Alfredo Villarroel

Dimensions and ion selectivity of recombinant AMPA and kainate receptor channels and their dependence on Q/R site residues

1. Recombinant alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate receptor (AMPAR) subunits (GluR‐A or GluR‐B) and kainate receptor (KAR) subunit (GluR‐6) in their unedited (Q)‐ and edited (R)‐forms were expressed in HEK 293 cells. To estimate the dimensions of the narrow portion of these channels, biionic reversal potentials for organic cations of different mean diameters were determined with Cs+ as the internal reference ion. 2. Homomeric channels assembled from Q‐form subunits were cation selective. The relation between the relative permeability and the mean size of different organic cations suggests that the diameter of the narrow portion of Q‐form channels is approximately 0.78 nm for AMPAR and 0.75 nm for KAR channels. 3. Homomeric channels assembled from R‐form subunits were permeant for anions and cations. When probed with CsC1 gradients the relative chloride permeability (PC1/PCs) was estimated as 0.14 for GluR‐B(R) and 0.74 for GluR‐6(R)‐subunit channels. The permeability versus mean size relation for large cations measured with the weakly permeant F‐ as anion, indicates that for the R‐form KAR channels the apparent pore diameter is close to 0.76 nm. 4. Heteromeric AMPAR and KAR channels co‐assembled from Q‐ and R‐form subunits were cation selective. The diameter of the narrow portion of these channels is estimated to be in the range between 0.70 and 0.74 nm. 5. The results indicated that the diameters of the narrow portion of AMPAR and KAR channels of different subunit composition and of widely different ion selectivity are comparable. Therefore, the differences in the anion versus cation selectivity, in Ca2+ permeability and in channel conductance are likely to be determined by the difference in charge density of the channel.

An amino acid exchange in the second transmembrane segment of a neuronal nicotinic receptor causes partial epilepsy by altering its desensitization kinetics

The α4 subunit of the neuronal nicotinic acetylcholine receptor is the first gene shown to be involved in a human idiopathic epileptic disease. A missense mutation, leading to the replacement of serine 248 by phenylalanine in the second transmembrane segment, had been detected in patients with autosomal dominant nocturnal frontal lobe epilepsy. The properties of the wild type receptor composed of α4 and β2 subunits and the mutant receptor where α4 subunits carried the mutation at serine 248 were compared by means of cDNA manipulation and expression in Xenopus oocytes. The mutant receptor exhibited faster desensitization upon activation by acetylcholine and recovery from the desensitized state was much slower than in the wild type receptor. We conclude that the reported mutation causes seizures via a diminution of the activity of the α4β2 neuronal nicotinic acetylcholine receptor.

Location of a threonine residue in the α−subunit M2 transmembrane segment that determines the ion flow through the acetylcholine receptor channel

By the combination of cDNA manipulation and functional analysis of normal and mutant acetylcholine receptor (AChR) channels of Torpedo expressed in Xenopus laevis oocytes determinants of ion flow were localized in the bends bordering the putative M2 transmembrane segment (Imoto et al. 1988). We now report that in the rat muscle AChR, substitution of a threonine residue in the α-subunit localized in the M2 transmembrane segment increases or decreases the channel conductance, depending on the size of the amino acid side chain located at this position. This threonine residue (αT264) is located adjacent to the cluster of charged amino acids that form the intermediate anionic ring (Imoto et al. 1988). This effect is pronounced for the large alkali cations Cs+, Rb+, K+ whereas for Na+ the effect is much smaller. Taken together the results suggest that the threonine residues at position 264 in the two α-subunits together with the amino acids of the intermediate anionic ring form part of a narrow region close to the cytoplasmic mouth of the AChR channel.

Dimensions of the narrow portion of a recombinant NMDA receptor channel

Glutamate-activated single-channel and ensemble currents were recorded from Xenopus laevis oocytes and HEK 293 cells expressing a recombinant NMDA receptor, assembled from NR1 and NR2A subunits. Cesium was the main charge carrier, and organic cations were used to determine the presence of vestibules of this channel and to estimate its pore diameter. The large organic cations tris-(hydroxymethyl)-aminomethane (Tris), N-methyl-glucamine (NMG), arginine (NMG), arginine (Arg), choline, and tetramethylammonium (TMA), when added in millimolar concentrations to the extracellular or cytoplasmic side, produced a voltage-dependent blockade of single-channel Cs+ currents. These molecules behaved as impermeant ions that only partially traverse the channel from either side. The smaller cations trimethylammonium (TriMA) and dimethylammonium (DMA) produced a small and nearly voltage-independent reduction in current amplitude, suggesting that they are permeant. In biionic experiments with Cs+ as the reference ion, the large blocking cations NMG, Arg, Tris, TMA, choline, hexamethonium (Hme), triethylammonium (TriEA), and tetraethylammonium (TEA) showed no measurable permeability. TriMA and smaller ammonium derivatives were permeant. Both the permeability and single-channel conductance of organic cations, relative to Cs+, decreased as the ion size increased. The results suggest that the NMDA receptor has extracellular and cytoplasmic mouths that can accommodate large cations up to 7.3 A in mean diameter. The narrow portion of the pore is estimated to have a mean diameter of 5.5 A.

Threonine in the selectivity filter of the acetylcholine receptor channel

The acetylcholine receptor (AChR) is a cation selective channel whose biophysical properties as well as its molecular composition are fairly well characterized. Previous studies on the rat muscle alpha-subunit indicate that a threonine residue located near the cytoplasmic side of the M2 segment is a determinant of ion flow. We have studied the role of this threonine in ionic selectivity by measuring conductance sequences for monovalent alkali cations and bionic reversal potentials of the wild type (alpha beta gamma delta channel) and two mutant channels in which this threonine was replaced by either valine (alpha T264V) or glycine (alpha T264G). For the wild type channel we found the selectivity sequence Rb greater than Cs greater than K greater than Na. The alpha T264V mutant channel had the sequence Rb greater than K greater than Cs greater than Na. The alpha T264G mutant channel on the other hand had the same selectivity sequence as the wild type, but larger permeability ratios Px/PNa for the larger cations. Conductance concentration curves indicate that the effect of both mutations is to change both the maximum conductance as well as the apparent binding constant of the ions to the channel. A difference in Mg2+ sensitivity between wild-type and mutant channels, which is a consequence of the differences in ion binding, was also found. The present results suggest that alpha T264 form part of the selectivity filter of the AChR channel were large ions are selected according to their dehydrated size.

Calcium permeability increase of endplate channels in rat muscle during postnatal development

1. Patches of endplate membrane were isolated from rat flexor digitorum brevis muscle at different postnatal stages to measure the time course of development changes in conductance, deactivation time constant and relative Ca2+ permeability of endplate channels. 2. The predominant channel conductance was 40 +/‐ 1 pS (n = 9) at postnatal day 9 (P9) or younger whereas it was 59 +/‐ 3 pS (n = 5) at P21 or in older muscle. The deactivation time constant of ensemble patch currents evoked by brief ACh application, decreased from 8 +/‐ 3 ms (n = 45) at P5‐9 to 2.3 +/‐ 0.3 ms (n = 5) in P21‐28 muscle. 3. The relative Ca2+ permeability, measured by the shift of biionic (Ca2+/Cs+) reversal potential of ensemble patch currents upon the replacement of high [Cs+] by high [Ca2+] extracellular solution and with Cs+ as internal reference ion, increased during postnatal development. THe biionic reversal potential shift changed from ‐21 +/‐ 1 mV (n = 8) at P5 to ‐8 +/‐ 1 mV (n = 10) in P15 or older muscle. 4. Recombinant gamma‐AChR channels expressed in Xenopus laevis oocytes had a biionic (Ca2+/Cs+) reversal potential shift of ‐24.9 +/‐ 2 mV (n = 14) comparable to that of neonatal endplate channels whereas the reversal potential shift for recombinant epsilon‐AChR channels was ‐7.6 +/‐ 0.9 mV (n = 13), comparable to that of endplate channels in adult muscle. 5. It is concluded that an approximately 3‐fold increase in Ca2+ current through endplate channels during postnatal development is caused by replacement of the fetal gamma‐subunit by the epsilon‐subunit in juvenile and adult muscle.

Asymmetry of the Rat Acetylcholine Receptor Subunits in the Narrow Region of the Pore

The acetylcholine receptor (AChR) channel is a pentameric protein in which every subunit contributes to the conducting parts of the pore. Recent studies of rat nicotinic AChR channels mutated in the α-subunit revealed that a threonine residue (αT264) in the transmembrane segment M2 forms part of the narrow region of the channel. We have mutated the residues at homologous positions in the β-, γ-, and δ-subunits and measured the resulting change in channel conductance. For all subunits the conductance is inversely related to the volume of the amino acid residue, suggesting that they form part of the channel narrow region. Exchanges of residues between subunits do not alter the conductance, suggesting a ring-like structure formed by homologous amino acids. To investigate the relative contribution of amino acid residues at these positions in determining the channel conductance, receptors carrying the same amino acid in each subunit in the narrow region were constructed. They form functional channels in which the conductance is inversely related to the volume of the amino acids in the narrow region. Channels in which the narrow region is formed by four serines and one valine have the same conductance if the valine is located in the α-, β-, or γ-subunits, but it is smaller if the valine is located in the δ-subunit. The results suggest a structural asymmetry of the AChR channel in its narrow region formed by the hydroxylated amino acids of α-, γ- and δ-subunits, where the δ-subunit serine is a main determinant of the channel conductance.

Acetylcholine receptor channel subtype directs the innervation pattern of skeletal muscle

Acetylcholine receptors (AChRs) mediate synaptic transmission at the neuromuscular junction, and structural and functional analysis has assigned distinct functions to the fetal (α2βγδ) and adult types of AChR (α2βεδ). Mice lacking the ε‐subunit gene die prematurely, showing that the adult type is essential for maintenance of neuromuscular synapses in adult muscle. It has been suggested that the fetally and neonatally expressed AChRs are crucial for muscle differentiation and for the formation of the neuromuscular synapses. Here, we show that substitution of the fetal‐type AChR with an adult‐type AChR preserves myoblast fusion, muscle and end‐plate differentiation, whereas it substantially alters the innervation pattern of muscle by the motor nerve. Mutant mice form functional neuromuscular synapses outside the central, narrow end‐plate band region in the diaphragm, with synapses scattered over a wider muscle territory. We suggest that one function of the fetal type of AChR is to ensure an orderly innervation pattern of skeletal muscle.

Structural determinants of channel conductance in fetal and adult rat muscle acetylcholine receptors.

1. The structural basis of the developmentally regulated increase in endplate channel conductance in rat, where the gamma‐subunit of the fetal muscle acetylcholine receptor (gamma‐AChR) is replaced by the epsilon‐subunit in the adult muscle receptor (epsilon‐AChR), was investigated by analysing the structure of gamma‐ and epsilon‐subunit genes and by expressing recombinant AChR channels of different molecular composition in Xenopus oocytes and measuring their single‐channel conductance. 2. The gamma‐ and epsilon‐subunit genes each have twelve exons. In both subunits, the four homologous segments, designated M1, M2, M3 and M4, which are thought to contribute to the formation of the pore, are encoded by four separate exons, E7, E8, E9 and E12. 3. Chimaeric epsilon(gamma)‐ or gamma(epsilon)‐subunits were constructed from the parental epsilon‐ and gamma‐subunits, respectively. Exchanging the four hydrophobic segments (M1‐M4) of the gamma‐subunit for those of the epsilon‐subunit and vice versa completely reversed the difference in conductance between gamma‐AChR and epsilon‐AChR channels. 4. Effects of single‐ and multiple‐point mutations in M1‐M4 segments of gamma‐ and epsilon‐subunits indicate that the major determinants of the difference in conductance between fetal and adult endplate channels are located in the M2 segment. The key differences are the exchange of alanine/threonine (gamma‐subunit) for serine/isoleucine (epsilon‐subunit) in M2, and the lysine (gamma‐subunit) for glutamine (epsilon‐subunit) exchanges in the regions flanking the M2 segment.

Organisation of the murine 5‐HT3 receptor gene and assignment tohuman chromosome 11

We have isolated the murine gene encoding the 5‐HT3 receptor (5‐HT3R), a member of the ligand‐gated ion channels, that mediates a variety of physiological effects in central and peripheral neurons. DNA sequence analysis of the 5‐HT3R gene revealed its organisation in 9 exons distributed over approximately 12 kbp of DNA. Alternative use ofexon 9 splice acceptor sites generated two 5‐HT3R variants. The 5‐HT3R gene, whose structure is closely related to neuronal and muscle AChRα genes, as demonstrated by four common splice junctions, was localised on human chromosome 11.