Functional Consequences of P/Q-type Ca2+Channel Cav2.1 Missense Mutations Associated with Episodic Ataxia Type 2 and Progressive Ataxia

Abstract

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 ...

Ubiquitin Ligase RNF138 Promotes Episodic Ataxia Type 2-Associated Aberrant Degradation of Human Cav2.1 (P/Q-Type) Calcium Channels

“…The cDNAs for Ca 2ϩ channel ␣ 1 subunits used in the study include human long-isoform ␣ 1A (Ca V 2.1; AF004884; kindly provided by Dr. Joanna Jen, UCLA; , human short-isoform ␣ 1A (Ca V 2.1-Short; AF004883; kindly provided by Dr. Jörg Striessnig, University of Innsbruck; Wappl et al, 2002), rat ␣ 1C (Ca V 1.2; M67515; kindly provided by Dr. Gerald Obermair, Medical University of Innsbruck; Obermair et al, 2004), and bovine ␣ 1B (Ca V 2.2; AF173882; kindly provided by Dr. Aaron Fox, University of Chicago, and Dr. Chien-Yuan Pan, National Taiwan University; Cahill et al, 2000). Myc-tagged (C terminus) Ca V 2.1 constructs (Ca V 2.1-6myc) was created by subcloning Ca V 2.1 cDNA into a modified pcDNA3 vector (Invitrogen) with six repeats of Myc sequences right before the stop codon.…”

“…Previous experimental evidence clearly demonstrates that the EA2-causing human Ca V 2.1 nonsense (truncation) mutant R1281X and the missense mutant F1406C display prominent loss-offunction phenotypes Wappl et al, 2002;Jeng et al, 2006). The Ca V 2.1 R1281X mutation is caused by a premature stop codon in the extracellular loop linking transmembrane S1-S2 segments of domain III, whereas Ca V 2.1 F1406C involves a point mutation at the pore-loop region of domain III.…”

“…Rat Ca V 2.1 protein detection was prevented by preabsorbing the immunoblot with a control antigen peptide. Endogenous Ca V 2.1 in the rat brain comprises a major isoform with an apparent molecular weight of ϳ190 kDa (labeled with black arrows), as well as a minor isoform of ϳ220 kDa (Sakurai et al, 1995;Westenbroek et al, 1995). Bottom, Coimmunoprecipitation of endogenous RNF138 with Ca V 2.1.…”

Familial hemiplegic migraine mutations increase Ca 2+ influx through single human Ca V 2.1 channels and decrease maximal Ca V 2.1 current density in neurons

“…Finally, the SplI-KpnI (nucleotides 3039-4450) fragment of this new subclone was ligated into the corresponding sites of Ca V 2.1␣ 1 in pGFP Ϫ (21). Cells transfected with ␣ 1A-2 were incubated at 28°C for 14-20 h before recording, because this procedure increased expression (18).…”

Silver nanoparticle exposure induces rat motor dysfunction through decrease in expression of calcium channel protein in cerebellum

“…As one of the crucial proteins, voltage-gated calcium ion channel (Cav2.1 channel) is involved in neurotransmission at central synapses. Loss-of-function CACNA1A gene mutants often showed early-onset motor dysfunction associated with distinct alterations of Ca 2+ channel properties, and impaired function of the cerebellar calcium channel Cav2.1 might have a central role in the pathogenesis of certain cases of ataxia (Mori et al, 2000;Wappl et al, 2002). Some types of ataxia, such as spinocerebellar ataxia type 6 were associated with decreased expression or impaired function of CACNA1A protein (Watase et al, 2008).…”