-subunits that are clustered on the postsynaptic membrane of inhibitory glycinergic neurons. Dominant and recessive mutations in GLRA1 subunits have been associated with a proportion of individuals and families with startle disease or hyperekplexia (MIM: 149400). Following SSCP and bi-directional di-deoxy fingerprinting mutational analysis of 22 unrelated individuals with hyperekplexia and hyperekplexia-related conditions, we report further novel missense mutations and the first nonsense point mutations in GLRA1, the majority of which localise outside the regions previously associated with dominant, disease-segregating mutations. Population studies reveal the unique association of each mutation with disease, and reveals that a proportion of sporadic hyperekplexia is accounted for by the homozygous inheritance of recessive GLRA1 mutations or as part of a compound heterozygote.

Role of Charged Residues in Coupling Ligand Binding and Channel Activation in the Extracellular Domain of the Glycine Receptor

The glycine receptor is a member of the ligand-gated ion channel receptor superfamily that mediates fast synaptic transmission in the brainstem and spinal cord. Following ligand binding, the receptor undergoes a conformational change that is conveyed to the transmembrane regions of the receptor resulting in the opening of the channel pore. Using the acetylcholine-binding protein structure as a template, we modeled the extracellular domain of the glycine receptor α1-subunit and identified the location of charged residues within loops 2 and 7 (the conserved Cys-loop). These loops have been postulated to interact with the M2-M3 linker region between the transmembrane domains 2 and 3 as part of the receptor activation mechanism. Charged residues were substituted with cysteine, resulting in a shift in the concentration-response curves to the right in each case. Covalent modification with 2-(trimethylammonium) ethyl methanethiosulfonate was demonstrated only for K143C, which was more accessible in the open state than the closed state, and resulted in a shift in the EC50 toward wild-type values. Charge reversal mutations (E53K, D57K, and D148K) also impaired channel activation, as inferred from increases in EC50 values and the conversion of taurine from an agonist to an antagonist in E53K and D57K. Thus, each of the residues Glu-53, Asp-57, Lys-143, and Asp-148 are implicated in channel gating. However, the double reverse charge mutations E53K:K276E, D57K:K276E, and D148K:K276E did not restore glycine receptor function. These results indicate that loops 2 and 7 in the extracellular domain play an important role in the mechanism of activation of the glycine receptor although not by a direct electrostatic mechanism.

Characterization of the Effects of Charged Residues in the Intracellular Loop on Ion Permeation in α1 Glycine Receptor Channels

The Cys loop receptor channels mediate fast synaptic transmission in the nervous system. The M2-demarcated transmembrane pore is an important determinant of their ion permeation properties. Portals within the intracellular domain are also part of the permeation pathway in cationic Cys loop receptors, with charged residues in a helical MA stretch partially lining these openings profoundly affecting channel conductance. It is unknown whether analogous portals contribute to the permeation pathway in anionic Cys loop receptors. We therefore investigated the influence of charged residues within the proposed MA stretch on functional properties of the homomeric glycine α1 receptor. Up to eight basic residues in the MA stretch were concurrently mutated to a negatively charged glutamate, and wild-type and mutant subunits were expressed in HEK-293 cells. Mutation of all eight residues produced a non-functional receptor. The greatest reduction in conductance at negative membrane potentials (from 92.2 ± 2.8 to 60.0 ± 2.2 picosiemens) was observed with glutamate present at the 377, 378, 385, and 386 positions (the 4E subunit). Inclusion of additional glutamate residues within this subunit did not decrease conductance further. Neutralizing these residues (the 4A subunit) caused a modest decrease in conductance (80.5 ± 2.3 picosiemens). Outward conductance at positive potentials was not markedly affected. Anion to cation selectivity and concentration-response relationships were unaffected by the 4A or 4E mutations. Our results identify basic residues affecting conductance in the glycine receptor, suggesting that portals are part of the extended permeation pathway but that the M2-demarcated channel pore is the dominant determinant of permeation properties in glycine receptors.

Kinetic determinants of agonist action at the recombinant human glycine receptor.

The amino acids glycine, β‐alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady‐state single‐channel recordings was used to compare the actions of glycine, β‐alanine and taurine upon homomeric α1 human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10–90 % rise times determined from rapid application of 100 μm of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 mm) the rise time for glycine (0.26 ms) was 1.8‐fold faster than that for β‐alanine (0.47 ms) and 3.9‐fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for β‐alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > β‐alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single‐opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor.

Mechanisms of channel gating of the ligand‐gated ion channel superfamily inferred from protein structure

The nicotinic‐like ligand‐gated ion channel superfamily consists of a group of structurally related receptors that activate an ion channel after the binding of extracellular ligand. The recent publications of the crystal structure of an acetylcholine binding protein and a refined electron micrograph structure of the membrane‐bound segment of an acetylcholine receptor have led to insights into the molecular determinants of receptor function. Although the structures confirmed much biochemical and electrophysiological data obtained about the receptors, they also provide opportunities to study further the mechanisms that allow channel activation stimulated by ligand‐binding. Here we review the mechanisms of channel gating that have been elucidated by information gained from the structures of the acetylcholine binding protein and membrane‐bound segment of the acetylcholine receptor.

Gating mechanisms in Cys-loop receptors

The Cys-loop receptor superfamily of ligand-gated ion channels has a prominent role in neuronal signalling. These receptors are pentamers, each subunit containing ten β-strands in the extracellular domain and four α-helical transmembrane domains (M1–M4). The M2 domain of each subunit lines the intrinsic ion channel pore and residues within the extracellular domain form ligand binding sites. Ligand binding initiates a conformational change that opens the ion-selective pore. The coupling between ligand binding in the extracellular domain and opening of the intrinsic ion channel pore located in the membrane is not fully understood. Several loop structures, such as loop 2, the Cys-loop, the pre-M1 region and the M2–M3 loop have been implicated in receptor activation. The current “conformational change wave” hypothesis suggests that binding of a ligand initiates a rotation of the β-sheets around an axis that passes through the Cys-loop. Due to this rotation, the Cys-loop and loop 2 are displaced. Movement of the M2–M3 loop then twists the M2 domain leading to a separation of the helices and opening of the pore. The publication of a crystal structure of an acetylcholine binding protein and the refined structure of the Torpedo marmorata acetylcholine receptor have improved the understanding of the mechanisms and structures involved in coupling ligand binding to channel gating. In this review, the most recent findings on some of these loop structures will be reported and discussed in view of their role in the gating mechanism.

Immunolabelling for VDAC, the mitochondrial voltage-dependent anion channel, on sarcoplasmic reticulum from amphibian skeletal muscle

Patch-clamp studies of ion channels in the sarcoball membrane, a relatively pure preparation of sarcoplasmic reticulum, had earlier revealed a high-conductance anion channel with some properties similar to the mitochondrial voltage-dependent anion channel (VDAC). Using post-embedding immunolabelling, the presence of VDAC was investigated in sarcoball preparations from the semitendinosus muscle of the cane toad Bufo marinus. As expected, the outer membrane of mitochondria found within the interior of skinned fibres was decorated with gold label. Surprisingly, sarcoplasmic reticulum membrane was also labelled. The sarcoball membranes, which could arise from either the sarcoplasmic reticulum or from mitochondria, were also labelled. These results indicate the presence of a VDAC-like protein in the sarcoplasmic reticulum.