title: A molecular link between inward rectification and calcium permeability of neuronal nicotinic acetylcholine alpha3beta4 and alpha4beta2 receptors.
abstract: Many nicotinic acetylcholine receptors (nAChRs) expressed by central neurons are located at presynaptic nerve terminals. These receptors have high calcium permeability and exhibit strong inward rectification, two important physiological features that enable them to facilitate transmitter release. Previously, we showed that intracellular polyamines act as gating molecules to block neuronal nAChRs in a voltage-dependent manner, leading to inward rectification. Our goal is to identify the structural determinants that underlie the block by intracellular polyamines and govern calcium permeability of neuronal nAChRs. We hypothesize that two ring-like collections of negatively charged amino acids (cytoplasmic and intermediate rings) near the intracellular mouth of the pore mediate the interaction with intracellular polyamines and also influence calcium permeability. Using site-directed mutagenesis and electrophysiology on alpha(4)beta(2) and alpha(3)beta(4) receptors expressed in Xenopus oocytes, we observed that removing the five negative charges of the cytoplasmic ring had little effect on either inward rectification or calcium permeability. However, partial removal of negative charges of the intermediate ring diminished the high-affinity, voltage-dependent interaction between intracellular polyamines and the receptor, abolishing inward rectification. In addition, these nonrectifying mutant receptors showed a drastic reduction in calcium permeability. Our results indicate that the negatively charged glutamic acid residues at the intermediate ring form both a high-affinity binding site for intracellular polyamines and a selectivity filter for inflowing calcium ions; that is, a common site links inward rectification and calcium permeability of neuronal nAChRs. Physiologically, this molecular mechanism provides insight into how presynaptic nAChRs act to influence transmitter release.
authors.affiliation: Department of Physiology, McGill University, Montréal, Québec, Canada H3G 1Y6.
authors.forename: A P
authors.initials: AP
authors.lastname: Haghighi
authors.forename: E
authors.initials: E
authors.lastname: Cooper
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002463:Cell Membrane Permeability; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009474:Neurons / Q000502:physiology*; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D051381:Rats; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013096:Spermine / Q000494:pharmacology; D014982:Xenopus laevis
chemical_list: D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; C403530:nicotinic receptor alpha3beta4; C403531:nicotinic receptor alpha4beta2; D013096:Spermine; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium