Two mutations in the M2 region of the human á4 neuronal nicotinic subunit -á4(S248F) and á4(776ins3) -have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) (Steinlein et al. 1995(Steinlein et al. , 1997. The á4(S248F) mutation is a serine to phenylalanine substitution at position 248 in the human á4 nicotinic subunit. The á4(776ins3) mutation is a 3-base pair insertion that adds a leucine at position 259 in the amino acid sequence of the human á4 subunit. Photo-affinity labelling and structurefunction experiments show that the M2 region of the nicotinic subunits forms the conducting pore of the receptor (reviewed in Karlin & Akabas, 1995). Thus, both ADNFLE mutations lie in the conducting pore of the nicotinic receptor. ADNFLE patients suffer from brief and occasionally violent nocturnal seizures . However, the physiological mechanism responsible for these seizures has not been established. Previous studies show that the predominant brain nicotinic receptor subtype is á4â2 (Whiting & Lindstrom, 1987;Flores et al. 1991;Whiting et al. 1991). Receptors formed by co-expressing á4(S248F) or á4(776ins3) subunits with wild-type (WT) â2 subunits in Xenopus oocytes (Weiland et al. 1996; Steinlein et al. 1997;Kuryatov et al. 1997) differ from the WT receptor in several ways but no common effects of the two mutations on the acetylcholine (ACh) response have been reported previously. To determine whether the ADNFLE mutations have any common effects on the ACh response, we constructed two rat homologues (S252F and +L264) of the human ADNFLE mutations á4(S248F) and á4(777ins3), co-expressed them with rat â2 subunits in Xenopus oocytes, and studied the properties of the expressed receptors. We also constructed the rat double mutation V247I:S252F, which combined the S252F mutation with a second V247I mutation that converted the only rat/human residue substitution in the á4 M2 region to the corresponding human residue. All three Journal of Physiology (1998) 1. We constructed rat homologues (S252F and +L264) of two human á4 nicotinic mutations -á4(S248F) and á4(777ins3) -that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat â2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nÒ ACh pulses. Second, they delayed the rise times of the 5-15 nÒ (+L264) and 30 nÒ (S252F) ACh responses. Third, they reduced extracellular Ca¥-induced increases in the 30 ìÒ ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured b...

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.

"Divisian of Chemi~'uy mtd nDit'isin. ~f Bialagv 156.29, Califaroir, i, stintW of TechnnleJll.v, Pasadena. CA 9/125, USA gc~ivcd 26 June 1992 Fifteen chimeric nicotinic receptor ,6 subunits were constructed consistinM of N-terminal neuronal ~4 sequences and C-terminal ~ sequanc¢~.flcsponscs to c~'tisin¢, nicotine, or tot ramcthylammonium wcrt compared to acetylcholine rcspo::scs {'or these subunits cxpre,,~l in Xenapu.s oocytes with =3 subunits, The results show that (i) two residues in the ¢xtracellular domain of chimeric.~4,j~2 subunits (IOII,~2FIfl4V, 110.~.S/,~4T) acamunt for much of the relative cytisine sensitivity; and (it) four extr'acellular residu~ ofehimeric,64,J12 subunits (112~2A/,~4V. 1 ! ~l.~.V/fl4l and I 15,P,.$/.a4R, 116/J2YI/~4S) account for most of th." relative tetramcthyl:tmmonium sensitivity. The ~ata did not r~rmit localization of nicotine ~flsitivity to any ~trticular r¢ilion. shows that both subunits contribute to the relative sensitivity to ganglionic stimulants and ncurotoxins [1.2], channel conductance and gating p+'operties [3-5] of nettronal nAChR's. Receptors containing the f14 subtype generally give larger rcsponst;s than receptors containing the ~2 subtype to ganglionic stimulants such as cytisine (CYT) and nicotine (NIC) [2]. To localize the regions of~4 and f12 that contribute to agonist selectivity, we constructed chimeras of~'4 and ~2 consisting of N.terminal sequences from ,84 varying length and appropriate C-terminal counterparts from f12. These were expressed in combination with ct3 in Xenopus oocytes. 2, MATERIALS AND METHODSFifteen f14,,f12 chimeras were constructed using a previously de. scribed PCR method [6] so that they cont~.ined an N-terminal end from.t/4 and a C-terminal end from B2, For example, the arrow labeled ? in Fig, 1 denotes the chimera/24(?).,~2 that comains the ? most N-terminal residues from/'/4 and the remaininll 4?0 C-terminal rest. dues tram B2. mRNA for a3 and the fl subunits wa~ transcribed in vitro [9] and L8 ng of each subunit was coinjected into stage V or VI Xen.pus laevis oocytes, The oocytes were incubated 2-7 days in a modified Barth's solution containing 5% horse.serum. We measured the peak current produced by bath application of30,uM acetylcholin¢ (ACh), 30 #aM cytisin¢ (CY'I'), 30/JNI nicotine (NIC), and 100/JM Correspondence address: H,A, l.,¢~tcr, Division of Biology 156-29.Pasadena, CA 91 I25, USA. Fax: (1) (tile) 564 8709, tetrumethylammonium (TMA) at u typi~l holdinll potential of -ISO mY usinll ;s two.electrode voltall¢ clamp, Receptors that lave ACh r~ponscs too larl~¢ for accurate rccordinli at -80 mV wcrt m~sured at more depolarized potentials (-70 to -~0 nzV), ACh responsms ofthe chimeric and wild-tyl~ (WT) rc~ptors were typically in tfl~ 100-2,000 nA ranlle. The recordinil solution contained 96 mM NaCI. 2 mM NaOH. I mM MgCI.., and S mM HEPF..5 (pH 7,4), External Ca"" was omitted to minimize the activation of the Ca =-activated CI" conduct. ance [8]. AlL recordings wart mad© at ambient tcmpcraturt (23-25'C1, 3, RESULTSThe ...

We constructed chimeras of the rat [32 and [34 neuronal nicotinic subunits to locate the regions that contribute to differences between the acetylcholine (ACh) dose-response relationships of the cx3132 and cx3134 receptors. Expressed in Xenopus oocytes, the r receptor displays an ECs0 for ACh ~ 20-fold less than the ECs0 of the ct3134 receptor. The apparent Hill slope (napp) of ~t3132 is near one whereas the ct3134 receptor displays an nap p near two. Substitutions within the first 120 residues convert the EC50 for ACh from one wild-type value to the other. Exchanging just [32:104-120 for the corresponding region of [34 shifts the ECs0 of ACh dose-response relationship in the expected direction but does not completely convert the ECs0 of the dose-response relationship from one wild-type value to the other. However, substitutions in the [32:104-120 region do account for the relative sensitivity of the ~t3132 receptor to cytisine, tetramethylammonium, and ACh. The expression of [34-like (strong) cooperativity requires an extensive region of [34 ([34:1-301). Relatively short [32 substitutions ([32:104-120) can reduce cooperativity to [32-like values. The results suggest that amino acids within the first 120 residues of [32 and the corresponding region of [34 contribute to an agonist binding site that bridges the et and [3 subunits in neuronal nicotinic receptors.

1 We studied the pharmacological properties of native rat brain and heterologously expressed rat a4b2 nicotinic receptors immunoprecipitated onto a ®xed substrate with the anti-a4 antibody mAb 299. 2 Immunodepletion with the anti-b2 antibody mAb 270 showed that 89% of the mAb-299-precipitated rat brain receptors contained b2.

Extracellular Ca2ϩ robustly potentiates the acetylcholine response of ␣4␤2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-␣4(S252F), ␣4(S256L), ␣4(ϩL264), ␤2(V262L), and ␤2(V262M)-reduced 2 mM Ca 2ϩ potentiation of the ␣4␤2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca 2ϩ activation or enhanced Ca 2ϩ block caused this reduction, we coexpressed the rat ADNFLE mutations with an ␣4 N-terminal mutation, ␣4(E180Q), that abolished ␣4␤2 allosteric Ca 2ϩ activation. In each case, Ca 2ϩ inhibition of the double mutants was less than that expected from a Ca 2ϩ blocking mechanism. In fact, the effects of Ca 2ϩ on the ADNFLE mutations near the intracellular end of the M2 region-␣4(S252F) and ␣4(S256L)-were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca 2ϩ on the ADNFLE mutations near the extracellular end of the M2 region-␣4(ϩL264)␤2, ␤2(V262L), and ␤2(V262M)-were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca 2ϩ on the ␣4␤2, ␣4(ϩL264)␤2, and ␣4␤2(V262L) single-channel conductances, the effects of membrane potential on the ␤2(V262L)-mediated reduction in Ca 2ϩ 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 Ca 2ϩ block. Moreover, analyses of the ␣4␤2, ␣4(S256L), and ␣4(ϩL264) Ca 2ϩ concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca 2ϩ potentiation of the ␣4␤2 acetylcholine response by altering allosteric activation rather than by enhancing block.

We have studied the voltage-jump relaxation currents for a series of neuronal nicotinic acetylcholine receptors resulting from the coexpression of wild-type and chimeric [34/[32 subunits with et3 subunits in Xenopus oocytes. With acetylcholine as the agonist, the wild-type ct3~4 receptors displayed five-to eightfold slower voltage-jump relaxations than did the wild-type ct3132 receptors. In both cases, the relaxations could best be described by two exponential components of approximately equal amplitudes over a wide range of [ACh]'s. Relaxation rate constants increased with [ACh] and saturated at 20-to 30-fold lower concentrations for the t~3132 receptor than for the cr receptor, as observed previously for the peak steady state conductance. Furthermore, the chimeric 134/132 subunits showed a transition in the concentration dependence of the rate constants in the region between residues 94 and 109, analogous to our previous observation with steady state conductances. However, our experiments with a series of [3-subunit chimeras did not localize residues that govern the absolute value of the kinetic parameters. Hill coefficients for the relaxations also differed from those previously measured for steady state responses. The data reinforce previous conclusions that the region between residues 94 and 109 on the [3 subunit plays a role in binding agonist but also show that other regions of the receptor control gating kinetics subsequent to the binding step.

No abstract