Inward rectification of neuronal nicotinic acetylcholine receptors investigated by using the homomeric alpha 7 receptor.
The strong inward rectification observed in neuronal nicotinic ACh receptors was examined by using alpha-bungarotoxin sensitive, homomeric alpha 7 neuronal nicotinic ACh receptors derived from chick brain. Receptors were expressed in Xenopus laevis oocytes and functionally assessed by the two electrode voltage clamp technique. Site directed mutagenesis of residues thought to line the putative ion pore revealed that negatively charged glutamate residues located at the inner mouth of the channel are essential for rectification. This finding was confirmed both for the active open state and for receptors mutated to conduct in one of the desensitized states. No outward tail relaxations were observed with voltage jumps to depolarizing potentials, suggesting that rectification was not due to intrinsic gating. For the wild type receptor, intracellular injection of CDTA, a chelating agent having a high affinity for Mg2+, reduced rectification in a dose dependent manner, suggesting that rectification originates, in part, from open channel block by internal free Mg2+. These findings support the hypothesis that charged residues at the inner mouth of the pore influence the Mg2+ affinity of the blocking site.
Forster
I
I
Physiologisches Institut, Universität Zürich, Switzerland.
Bertrand
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D004594:Electrophysiology; D018698:Glutamic Acid / Q000378:metabolism; D017136:Ion Transport; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000502:physiology*; D014982:Xenopus laevis
D011978:Receptors, Nicotinic; D018698:Glutamic Acid; D008274:Magnesium