Site-specific, photochemical proteolysis applied to ion channels in vivo

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the “signature” Cys128–Cys142 disulfide loop of the nAChR α subunit.

Unmasking the functions of the chromaffin cell α7 nicotinic receptor by using short pulses of acetylcholine and selective blockers

Methyllycaconitine (MLA), α-conotoxin ImI, and α-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 μM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC50s of 100 nM for MLA and 300 nM for α-conotoxin ImI and α-bungarotoxin. MLA (100 nM), α-conotoxin ImI (1 μM), and α-bungarotoxin (1 μM) halved the entry of 45Ca2+ stimulated by 5-s pulses of 300 μM ACh applied to incubated cells. These supramaximal concentrations of α7 nicotinic receptor blockers depressed by 30% (MLA), 25% (α-bungarotoxin), and 50% (α-conotoxin ImI) the inward current generated by 1-s pulses of 100 μM ACh, applied to voltage-clamped chromaffin cells. In Xenopus oocytes expressing rat brain α7 neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, α-conotoxin ImI, and α-bungarotoxin with IC50s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through α3β4 nAChR was unaffected by α-conotoxin ImI and α-bungarotoxin, and weakly blocked by MLA (IC50 = 1 μM). The functions of controlling the electrical activity, the entry of Ca2+, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to α3β4 nAChR; the present results constitute the first evidence to support a prominent role of α7 nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.

α10: A determinant of nicotinic cholinergic receptor function in mammalian vestibular and cochlear mechanosensory hair cells

We report the cloning and characterization of rat α10, a previously unidentified member of the nicotinic acetylcholine receptor (nAChR) subunit gene family. The protein encoded by the α10 nAChR subunit gene is most similar to the rat α9 nAChR, and both α9 and α10 subunit genes are transcribed in adult rat mechanosensory hair cells. Injection of Xenopus laevis oocytes with α10 cRNA alone or in pairwise combinations with either α2-α6 or β2-β4 subunit cRNAs yielded no detectable ACh-gated currents. However, coinjection of α9 and α10 cRNAs resulted in the appearance of an unusual nAChR subtype. Compared with homomeric α9 channels, the α9α10 nAChR subtype displays faster and more extensive agonist-mediated desensitization, a distinct current–voltage relationship, and a biphasic response to changes in extracellular Ca2+ ions. The pharmacological profiles of homomeric α9 and heteromeric α9α10 nAChRs are essentially indistinguishable and closely resemble those reported for endogenous cholinergic eceptors found in vertebrate hair cells. Our data suggest that efferent modulation of hair cell function occurs, at least in part, through heteromeric nAChRs assembled from both α9 and α10 subunits.

An inhalational anesthetic binding domain in the nicotinic acetylcholine receptor.

To determine inhalational anesthetic binding domains on a ligand-gated ion channel, I used halothane direct photoaffinity labeling of the nicotinic acetylcholine receptor (nAChR) in native Torpedo membranes. [14C]Halothane photoaffinity labeling of both the native Torpedo membranes and the isolated nAChR was saturable, with Kd values within the clinically relevant range. All phospholipids were labeled, with greater than 95% of the label in the acyl chain region. Electrophoresis of labeled nAChR demonstrated no significant subunit selectivity for halothane incorporation. Within the alpha-subunit, greater than 90% of label was found in the endoprotease Glu-C digestion fragments which contain the four transmembrane regions, and the pattern was different from that reported for photoactivatable phospholipid binding to the nAChR. Unlabeled halothane reduced labeling more than did isoflurane, suggesting differences in the binding domains for inhalational anesthetics in the nAChR. These data suggest multiple similar binding domains for halothane in the transmembrane region of the nAChR.