S.D. Jane

Membrane Clustering and Bungarotoxin Binding by the Nicotinic Acetylcholine Receptor: Role of the β Subunit

Abstract: Nicotinic acetylcholine receptors (nAChRs) are localised at morphologically distinct regions of the postsynaptic membrane by interactions between the receptor subunits and cytoskeletal proteins, such as the 43‐kDa protein. We have used Xenopus oocytes to examine the localisation and pharmacological properties of muscle nAChRs associated with 43‐kDa protein and to compare them with hybrid muscle nAChRs containing a β subunit derived from a neuronal source. Receptors expressed on the oocyte outer membrane were visualised using confocal scanning laser microscopy. Coexpression of mouse muscle subunit α1β1γδ and 43‐kDa protein transcripts produced discrete receptor aggregates with a diameter of 1–5 µm whose function was partially blocked by application of neuronal bungarotoxin (NBT) at 100 nM. Substitution of the β1 subunit by the neuronal β2 protein produced a functioning receptor that did not aggregate in the presence of 43‐kDa protein and was substantially blocked by the same concentration of NBT. Hybrid α1β4γδ receptors exhibited a combination of characteristics in that they clustered like normal muscle subunits in the presence of 43‐kDa protein, but showed a sensitivity to NBT intermediate between that of muscle receptors and that of hybrids containing β2. These results suggest that the β subunit is an important determinant in receptor localisation and sensitivity to NBT.

Effects of Mg2+ and ATP on the phosphate transporter of sarcoplasmic reticulum.

The extra uptake of Ca2+ by vesicles of sarcoplasmic reticulum (SR) observed in the presence of Pi, attributable to transport of Pi by the Pi-transporter, has been studied. It has been shown that the Pi transporter is stimulated by ATP. Single channel conductance measurements have shown that the Cl- channel in the SR membrane is impermeable to Pi. It is suggested that the transporter could be an ion antiporter system. Studies of uptake as a function of pH and Mg2+ concentration suggest that transport of MgHPO4 and H2PO-4 are faster than transport of HPO2-4. For oxalate and pyrophosphate, Mg2+ binding inhibits transport. It is suggested that protonation of lysine residue(s) at the anion binding site increase the rate of transport.

An electrophysiological and spectroscopic study of the properties and structure of biological calcium channels. Investigations of a model ion channel

The N- and C-terminally protected peptide N-acetyl-Asp-Phe-Ala-Asn-Arg-Val-Leu-Leu-Ser-Leu-Phe-Thr-Ile-Glu-Met-Leu -Leu-Lys-Met-Leu-NH2, closely based on the sequence of the putative S2 membrane spanning helix of domain II of the dihydropyridine receptor calcium channel of the T-system of skeletal muscle, residues 465-486 (Tanabe et al. (1987) Nature 328, 313-318) has been synthesised. Conductance measurements in planar lipid bilayers show that the peptide is capable of inducing the transmembrane passage of calcium and barium ions, in preference to monovalent cations. No anion conductance is observed. 1H-NMR spectroscopy demonstrates that in an amphilic solvent, methanol, the peptide forms highly stable structures characterised by very slow exchange with solvent of peptide N-H protons. Double-quantum filtered phase-sensitive COSY shows that, on the basis of NH-CH alpha scalar coupling constants, most peptide torsion angles are appropriate to an overall alpha-helical conformation; the presence of some alpha-helix is also supported by CD measurements. Most side-chain connectivities have been identified in a DIPSI-TOCSY experiment. This evidence has been used to construct a low-resolution model of the ion-conducting channel of the muscle T-system dihydropyridine receptor from the sequences of the four homologous putative channel-lining stretches. It is characterised by an association of acidic residues at the putative extra-membranous face of the channel, followed by a predominantly hydrophobic band. The next prominent feature of the model is an ordered array of four acidic residues (glutamates 100, 478, 846 and 1164), followed by four lysines (104, 482, 850 and 1168) which may play a gating role.

Activation of nicotinic acetylcholine receptors expressed in quail fibroblasts: effects on intracellular calcium.

1 The aim of these experiments was to determine the ability of the muscle‐type nicotinic acetylcholine receptor (AChR) stably expressed in quail fibroblasts (QF18 cells) to elevate intracellular calcium ([Ca2+]i) upon activation. Ratiometric confocal microscopy, with the calcium‐sensitive fluorescent dye Indo‐1 was used. 2 Application of the nicotinic agonist, suberyldicholine (SDC), to the transfected QF18 cells caused an increase in [Ca2+]i. Control [Ca2+]i levels in QF18 cells were found to be 164 ± 22 nM (mean ± s.e.mean; n = 40 cells) rising to 600 ± 81 nM on addition of SDC (10 μm; n=15 cells), whereas no increase in [Ca2+]i was seen in non‐transfected control QT6 fibroblasts (before: 128 ± 9 nM, n = 40; after: 113±13 nM, n= 15). 3 The increase in [Ca2+]i caused by application of SDC was dose‐dependent, with an EC50 value of 12.7 ± 5.9 μm (n=14). 4 The responses to SDC in QF18 cells were blocked by prior application of α‐bungarotoxin (200 nM), by the addition of Ca2+ (100 μm), by removal of Na+ ions from the extracellular solution, or by the voltage‐sensitive calcium channel blockers nifedipine and ω‐conotoxin, which act with IC50 values of 100 nM and 100 pM respectively. 5 We conclude that activation of the nicotinic AChRs leads to a Na+‐dependent depolarization and hence activation of endogenous voltage‐sensitive Ca2+ channels in the plasma membrane and an increase in [Ca2+]i. There is no significant entry of Ca2+ through the nicotinic receptor itself.