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 decay during a 3-s pulse applied from a holding potential of −80 mV to +10 mV is illustrated in Fig. 1, B and C. For wild type and mutant channels, current decay could be well described by a double exponential time course. The fast component of current decay was significantly (p < 0.01) faster for mutants T666M and V714A but not for I1819L and R192Q (Fig. 1 C). No significant changes were found for the slow component (see legend to Fig. 1 C).Table IEffects of mutations on activation and inactivation propertiesMutantV0.5 actK-actV0.5 inactKinactBI4.1 ± 0.8 mV−4.1 ± 0.2−20.2 ± 1.8 mV6.7 ± 0.4R192Q1.9 ± 0.8 mV−3.9 ± 0.1−21.3 ± 0.6 mV7.2 ± 0.3T666M−1.5 ± 0.9 mV1-aIndicates a statistically significant (p < 0.01) difference from wild type; means ± S.E. (n = 7–27).−4.9 ± 0.11-aIndicates a statistically significant (p < 0.01) difference from wild type; means ± S.E. (n = 7–27).−24.0 ± 1.2 mV6.7 ± 0.3V714A−5.4 ± 0.6 mV1-aIndicates a statistically significant (p < 0.01) difference from wild type; means ± S.E. (n = 7–27).−3.6 ± 0.1−20.2 ± 0.6 mV5.6 ± 0.21-aIndicates a statistically significant (p < 0.01) difference from wild type; means ± S.E. (n = 7–27).I1811L−1.5 ± 1.4 mV1-aIndicates a statistically significant (p < 0.01) difference from wild type; means ± S.E. (n = 7–27).−4.4 ± 0.2−18.0 ± 2.4 mV6.3 ± 0.51-a Indicates a statistically significant (p < 0.01) difference from wild type; means ± S.E. (n = 7–27). Open table in a new tab The mutational effects on current inactivation could affect the accumulation of channels in inactivation during frequent depolarizations at high firing rates in neurons. To test this possibility we applied trains of pulses (1 Hz) from a holding potential of −60 mV to a test potential of +10 mV. As illustrated in Fig. 2 A, IBadecreased by 18 ± 1% (n = 14) during a train of 15 pulses in wild type channels. In mutants T666M, V714A, I1819L, but not in R192Q, the amount of accumulation in an inactivated state was significantly (p < 0.01) different from wild type. Peak IBa decrease during the pulse train was about 2-fold larger in T666M (34 ± 2%, n = 15) and about 2-fold smaller in I1819L (7.2 ± 0.8%,n = 6) than in wild type. Less accumulation in inactivation was also found for mutant V714A (13.2 ± 0.5%,n = 14). At the more negative holding potential of −80 mVIBa current decay of mutants T666M (12.7 ± 1%, n = 15) and I1819L (4.5 ± 0.6%,n = 6) was also significantly different from wild type (7.5 ± 0.4%, n = 14). The accumulation of channels in inactivation during a pulse train depends on how fast inactivation is removed between pulses. We therefore investigated the effects of the mutations on the time course of recovery by employing a double pulse protocol (Fig. 3). Wild type and mutant channels were inactivated by a 3-s conditioning prepulse from −80 mV to +10 mV (Fig. 1 B). The time course of recovery from inactivation at −60 mV was determined by subsequent test pulses applied at various times after the prepulse as described under "Experimental Procedures." In wild type and mutant channels about 90% of IBarecovered within 20 s. The time course followed a biexponential function suggesting that recovery occurs from more than one inactivated state. In wild type the fast component (τfast = 0.432 ± 0.033 s, n = 7) accounted for 68 ± 2% of recovered IBa and was about 8-fold faster than the slow component (τslow = 3.1 ± 0.34 s). Mutations T666M, V714A, and I1819L profoundly altered the time course of channel recovery from inactivation (Fig. 3, A and B). Mutant T666M slowed IBa recovery mainly by significantly increasing τfast and, to a smaller extent, also τslow (Fig. 3 B). In contrast to T666M, mutants V714A and I1819L recovered much faster than the wild type channel. These mutations significantly increased the contribution of the fast recovering component, decreased τfast 3–4-fold (Fig. 3 B) and slightly reduced τslow. The slower recovery from inactivation of T666M explains the enhanced accumulation of these channels in inactivation during pulse trains, whereas accelerated recovery of I1819L decreases such accumulation (Fig. 2). Mutation V714A also accelerated recovery from inactivation but yet exhibited a larger IBadecrease during the train than I1819L (Fig. 2 B). This can be explained by the finding that mutation V714A, but not I1819L, accelerated inactivation during a single test pulse (Fig. 1 B), which can also favor accumulation in inactivation during pulse trains. Taken together our data clearly demonstrate that FHM mutations T666M, V714A, and I1819L affect α1A Ca2+ channel inactivation kinetics. This can cause significant changes in channel availability at higher depolarization frequencies. Our data provide convincing evidence 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) affect the kinetic properties and the voltage dependence of α1A Ca2+channel activation. These mutations especially changed channel recovery from inactivation and thereby altered the extent to which mutant channels accumulate in an inactivated state during rapid depolarizations. Our experiments show that these mutations yield at least two functional phenotypes, leading to either an increase or a decrease in Ca2+ channel availability. Mutants V714A and I1819L are located at almost identical positions at the intracellular end of the homologous helices S6 in repeats II (V714) and IV (I1819). Both accelerated recovery from inactivation and slightly shifted the activation curve toward more negative potentials. Therefore these mutations should increase Ca2+ channel availability and promote voltage-dependent Ca2+ influx into neurons. Mutation T666M is located in the linker IIS5-S6. It also slightly shifted the voltage dependence of activation toward more negative potentials but simultaneously slowed channel recovery from inactivation. The latter effect can decrease channel availability at high stimulation frequency (Fig. 2). Our data are in accordance with previous findings describing changes in the inactivation properties by site-directed mutations in the S6 segments of voltage-gated Ca2+ (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, 23Zhang J.F. Ellinor P.T. Aldrich R.W. Tsien R.W. 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According to present folding models of voltage-gated cation channels (30Durell S.R. Guy H.R. Neuropharmacology. 1997; 35: 761-773Crossref Scopus (62) Google Scholar), the S5-S6 linkers (containing mutation T666M) and S6 segments (containing V714A and I1819L) participate in the formation of the ion pore. Our data obtained with the FHM mutations therefore further support the hypothesis (24Hering S. Aczel S. Grabner M. Döring F. Berjukow S. Mitterdorfer J. Sinnegger M.J. Striessnig J. Degtiar V.E. Wang Z. Glossmann H. J. Biol. Chem. 1996; 271: 24471-24475Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) that pore-forming residues play an important role for Ca2+ channel inactivation. Mutation R192Q eliminates a conserved positive charge within the amphipathic helix IS4, one of the putative voltage sensors (31Hille B. Ionic Channels of Excitable Membranes. Sinauer, Sunderland1991Google Scholar). The finding that its mutation does not cause detectable functional changes under our experimental conditions is surprising. A recent analysis of charge neutralizing S4 mutations in a L-type Ca2+ channel α1 subunit (32Garcia J. Nakai J. Imoto K. Beam K.G. Biophys. J. 1997; 72: 2515-2523Abstract Full Text PDF PubMed Scopus (54) Google Scholar) has demonstrated that a large number but not all conservative positive charges in S4 contribute to channel gating. Charge neutralizations with no effect onV0.5 were mainly seen in IIS4 and IVS4 but also included a residue in IS4. Our data also do not rule out possible effects of R192Q on the time course of current activation, which was not analyzed in our study. Our data obtained with FHM mutations T666M, V714A, and I1819L agree with the hypothesis (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 α1Asubunit underly the neuronal instability which renders patients susceptible to migraine attacks that can be triggered by neural stimuli, such as stress or sensory afferentiation (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 (and other more common hereditary forms of migraine) is an autosomal dominant disease (15Färkkilä M. Ann. Med. 1994; 26: 7-8Crossref PubMed Scopus (10) Google Scholar), only a fraction of the channels should be affected by the mutations. With the exception of cerebellar degeneration, FHM (and other migraine) patients show no other major disturbances in neurological function, suggesting that the functional consequences of the mutations only become physiologically relevant under certain conditions. This assumption agrees with our observation that the functional consequences of altered inactivation properties become especially obvious only at higher stimulation frequency. Presynaptic V714A and I1819L channels can be considered "gain-of-function" mutants under these conditions. Their relative contribution to Ca2+ entry would gradually increase with firing rate because they are expected to accumulate to a smaller extent in inactivation than wild type α1A. This could result in higher than normal Ca2+ entry with even more pronounced effects on neurotransmitter release, which can rise with the fourth power of intracellular Ca2+ concentrations. Such an enhanced Ca2+ entry could eventually also lead to episodes of neuronal Ca2+ overloading and explain the cerebellar neurodegeneration observed in some FHM patients, including patients with the I1811L (rabbit 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 situation is even more complex because mutant P/Q-type Ca2+ channels must also be localized 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). There alterations in Ca2+ entry at high firing rates could affect neuronal Ca2+-dependent processes, such as Ca2+-dependent phosphorylation/dephosphorylation and gene transcription. Decreased Ca2+ entry through Ca2+ channels (such as expected for mutant T666M) into cell bodies can also increase the firing rate of a neuron. Inhibition of Ca2+ current can diminish neuronal spike after hyperpolarizations (e.g. by decreased activation of Ca2+-activated K+-channels, 33Storm J.F. Pedarzani P. Eur. J. Neurosci. 1996; 8: 2098-2110Crossref PubMed Scopus (39) Google Scholar) as has recently been shown,e.g. for P/Q-type channels in caudal raphe neurons (34Bayliss D.A. Li Y.-W. Talley E.M. J. Neurophysiol. 1997; 77: 1362-1374Crossref PubMed Scopus (76) Google Scholar). Among others, these neurons seem to play a crucial role 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 prompt further experiments to assess the pathophysiological consequences of the FHM mutations under conditions that more closely resemble neuronal activity in vivo. It will be especially important to study the effects of the mutations at 37 °C. At this temperature faster channel kinetics would allow higher stimulation rates than in our experiments in X. laevis oocytes. Higher stimulation frequency may lead to considerable accumulation in inactivation even during trains of much shorter pulses than used in our study. Although our data clearly demonstrate that three of the four FHM mutations lead to significant alterations in α1A subunit function, several questions remain to be answered. The rabbit and human α1A share high sequence identity (93.5%) with sequence heterology limited to the C-terminal tail and the long cytoplasmic loops (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, 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). Only a total of 4 amino acid differences exists in the putative pore forming region (consisting of S5, S5-S6 linkers, S6 of the four repeats). Despite this high conservation quantitative or qualitative differences of mutational effects on rabbit and human α1A cannot be excluded. The three mutations affecting channel gating are located in conserved regions participating in the formation of the channel pore. They are not located within other known functional domains of the channel, such as α1 subunit interaction domains for accessory subunits (35Pragnell M. De Waard M. Tanabe T. Mori Y. Snutch T.P. Campbell K.P. Nature. 1994; 368: 67-70Crossref PubMed Scopus (553) Google Scholar) or G-proteins (36De Waard M. Liu H. Walker D. Scott V.E. Gurnett C.A. Campbell K.P. 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Our data demonstrate that residues in putative pore-forming regions of Ca2+ channel α1A subunits determine inactivation properties. Further experiments are required to prove that FHM mutations alter Ca2+ entry and neurotransmitter release preferentially at high firing rates in intact neurons. We thank Dr. Y. Mori for rabbit BI-II cDNA, Dr. Charles Cohen, Gregory Kaczorowski, and J. Mitterdorfer for valuable comments on the manuscript, Dr. S. Berjukow for helpful discussion, and D. Kandler and B. Kurka for expert technical assistance.