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Missense mutations in the pore-forming human ␣ 1A subunit of neuronal P/Q-type Ca 2؉ 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 ␣ 1A subunit into the conserved rabbit ␣ 1A (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. Ba 2؉ current (I Ba ) 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.␣ 1A subunits, in a complex with a ␤ and ␣ 2 ␦ subunit (1, 2), constitute the pore-forming subunit of neuronal voltage-gated P/Q-type Ca 2ϩ channels. This channel type is not only located on nerve cell bodies and dendrites but is also present in presynaptic terminals (3) where it controls depolarization-induced Ca 2ϩ influx tightly coupled to neurotransmitter release (4). Its gating properties are modulated by neurotransmitters (5, 6) and affected by ␤ subunits in an isoform-specific manner (7,8). This suggests that a tight control of P/Q-type Ca 2ϩ channel activity is a prerequisite to fine tune its physiological function.Missense mutations in the gene encoding human ␣ 1A (CACNL1A4) have recently been found to segregate with patients suffering from familial hemiplegic migraine (FHM) 1 (9), 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 (10).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 (11)(12)(13)(14)(15). This may be the underlying cause of cortical spreading depression and hypoperfusion, phenomena associated with migraine attacks (12, 13). 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 ...

We have investigated the functional consequences of three P/Q-type Ca 2؉ channel ␣1A (Ca v 2.1␣ 1 ) subunit mutations associated with different forms of ataxia (episodic ataxia type 2 (EA-2), R1279Stop, AY1593/1594D; progressive ataxia (PA), G293R). Mutations were introduced into human ␣1A cDNA and heterologously expressed in Xenopus oocytes or tsA-201 cells (with ␣ 2 ␦ and ␤1a) for electrophysiological and biochemical analysis. G293R reduced current density in both expression systems without changing single channel conductance. R1279Stop and AY1593/1594D protein were expressed in tsA-201 cells but failed to yield inward barium currents (I Ba ). However, AY1593/1594D mediated I Ba when expressed in oocytes. G293R and AY1593/1594D shifted the current-voltage relationship to more positive potentials and enhanced inactivation during depolarizing pulses (3 s) and pulse trains (100 ms, 1 Hz). Mutation AY1593/ 1594D also slowed recovery from inactivation. Single channel recordings revealed a change in fast channel gating for G293R evident as a decrease in the mean open time. Our data support the hypothesis that a pronounced loss of P/Q-type Ca 2؉ channel function underlies the pathophysiology of EA-2 and PA. In contrast to other EA-2 mutations, AY1593/1594D and G293R form at least partially functional channels.Genetic defects within the pore-forming Ca v 2.1␣ 1 (␣1A) subunit of neuronal voltage-gated P/Q-type Ca 2ϩ channels are associated with inherited human neurological diseases, such as Familial Hemiplegic Migraine (FHM), 1 Episodic Ataxia Type 2 (EA-2), progressive cerebellar ataxia, and epilepsy (1-6). Therefore, neuronal Ca 2ϩ channel dysfunction represents an important pathophysiological mechanism that may also underlie more common forms of migraine, epilepsy, and neurodegenerative processes.By mediating depolarization-induced Ca 2ϩ influx into dendrites, cell bodies, and nerve terminals, neuronal voltage-gated Ca 2ϩ channels control important neuronal processes. This includes fast neurotransmitter release, gene expression, neuronal plasticity, migration, and differentiation (7). P/Q-type Ca 2ϩ channels are very tightly coupled to neurotransmitter release in many neurons (see e.g. Refs. 8 and 9) and mediate most of the depolarization-induced Ca 2ϩ current in cerebellar Purkinje cells (10). They exist as hetero-oligomeric complexes of ␣1A subunits together with accessory subunits (especially ␣2␦ and ␤ (11)). ␣1A subunit-mediated neurotransmitter release is tightly controlled by other neurotransmitters (e.g. via protein kinase C phosphorylation and G-protein ␤␥ subunits (12-14)) and by their direct association with synaptic vesicles through soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (15). Hence, these channels are ideally suited to fine-tune synaptic strength. This also explains why genetic defects, despite causing only minor changes in channel gating or expression, can lead to the above-mentioned clinical symptoms in humans and to the severe neurological abnormalities ...

Missense mutations in the pore-forming human ␣ 1A subunit of neuronal P/Q-type Ca 2؉ channels are associated with familial hemiplegic migraine. We studied the functional consequences on P/Q-type Ca 2؉ channel function of three recently identified mutations, R583Q, D715E, and V1457L after introduction into rabbit ␣ 1A and expression in Xenopus laevis oocytes. The potential for half-maximal channel activation of Ba 2؉ inward currents was shifted by > 9 mV to more negative potentials in all three mutants. The potential for half-maximal channel inactivation was shifted by > 7 mV in the same direction in R583Q and D715E. Biexponential current inactivation during 3-s test pulses was significantly faster in D715E and slower in V1457L than in wild type. Mutations R583Q and V1457L delayed the time course of recovery from channel inactivation. The decrease of peak current through R583Q (30.2%) and D715E (30.1%) but not V1457L (18.7%) was more pronounced during 1-Hz trains of 15 100-ms pulses than in wild type (18.2%). Our data demonstrate that the mutations R583Q, D715E, and V1457L, like the previously reported mutations T666M, V714A, and I1819L, affect P/Q-type Ca 2؉ channel gating. We therefore propose that altered channel gating represents a common pathophysiological mechanism in familial hemiplegic migraine.Voltage-gated P/Q-type Ca 2ϩ channels are expressed on cell bodies and dendrites of cerebellar Purkinje cells and other neurons (1-3) where they are thought to control neuronal excitability, gene expression, neuronal plasticity, and differentiation. These channels are also expressed on presynaptic terminals (3) mediating depolarization-induced Ca 2ϩ influx tightly coupled to neurotransmitter release (4). The Ca 2ϩ -selective pore of P/Q-type Ca 2ϩ channels is formed by ␣ 1A subunits, which also contain the voltage sensors. ␣ 1A is encoded by the human gene CACNA1A on chromosome 19p13 (5).P/Q-type Ca 2ϩ channels have received much attention recently because CACNA1A mutations have been described which are responsible for at least three different neurological human diseases: episodic ataxia type 2 (EA-2), 1 spinocerebellar ataxia type 6, and familial hemiplegic migraine (FHM) with and without cerebellar ataxia. These mutations may provide important insight into how altered Ca 2ϩ signaling and neuronal excitability can lead to neurodegeneration and episodic neurological diseases such as migraine.Four nonsense mutations (6 -8), three splice site mutations, and four deletions in CACNA1A (5, 7) have been found to segregate in patients with EA-2. Small CAG expansions were observed in a large series of patients with spinocerebellar ataxia type 6 (9), and a further CACNA1A missense mutation was identified in a patient with severe progressive ataxia (10). At least seven missense mutations have been identified in families with FHM (5, 11-13). Defects in the ␣ 1A gene are also responsible for the phenotypes (absence epilepsy and ataxia) of tottering (tg) and leaner (tg la ) mutant mice (14) and may also occur in more common for...

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (␣ 1S or ␣ 1C ) to a weakly sensitive, nonstereoselective class A (␣ 1A ) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type ␣ 1 into the ␣ 1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (؊)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (؊)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.L-type calcium (Ca 2ϩ ) channels (classes C (formed by ␣ 1C subunits), D (␣ 1D ), and S (␣ 1S )) possess high affinity stereoselective drug receptors for Ca 2ϩ antagonists such as 1,4-dihydropyridines (DHPs), 1 phenylalkylamines (PAAs), and benzothiazepines (BTZs) (reviewed in Refs. 1-5) located on their pore-forming ␣ 1 channel subunit (6). Classes A (␣ 1A ), B (␣ 1B ), and E (␣ 1E ) Ca 2ϩ channels are insensitive for DHPs (2, 5, 7-9) and only weakly sensitive for . Essential parts of the high affinity binding sites for DHPs and PAAs on L-type Ca 2ϩ channels have been identified by replacing sequence stretches in ␣ 1C or ␣ 1S subunits by corresponding non-L-type sequences (8, 13) or by mutating single amino acids in these subunits (10, 13). Alternatively, molecular determinants of the high affinity DHP and PAA receptor sites could be localized in pore-lining regions of repeats III and/or IV by transferring L-type ␣ 1 sequences into the ␣ 1A subunit (9, 12). Transfer of segment IVS6 from ␣ 1S to ␣ 1A enhanced PAA sensitivity of the resulting ␣ 1A /␣ 1S chimera to the level of L-type ␣ 1 subunits (12).The efficacy of the BTZ diltiazem as an antiarrhythmic and antihypertensive drug is due to its voltage-and use-dependent block of L-type Ca 2ϩ channels (14). Studies on cloned ␣ 1 subunits of different Ca 2ϩ channel classes (C, B, A, and E) have enabled a more precise characterization of their pharmacological features (15). To identify the molecular determinants of the high affinity BTZ interaction domain of L-type Ca 2ϩ channels, we introduced corresponding L-type sequence stretches into ␣ 1A . The diltiazem sensitivity of the resulting ␣ 1 chimeras was measured as use-dependent barium current (I Ba ) block after coexpression with ␤ 1a (16) and ␣ 2 /␦ (17) in Xenopus oocytes. EXPERIMENTAL PROCEDURESMolecular Biology-The construction of L-type chimera Lh (repeats I-IV from ␣ 1C-a (18)) with the N terminus replaced with ␣ 1S (19), as well as construction of chimeras AL12h and AL22, were described previously (9, 12). Chimera AL20 was generated by replacing the ClaI (nucleotide position, 4925)-XbaI (3Ј-polylinker) fragment of AL9 (9) by ...