Andrew Palma

Effects of diverse ω-conopeptides on the in vivo release of glutamic and γ-aminobutyric acids

ω-Conopeptides are antagonists of subtypes of neuronal calcium channels. Two ω-conopeptides, SVIB and MVIIC, have recently been identified which have a novel specificity for these ionophores. We have tested the actions these peptides, as well as the more selective MVIIA, on the release of glutamic acid and γ-aminobutyric acid (GABA) in the hippocampus in vivo. For the assay of peptide effects on release, we used microdialysis to deliver multiple pulses of elevated potassium to the brain extracellular fluid. Peptide effects were quantitated from the decrement of the release with peptide perfused through the probes, in comparison to that in control experiments. Synthetic MVIIC caused a 40–50% decrement in the release of both glutamate and GABA at a probe concentration of about 200 nM. Synthetic SVI-B caused a 50% block at about 20–40 μM, while about 200 μM of MVIIA was required for 50% block. Chromatographic experiments showed that differences in potency between MVIIC and MVIIA were not explained by differential degradation. Blockade of release was also observed in the thalamus. MVIIC provides a tool for investigating the role of calcium mediated release of glutamate and GABA in physiological and pathological processes in the mammalian brain in vivo.

Site-specific mutations of nicotinic acetylcholine receptor at the lipid-protein interface dramatically alter ion channel gating

McNamee, 1991). cDNA cloning reveals that each subunit has four homologous hydrophobic segments (Ml, M2, M3, and M4) that are believed to span the lipid bilayer. There is strong evidence that M2 is the ion channel-lining domain, but the specific functions of the other hydrophobic segments are not well characterized. In this report, the role ofthe M4 segment in ion channel function was investigatedby usingsite-directedmutagenesis of a specific cysteine residue (aCys418) in the M4 regionofTorpedo californica a subunit. The M4 region is believed to be located at the lipid-protein interface based primarily on chemical labeling studies (Giraudat et aI., 1985), and we have examined both the labeling and mutation of cysteine residues in these domains (Pradier et aI., 1989; Li et aI., 1990). We show here that the mutation on the a subunit alters ion channel gating in a dramatically different way than previous mutations.

Regulation of nicotinic acetylcholine receptor function by adenine nucleotides

Summary1.Nicotinic acetylcholine receptors (nAChR) from BC3H1 cells (which express a skeletal muscle-type receptor) and fromTorpedo californica electric organ were expressed inXenopus laevis oocytes and studied with a voltage-clamp technique.2.We found that bath application of ATP in the micromolar to millimolar range increased the ACh-elicited current in both muscle and electrocyte receptors. The effect of ATP increased with successive applications. This “use-dependent” increase in potentiation was Ca2+ dependent, while the potentiation itself was not.3.Four other nucleotides were tested on muscle nAChR: ADP, AMP, adenosine, and GTP. Of these, only ADP was a potentiator, but its effect was not use dependent. Neither ATP nor ADP affected the resting potential of the oocyte membrane.4.ADP potentiated the response to suberyldicholine and nicotine, as well as ACh.5.Finally, ADP reversed the phencyclidine-induced block of ACh currents in oocytes expressing muscle nAChR.

Effects of pH on acetylcholine receptor function

SummaryWe have examined the effects of changing extracellular pH on the function of nicotinic acetylcholine receptors fromTorpedo californica using ion flux and electrophysiological methods. Agonist-induced cation efflux from vesicles containing purified, reconstituted receptors showed a monotonic dependence on external hydrogen ion concentration with maximal fluxes at alkaline pH and no agonist-induced efflux at pHs less than ∼5. A similar pH dependence was measured for the peak agonist-activated membrane currents measured in microelectrode voltage-clampedXenopus oocytes induced to expressTorpedo receptor through mRNA injection. Half-maximal inhibition occurred at a similar pH in both systems, in the range of pH 6.5–7.0. Single-channel currents fromTorpedo ACh receptors measured in patch-clamp recordings were also reduced in amplitude at acid pH with an apparent pKa for block of <5. Measurements of channel kinetics had a more complicated dependence on pH. The mean channel open time determined from patch-clamp measurements was maximal at neutral pH and decreased at both acid and alkaline pHs. Thus, both channel permeability properties and channel gating properties are affected by the extracellular pH.