Structure-Function Analysis of the β Subunit of Neuronal Nicotinic Acetylcholine Receptors

Nicotinic receptors belong to the superfamily of ligand-gated ion channels. Since evidence was rapidly accumulating implicating the non-α subunits in ligand-binding events, we decided to investigate eventual contributions of the neuronal β subunit to these events by performing a series of increasingly detailed experiments on a series of chimeric β subunits. In the first set of experiments, we constructed a variety of chimeric β subunits consisting of NH2-terminal neuronal β4 sequences and COOH-terminal β2 sequences and expressed them with the α3 subunit in Xenopus oocytes. The results showed that (a) two residues in the extracellular domain of chimeric β4•β2 subunits (108β2Phe↔β4Val, 110β2Ser↔β4Thr) account for much of the relative cytisine sensitivity; and (b) four extracellular residues of chimeric β4•β2 subunits (112β2Ala↔β4Val, 113β2Val↔β4Ile and 115β2Ser↔β4Arg, 116β2Tyr↔β4Ser) account for most of the relative tetramethylammonium sensitivity. Encouraged by the above results, we continued our experiments with additional chimeras of the β2 and β4 neuronal nicotinic subunits to locate regions that contribute to differences between the acetylcholine dose-response relationships of α3β2 and α3β4 receptors. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other, suggesting that amino acids within the first 120 residues of β2 and the corresponding region of β4 contribute to an agonist binding site that bridges the α and β subunits in neuronal nicotinic receptors. Since the EC50 phenotypes caused by the β2 and β4 subunits could be due to a difference in gating or binding properties, we attempted to unravel this question by performing voltage-jump relaxations for the series of neuronal nicotinic acetylcholine receptors we constructed previously. The chimeric β4/β2 subunits showed a transition in the concentration dependence of the relaxation rate constants in the region between residues 94 and 109, analogous to our previous observation with steady-state dose-response relationships. The data reinforce previous conclusions that the region between residues 94 and 109 on the β subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.</p

Regions of β4·β2 subunit chimeras that contribute to the agonist selectivity of neuronal nicotinic receptors

AbstractFifteen chimeric nicotinic receptors β subunits were constructed consisting of N-terminal neuronal β4 sequences and C-terminal β2 sequences. Responses to cytisine, nicotine, or tetramethylammonium were compared to acetylcholine responses for these subunits expressed in Xenopus oocytes with α3 subunits. The results show that (i) two residues in the extracellular domain of chimeric β4·β2 subunits (108β2F/β4V, 110β2S/β4T) account for much of the relative cytisine sensitivity; and (ii) four extracellular residues of chimeric β4·β2 subunits (112β2A/β4V, 113β2V/β4I and 115β2S/β4R, 116β2Y/β4S) account for most of the relative tetramethylammonium sensitivity. The data did not permit localization of nicotine sensitivity to any particular region

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Extracellular Ca²⁺ robustly potentiates the acetylcholine response of α4β2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)—α4(S252F), α4(S256L), α4(+L264), β2(V262L), and β2(V262M)—reduced 2 mM Ca²⁺ potentiation of the α4β2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca²⁺ activation or enhanced Ca²⁺ block caused this reduction, we coexpressed the rat ADNFLE mutations with an α4 N-terminal mutation, α4(E180Q), that abolished α4β2 allosteric Ca²⁺ activation. In each case, Ca²⁺ inhibition of the double mutants was less than that expected from a Ca²⁺ blocking mechanism. In fact, the effects of Ca²⁺ on the ADNFLE mutations near the intracellular end of the M2 region—α4(S252F) and α4(S256L)—were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca²⁺ on the ADNFLE mutations near the extracellular end of the M2 region—α4(+L264)β2, β2(V262L), and β2(V262M)—were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca²⁺ on the α4β2, α4(+L264)β2, and α4β2(V262L) single-channel conductances, the effects of membrane potential on the β2(V262L)-mediated reduction in Ca²⁺ potentiation, and the effects of eliminating the negative charges in the extracellular ring on this reduction failed to provide any direct evidence of mutant-enhanced Ca²⁺ block. Moreover, analyses of the α4β2, α4(S256L), and α4(+L264) Ca²⁺ concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca²⁺ potentiation of the α4β2 acetylcholine response by altering allosteric activation rather than by enhancing block

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

The β subunit dominates the relaxation kinetics of heteromeric neuronal nicotinic receptors

The ACh-induced voltage-jump relaxation currents of the nicotinic receptors formed by pair-wise expression of the rat α2, α3, or α4 subunits with the β2 or β4 subunit in Xenopus oocytes were fitted best by the sum of two exponentials and a constant between -60 and -150 mV.As the ACh concentration approached zero, the relaxation time constants approached limiting values that should equal the single-channel burst duration at low ACh concentrations and the synaptic current decay time constants. β4 co-expression prolonged the zero ACh concentration limits for the relaxation time constants. The fast β4 zero ACh concentration limits ranged from 40 to 121 ms between -60 and -150 mV, and the slow β4 zero ACh concentration limits ranged from 274 to 1039 ms. In contrast, the fast β2 limits were 4–6 ms over the same voltage range and the slow β2 limits were 30–53 ms.Expression with the β4 subunit increased the voltage sensitivity of the α2, α3 and slow α4 relaxation time constants but not that of the fast α4 relaxation time constant.Reducing the temperature from 22°C to 8–9°C increased the α4β2 and α3β4 relaxation time constants 2.3- to 6.6-fold and reduced the fractional amplitude of the fast relaxation component. It also increased the voltage dependence of the fast α3β4 relaxation time constant and decreased that of the slow time constant. The Q10 for α4β2 and α3β4 relaxation time constants ranged from 1.9 to 3.9 between 10 and 20°C.The β subunit appears to have a dominant influence on the voltage-jump relaxation kinetics of heteromeric neuronal nicotinic receptors

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