Two Domains of the Beta Subunit of Neuronal Nicotinic Acetylcholine Receptors Contribute to the Affinity of Substance P 1

ABSTRACT Substance P is known to noncompetitively inhibit activation of muscle and neuronal nicotinic acetylcholine receptors. Neuronal nicotinic receptors formed from different combinations of ␣ and ␤ subunits exhibited differential sensitivity to substance P, with those containing ␤-4 subunits having a 25-fold higher affinity than those having ␤-2 subunits. To identify the regions and/or amino acid residues of the ␤ subunit responsible for this difference, chimeric ␤ subunits were coexpressed with ␣-3 in Xenopus oocytes and the IC 50 values for substance P were determined. Amino acid residues between 105 and 109 (␤4 numbering), in the middle of the N-terminal domain, and between 214 and 301, between the extracellular side of M1 and the intracellular side of M3, were identified as major contributors to the apparent affinity of substance P. The affinity of acetylcholine was only affected by residue changes between 105 and 109. Site-directed mutagenesis revealed two amino acids that are important determinants of the affinity of substance P, ␤4(V108)/␤2(F106), which is in the middle of the first extracellular domain, and ␤4(F255)/␤2(V253), which is within the putative channel lining transmembrane domain M2. However, other residues within these domains must be making subtle but significant contributions, since simultaneous mutation of both these amino acids did not cause complete interconversion of the ␤ subunit-dependent differences in the receptor affinity for substance P. The tachykinin SP is a neurotransmitter and neuromodulator in the central and peripheral nervous systems Muscle and neuronal nAChRs are pentameric proteins forming ligand-gated ion channel

Five ADNFLE mutations reduce the Ca2+ dependence of the mammalian α4β2 acetylcholine response

Five nicotinic acetylcholine receptor (nAChR) mutations are currently linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). The similarity of their clinical symptoms suggests that a common functional anomaly of the mutations underlies ADNFLE seizures. To identify this anomaly, we constructed rat orthologues (S252F, +L264, S256L, V262L, V262M) of the human ADNFLE mutations, expressed them in Xenopus oocytes with the appropriate wild-type (WT) subunit (α4 or β2), and studied the Ca2+ dependence of their ACh responses. All the mutations significantly reduced 2 mM Ca2+-induced increases in the 30 μM ACh response (P < 0.05). Consistent with a dominant mode of inheritance, this reduction persisted in oocytes injected with a 1:1 mixture of mutant and WT cRNA. BAPTA injections showed that the reduction was not due to a decrease in the secondary activation of Ca2+-activated Cl− currents. The S256L mutation also abolished 2 mM Ba2+ potentiation of the ACh response. The S256L, V262L and V262M mutations had complex effects on the ACh concentration-response relationship but all three mutations shifted the concentration-response relationship to the left at [ACh]≥ 30 μM. Co-expression of the V262M mutation with a mutation (E180Q) that abolished Ca2+ potentiation resulted in 2 mM Ca2+ block, rather than potentiation, of the 30 μM ACh response, suggesting that the ADNFLE mutations reduce Ca2+ potentiation by enhancing Ca2+ block of the α4β2 nAChR. Ca2+ modulation may prevent presynaptic α4β2 nAChRs from overstimulating glutamate release at central excitatory synapses during bouts of synchronous, repetitive activity. Reducing the Ca2+ dependence of the ACh response could trigger seizures by increasing α4β2-mediated glutamate release during such bouts