Familial Hemiplegic Migraine Mutations Change α1ACa2+ Channel Kinetics

Missense mutations in the pore-forming human α1A subunit of neuronal P/Q-type Ca2+channels are associated with familial hemiplegic migraine (FHM). The pathophysiological consequences of these mutations are unknown. We have introduced the four single mutations reported for the human α1A subunit into the conserved rabbit α1A(R192Q, T666M, V714A, and I1819L) and investigated possible changes in channel function after functional expression of mutant subunits in Xenopus laevis oocytes.Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba2+ current (IBa) inactivation was slightly faster in mutants T666M and V714A than in wild type. The time course of recovery from channel inactivation was slower than in wild type in T666M and accelerated in V714A and I1819L. As a consequence, accumulation of channel inactivation during a train of 1-Hz pulses was more pronounced for mutant T666M and less pronounced for V714A and I1819A. Our data demonstrate that three of the four FHM mutations, located at the putative channel pore, alter inactivation gating and provide a pathophysiological basis for the postulated neuronal instability in patients with FHM. Missense mutations in the pore-forming human α1A subunit of neuronal P/Q-type Ca2+channels are associated with familial hemiplegic migraine (FHM). The pathophysiological consequences of these mutations are unknown. We have introduced the four single mutations reported for the human α1A subunit into the conserved rabbit α1A(R192Q, T666M, V714A, and I1819L) and investigated possible changes in channel function after functional expression of mutant subunits in Xenopus laevis oocytes. Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba2+ current (IBa) inactivation was slightly faster in mutants T666M and V714A than in wild type. The time course of recovery from channel inactivation was slower than in wild type in T666M and accelerated in V714A and I1819L. As a consequence, accumulation of channel inactivation during a train of 1-Hz pulses was more pronounced for mutant T666M and less pronounced for V714A and I1819A. Our data demonstrate that three of the four FHM mutations, located at the putative channel pore, alter inactivation gating and provide a pathophysiological basis for the postulated neuronal instability in patients with FHM. α1A subunits, in a complex with a β and α2δ subunit (1Gurnett C.A. Campbell K.P. J. Biol. Chem. 1996; 271: 27958-27975Abstract Full Text Full Text PDF Scopus (59) Google Scholar, 2Isom L.L. Catterall W.A. Neuron. 1994; 12: 1183-1194Abstract Full Text PDF PubMed Scopus (494) Google Scholar), constitute the pore-forming subunit of neuronal voltage-gated P/Q-type Ca2+ channels. This channel type is not only located on nerve cell bodies and dendrites but is also present in presynaptic terminals (3Westenbroek R.E. Sakurai T. Elliott E.M. Hell J.W. Starr T.V. Snutch T.P. Catterall W.A. J. Neurosci. 1995; 15: 6403-6418Crossref PubMed Google Scholar) where it controls depolarization-induced Ca2+ influx tightly coupled to neurotransmitter release (4Wheeler D.B. Randall A. Tsien R.W. Science. 1994; 264: 107-111Crossref PubMed Scopus (842) Google Scholar). Its gating properties are modulated by neurotransmitters (5Bourinet E. Soong T.W. Stea A. Snutch T.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 431: 470-472Google Scholar, 6Herlitze S. Garcia D.E. Mackie K. Hille B. Scheuer T. Catterall W.A. Nature. 1996; 380: 258-262Crossref PubMed Scopus (706) Google Scholar) and affected by β subunits in an isoform-specific manner (7Stea A. Tomlinson W.J. Soong T.W. Bourinet E. Dubel S.J. Vincent S.R. Snutch T.P. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10576-10580Crossref PubMed Scopus (308) Google Scholar, 8Liu H. De Waard M. Scott V.E.S. Gurnett C.A. Lennon V.A. Campbell K.P. J. Biol. Chem. 1996; 271: 13804-13810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). This suggests that a tight control of P/Q-type Ca2+ channel activity is a prerequisite to fine tune its physiological function. Missense mutations in the gene encoding human α1A(CACNL1A4) have recently been found to segregate with patients suffering from familial hemiplegic migraine (FHM) 1The abbreviations were used: FHM, familial hemiplegic migraine; BAPTA, 1,2-bis-(O-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid. (9Ophoff R.A. Terwindt G.M. Vergouwe M.N. Eijk R.v. Oefner P.J. Hoffmann S.M.G. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J.B. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar), an autosomal dominant disorder. Although FHM represents a rare form of migraine, a detailed analysis of the functional consequences of this channelopathy may provide insight into the pathophysiology of migraine. Mutations in the CACNL1A4 gene could also underly more common forms of migraine with and without aura (10May A. Ophoff R.A. Terwindt G.M. Urban C. Eijk R.v. Haan J. Diener H.C. Lindhout D. Frants R.R. Sandkuijl L.A. Ferrari M.D. Hum. Genet. 1995; 96: 604-608Crossref PubMed Scopus (178) Google Scholar). The pathophysiology of migraine remains to be fully understood and the mechanisms triggering an attack are unknown. Recent advances in brain imaging techniques (positron emission tomography and magnetic resonance spectroscopy) support a “primary neuronal theory” where attacks originate on the basis of a neuronal hyperexcitability of unknown origin (11Schoenen J. Curr. Opin. Neurol. 1997; 10: 237-243Crossref PubMed Scopus (58) Google Scholar, 12Flippen C. Welch K.M.A. Curr. Opin. Neurol. 1997; 10: 226-230Crossref PubMed Scopus (18) Google Scholar, 13Zagami A.S. Curr. Opin. Neurol. 1994; 7: 272-277Crossref PubMed Scopus (25) Google Scholar, 14Tfelt-Hansen P. Pharmacol. Toxicol. 1994; 75: 72-75Crossref PubMed Scopus (3) Google Scholar, 15Färkkilä M. Ann. Med. 1994; 26: 7-8Crossref PubMed Scopus (10) Google Scholar). This may be the underlying cause of cortical spreading depression and hypoperfusion, phenomena associated with migraine attacks (12Flippen C. Welch K.M.A. Curr. Opin. Neurol. 1997; 10: 226-230Crossref PubMed Scopus (18) Google Scholar, 13Zagami A.S. Curr. Opin. Neurol. 1994; 7: 272-277Crossref PubMed Scopus (25) Google Scholar). Neuronal instability within central pain-modulating serotoninergic systems could not only serve as a “brainstem generator” of attacks but also initiate the headache and the events of neurogenic inflammation in the trigeminovascular system (12Flippen C. Welch K.M.A. Curr. Opin. Neurol. 1997; 10: 226-230Crossref PubMed Scopus (18) Google Scholar). It is therefore attractive to speculate that the four single α1A mutations found in FHM patients lead to such a neuronal instability by changes in P/Q-type Ca2+ channel function. As the direct analysis of changes in α1A Ca2+channel gating in human tissue samples is not feasible, we introduced the corresponding mutations into rabbit α1 subunits, which shares 94% sequence identity with the human α1A, and analyzed the biophysical properties of the mutant channels after heterologous expression in Xenopus laevis oocytes. Nucleotide numbering of restriction sites is given in parentheses. Mutants were constructed by applying the “geneSOEing” technique (16Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar) as described previously (17Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). A ClaI (5′-pSP polylinker region)-SphI (5858) fragment from rabbit class A calcium channel (BI-II) α1AcDNA in pSPCBI-2 (18Mori Y. Friedrich T. Kim M.-S. Mikami A. Nakai J. Ruth P. Bosse E. Hofmann F. Flockerzi V. Furuichi T. Mikoshiba K. Imoto K. Tanabe T. Numa S. Nature. 1991; 350: 398-402Crossref PubMed Scopus (709) Google Scholar) was subcloned into plasmid pSP72 (Promega) with modified polylinker. Mutation R192Q was constructed by using aHindIII (5′-pSPCBI-2 polylinker region)-NotI (894) cassette within the subclone. The mutation was subsequently introduced into BI-II by co-ligation of the mutated subclone fragmentHindIII (5′-pSPCBI-2 polylinker region)-XhoI (1689) with fragments XhoI (1689)-SphI (5858),SphI (5858)-XbaI (3′ of polyadenylation signal), and XbaI (3′ of polyadenylation signal)-HindIII (5′-pSPCBI-2 polylinker region) from pSPCBI-2, respectively. Single mutants T666M and V714A were constructed by using aXhoI (1689)-HindIII (2503) cassette in the subclone after elimination of the 5′-polylinker HindIII restriction site. The single mutations were subsequently introduced into pSPCBI-2 by exchanging a XhoI (1689)-NheI (3543) fragment in pSPCBI-2 for the respective mutant sequence. Mutation I1819L (corresponding to the human FHM mutation I1811L) was constructed by exchanging the KpnI-BglII cassette of construct AL22 (19Döring F. Degtiar V.E. Grabner M. Striessnig J. Hering S. Glossmann H. J. Biol. Chem. 1996; 271: 11745-11749Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) for the respective mutant BI-II sequence. All polymerase chain reaction-generated fragments were sequenced completely to confirm sequence integrity. Preparation of stage V-VI oocytes from X. laevis and injection of cRNA are described in detail elsewhere (17Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Capped run-off poly(A+) cRNA transcripts from XbaI-linearized cDNA templates were synthesized according to the procedures of Krieg and Melton (20Krieg P.A. Melton D.A. Nucleic Acids Res. 1984; 12: 7057-7070Crossref PubMed Scopus (1081) Google Scholar). α1cRNAs were coinjected with β1a (21Ruth P. Roehrkasten A. Biel M. Bosse E. Regulla S. Meyer H.E. Flockerzi V. Hofmann F. Science. 1989; 245: 1115-1118Crossref PubMed Scopus (257) Google Scholar) and α2δ (22Ellis S.B. Williams M.E. Ways N.R. Brenner R. Sharp A.H. Leung A.T. Campbell K.P. McKenna E. Koch W.J. Hui A. Schwartz A. Harpold M.M. Science. 1988; 241: 1661-1664Crossref PubMed Scopus (442) Google Scholar) subunit cRNAs. To exclude effects of endogenous Ca2+-activated Cl− currents on current kinetics experiments were also carried out in oocytes previously injected with 50–100 nl of a 0.1 mBAPTA solution. Inward Ba2+currents (IBa) through expressed channel complexes were measured using the two-microelectrode voltage-clamp technique as described previously (17Grabner M. Wang Z. Hering S. Striessnig J. Glossmann H. Neuron. 1996; 16: 207-218Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). Similar current amplitudes were obtained with mutant and wild type α1A subunits. Oocytes expressing peak IBa smaller than 400 nA or larger than 1.6 μA were excluded from analysis. Data analysis and acquisition was performed by using the pClamp software package (version 6.0, Axon Instruments). Recordings were carried out at room temperature in a bath solution containing 40 mm Ba(OH)2, 40 mm N-methyl-d-glucamine, 10 mm HEPES, 10 mm glucose, adjusted to a pH of 7.4 with methanesulfonic acid. Voltage recording and current injecting microelectrodes were filled with 2.8 m CsCl, 0.2 m CsOH, 10 mm EGTA, 10 mm HEPES (adjusted to pH 7.4 with HCl), and had resistances of 0.3–2 megohm. Recovery of IBa from inactivation was studied using a double-pulse protocol. After a 3-s depolarizing prepulse to +10 mV (holding potential −80 mV) the time course of IBa recovery was determined at −60 mV by applying 300-ms test pulses to +10 mV at various time intervals after the prepulse. Peak IBa was normalized to the peak current amplitude measured during the prepulse.IBa was then allowed to recover during 1 min at −100 mV. This double pulse protocol was repeated individually for each recovery time interval in the same oocyte. The voltage dependence of inactivation (steady state inactivation) was determined from normalized inward currents elicited during steps to +10 mV after 10-s steps to various holding potentials. The voltage dependence of activation was determined from I-V curves obtained by step depolarizations from a holding potential of −80 mV to various test potentials. The half-maximal voltage for activation (V0.5,act), the slope factor of the curve atV0.5,act (kact), the half-maximal voltage for steady state inactivation (kinact), and the slope factor of the curve (V0.5,inact) were obtained by fitting the data to the Boltzmann equation. Nonlinear least square fitting and statistical calculations were performed using OriginR(Microcal). Data are given as means ± S.E. for the indicated number of experiments. To study the functional consequences of single amino acid mutations associated with human FHM we introduced the corresponding mutations into the rabbit α1A subunit (BI-II, Ref. 18Mori Y. Friedrich T. Kim M.-S. Mikami A. Nakai J. Ruth P. Bosse E. Hofmann F. Flockerzi V. Furuichi T. Mikoshiba K. Imoto K. Tanabe T. Numa S. Nature. 1991; 350: 398-402Crossref PubMed Scopus (709) Google Scholar). Their positions are illustrated in Fig. 1 A. Human mutation I1811L corresponds to I1819L in rabbit α1A. Wild type and mutant α1A subunits were functionally expressed in X. laevis oocytes (together with accessory β1a and α2δ subunits) and macroscopic channel properties measured using the two microelectrode voltage-clamp technique. The half-maximal voltage for activation (V0.5act) was slightly, but significantly, shifted toward more negative potentials for mutants T666M, V714A, and I1819L (Table I). The midpoint voltage for steady-state inactivation was not significantly affected (Table I). The effects of mutations on IBa during a 3-s pulse from a holding potential of −80 mV to +10 mV is illustrated in Fig. and C. wild type and mutant current could be described by a double time The of current was significantly faster for mutants T666M and V714A but not for I1819L and R192Q 1 changes were found for the to Fig. 1 of mutations on activation and inactivation ± ± ± ± ± ± ± ± ± a from wild means ± S.E. ± a from wild means ± S.E. ± ± ± a from wild means ± S.E. ± ± ± a from wild means ± S.E. ± a from wild means ± S.E. ± ± ± a from wild means ± S.E. in a The effects on current inactivation could the accumulation of channels in inactivation during depolarizations at in To test this we of pulses from a holding potential of −60 mV to a test potential of +10 mV. As illustrated in Fig. by ± during a train of pulses in wild type channels. mutants T666M, V714A, but not in the of accumulation in an state was significantly from wild type. Peak IBa during the pulse train was larger in T666M ± and smaller in I1819L ± than in wild type. accumulation in inactivation was also found for mutant V714A ± the more negative holding potential of −80 current of mutants T666M ± and I1819L ± was also significantly from wild type ± The accumulation of channels in inactivation during a pulse train on inactivation is We therefore investigated the effects of the mutations on the time course of recovery by a double pulse protocol Wild type and mutant channels were by a 3-s prepulse from −80 mV to +10 mV 1 The time course of recovery from inactivation at −60 mV was determined by test pulses at various after the prepulse as described wild type and mutant channels of within The time course a function that recovery from more than wild type the ± for ± of IBa and was faster than the ± Mutations T666M, V714A, and I1819L the time course of channel recovery from inactivation A and T666M IBa recovery by significantly to a smaller also to T666M, mutants V714A and I1819L faster than the wild type mutations significantly the of the and slightly The slower recovery from inactivation of T666M the accumulation of these channels in inactivation during pulse accelerated recovery of I1819L such accumulation Mutation V714A also accelerated recovery from inactivation but a larger during the train than I1819L This be by the that mutation V714A, but not accelerated inactivation during a single test pulse 1 which also accumulation in inactivation during pulse data demonstrate that FHM mutations T666M, V714A, and I1819L α1A Ca2+ channel inactivation This cause changes in channel at Our data provide that three of the four mutations reported in FHM patients (9Ophoff R.A. Terwindt G.M. Vergouwe M.N. Eijk R.v. Oefner P.J. Hoffmann S.M.G. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J.B. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar) the properties and the voltage dependence of α1A Ca2+channel mutations channel recovery from inactivation and the to which mutant channels in an state during Our experiments that these mutations at least two functional to an or a in Ca2+ channel Mutants V714A and I1819L are located at positions at the of the in and accelerated recovery from inactivation and slightly shifted the activation curve toward more negative potentials. these mutations Ca2+ channel and Ca2+ influx into Mutation T666M is located in the It also slightly shifted the voltage dependence of activation toward more negative potentials but channel recovery from The channel at Our data are in with changes in the inactivation properties by mutations in the of voltage-gated Ca2+ (19Döring F. Degtiar V.E. Grabner M. Striessnig J. Hering S. Glossmann H. J. Biol. 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Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) that pore-forming an for Ca2+ channel Mutation R192Q a conserved within the of the putative voltage B. of Scholar). The that its mutation not cause functional changes is A analysis of mutations in a Ca2+ channel α1 subunit J. Nakai J. Imoto K. J. 1997; Full Text PDF PubMed Scopus Google Scholar) that a number but not in to channel with were in and but also a in Our data also not out possible effects of R192Q on the time course of current which was not analyzed in Our data obtained with FHM mutations T666M, V714A, and I1819L with the (9Ophoff R.A. Terwindt G.M. Vergouwe M.N. Eijk R.v. Oefner P.J. Hoffmann S.M.G. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J.B. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar, 12Flippen C. Welch K.M.A. Curr. Opin. Neurol. 1997; 10: 226-230Crossref PubMed Scopus (18) Google Scholar) that mutations in the underly the neuronal instability which patients to migraine attacks that be by such as or (12Flippen C. Welch K.M.A. Curr. Opin. Neurol. 1997; 10: 226-230Crossref PubMed Scopus (18) Google Scholar, 15Färkkilä M. Ann. Med. 1994; 26: 7-8Crossref PubMed Scopus (10) Google Scholar). As FHM more common forms of is an autosomal dominant M. Ann. Med. 1994; 26: 7-8Crossref PubMed Scopus (10) Google Scholar), only a of the channels be affected by the the of FHM patients in that the functional consequences of the mutations only This with that the functional consequences of inactivation properties only at V714A and I1819L channels be mutants these Their to Ca2+ with are to to a smaller in inactivation than wild type α1A. This could in than Ca2+ with more pronounced effects on neurotransmitter which with the of Ca2+ an Ca2+ could also lead to of neuronal Ca2+ and the observed in FHM patients with the I1811L I1819L) mutation (9Ophoff R.A. Terwindt G.M. Vergouwe M.N. Eijk R.v. Oefner P.J. Hoffmann S.M.G. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J.B. Hofker M.H. Ferrari M.D. Frants R.R. Cell. 1996; 87: 543-552Abstract Full Text Full Text PDF PubMed Scopus (2136) Google Scholar). The is more complex mutant P/Q-type Ca2+ channels also be on cell bodies and dendrites (3Westenbroek R.E. Sakurai T. Elliott E.M. Hell J.W. Starr T.V. Snutch T.P. Catterall W.A. J. Neurosci. 1995; 15: 6403-6418Crossref PubMed Google Scholar). in Ca2+ at could neuronal such as and gene Ca2+ through Ca2+ channels as for mutant into cell bodies also the of a of Ca2+ current neuronal after by activation of Ca2+-activated P. J. Neurosci. 1996; PubMed Scopus Google Scholar) as recently been for P/Q-type channels in D.A. E.M. J. 1997; 77: PubMed Scopus Google Scholar). these to a in the pathophysiology of migraine (12Flippen C. Welch K.M.A. Curr. Opin. Neurol. 1997; 10: 226-230Crossref PubMed Scopus (18) Google Scholar). Our data experiments to the pathophysiological consequences of the FHM mutations that more neuronal activity in It be to study the effects of the mutations at this temperature faster channel kinetics than in experiments in X. laevis oocytes. may lead to accumulation in inactivation during of pulses than in Although data demonstrate that three of the four FHM mutations lead to in α1A subunit to be The rabbit and human α1A sequence identity with sequence to the and the (9Ophoff R.A. Terwindt G.M. Vergouwe M.N. Eijk R.v. Oefner P.J. Hoffmann S.M.G. Lamerdin J.E. Mohrenweiser H.W. Bulman D.E. Ferrari M. Haan J. Lindhout D. van Ommen G.J.B. Hofker M.H. Ferrari M.D. Frants R.R. 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De Waard M. Scott V.E.S. Gurnett C.A. Lennon V.A. Campbell K.P. J. Biol. Chem. 1996; 271: 13804-13810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, M. D. H. R. D. Striessnig J. J. Biol. Chem. 1997; Full Text Full Text PDF Scopus Google Scholar) or the of The biophysical of the mutants may also be affected by the Our data demonstrate that in putative pore-forming of Ca2+ channel α1A subunits inactivation experiments are to that FHM mutations alter Ca2+ and neurotransmitter release at in We Y. for rabbit BI-II and J. for on the S. for and D. and B. for