CACANA1A mutations causing episodic and progressive ataxia alter channel trafficking and kinetics

Department of Neurology, University of California at Los Angeles, 710 Westwood Plaza, Los Angeles, CA 90095-1769, USA.
Neurology (Impact Factor: 8.29). 07/2005; 64(12):2090-7. DOI: 10.1212/01.WNL.0000167409.59089.C0
Source: PubMed


CACNA1A encodes CaV2.1, the pore-forming subunit of P/Q-type voltage-gated calcium channel complexes. Mutations in CACNA1A cause a wide range of neurologic disturbances variably associated with cerebellar degeneration. Functional studies to date focus on electrophysiologic defects that do not adequately explain the phenotypic findings.
To investigate whether some missense mutations might interfere with protein folding and trafficking, eventually leading to protein aggregation and neuronal injury.
The authors studied the functional consequences of two pore missense mutations, C287Y and G293R, in two families with EA2, one newly discovered and the other previously reported. Both mutations caused episodic and interictal ataxia. The biophysical properties of mutant and wild type calcium channels were examined by whole-cell patch-clamp recordings in transfected COS-7 cells. The plasma membrane targeting was visualized by confocal fluorescence imaging on CaV2.1 tagged with green fluorescent protein.
The mutant channels exhibited a marked reduction in current expression and deficiencies in plasma membrane targeting.
In addition to altered channel function, the deficiency in protein misfolding and trafficking associated with the C287Y and G293R mutants may contribute to the slowly progressive cerebellar ataxia.

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    • "Under in vitro conditions, lowering of the incubation temperature to 26°C or 27°C partially corrected the trafficking defects of numerous disease-related mutant channels [22], [36], [37], giving rise to detectable ionic currents and mature proteins in electrophysiological and immunoblotting analyses, respectively. The mechanism of low temperature effects is not well understood and may involve improved protein folding, inhibition of proteasomal degradation, or increased surface trafficking. "
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    ABSTRACT: Myotonia congenita is a hereditary muscle disorder caused by mutations in the human voltage-gated chloride (Cl(-)) channel CLC-1. Myotonia congenita can be inherited in an autosomal recessive (Becker type) or dominant (Thomsen type) fashion. One hypothesis for myotonia congenita is that the inheritance pattern of the disease is determined by the functional consequence of the mutation on the gating of CLC-1 channels. Several disease-related mutations, however, have been shown to yield functional CLC-1 channels with no detectable gating defects. In this study, we have functionally and biochemically characterized a myotonia mutant: A531V. Despite a gating property similar to that of wild-type (WT) channels, the mutant CLC-1 channel displayed a diminished whole-cell current density and a reduction in the total protein expression level. Our biochemical analyses further demonstrated that the reduced expression of A531V can be largely attributed to an enhanced proteasomal degradation as well as a defect in protein trafficking to surface membranes. Moreover, the A531V mutant protein also appeared to be associated with excessive endosomal-lysosomal degradation. Neither the reduced protein expression nor the diminished current density was rescued by incubating A531V-expressing cells at 27°C. These results demonstrate that the molecular pathophysiology of A531V does not involve anomalous channel gating, but rather a disruption of the balance between the synthesis and degradation of the CLC-1 channel protein.
    Full-text · Article · Feb 2013 · PLoS ONE
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    • "ther neurodegenerative diseases . Defec - tive protein trafficking has emerged as an important mechanism in many inheritable diseases ( Kim and Arvan , 1998 ) , and this applies to various channelopathies , including cystic fibrosis ( Gelman and Kopito , 2003 ) and long QT2 syndrome ( Delisle et al . , 2004 ) , as well as calcium channelopathies ( Wan et al . , 2005 ; Jeng et al . , 2008 ) . Within the Ca v 2 . 1 subunit , the ER quality con - trol machinery may recognize many different regions ( i . e . , trans - membrane domains and loops ) . At first glance , because the I - II loop contains multiple ER retention motifs ( Bichet et al . , 2000 ) , it is tempting to speculate that a misfolded I -"
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    ABSTRACT: Channelopathies are often linked to defective protein folding and trafficking. Among them, the calcium channelopathy episodic ataxia type-2 (EA2) is an autosomal dominant disorder related to mutations in the pore-forming Ca(v)2.1 subunit of P/Q-type calcium channels. Although EA2 is linked to loss of Ca(v)2.1 channel activity, the molecular mechanism underlying dominant inheritance remains unclear. Here, we show that EA2 mutants as well as a truncated form (D(I-II)) of the Ca(v)3.2 subunit of T-type calcium channel are misfolded, retained in the endoplasmic reticulum, and subject to proteasomal degradation. Pulse-chase experiments revealed that misfolded mutants bind to nascent wild-type Ca(v) subunits and induce their subsequent degradation, thereby abolishing channel activity. We conclude that this destructive interaction mechanism promoted by Ca(v) mutants is likely to occur in EA2 and in other inherited dominant channelopathies.
    Full-text · Article · May 2008 · The Journal of Neuroscience : The Official Journal of the Society for Neuroscience
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    • "More recently, it was reported that the suppressive effects of EA2 truncations on exogenous Ca v 2.1 currents from wild-type α 1 2.1 subunits were significantly alleviated by increasing the availability of β subunits (Jeng et al., 2006). However, the same approach failed to even modestly reverse the dominant-negative effects exerted by EA2 missense mutants (Jeng et al., 2006), which in addition to hypo-conduction exhibit defective intracellular trafficking (Wan et al., 2005b). These latter findings suggest that α 1 2.1 missense mutants may be more pathogenic than truncations. "
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    ABSTRACT: Episodic ataxia type 2 (EA2) is an autosomal dominant disorder arising from CACNA1A mutations, which commonly predict heterozygous expression of Ca(v)2.1 calcium channels with truncated alpha(1)2.1 pore subunits. We hypothesized that alpha(1)2.1 truncations in EA2 exert dominant-negative effects on the function of wild-type subunits. Wild-type and truncated alpha(1)2.1 subunits with fluorescent protein tags were transiently co-expressed in cells stably expressing Ca(v) auxiliary beta subunits, which facilitate alpha1 subunit functional expression through high-affinity interactions with the alpha interaction domain (AID). Co-expression of wild-type subunits with truncations often resulted in severely reduced whole-cell currents compared to expression of wild-type subunits alone. Cellular image analyses revealed that current suppression was not due to reduced wild-type expression levels. Instead, the current suppression depended on truncations terminating distal to the AID. Moreover, only AID-bearing alpha(1)2.1 proteins co-immunoprecipitated with Ca(v) beta subunits. These results indicate that Ca(v) beta subunits may play a prominent role in EA2 disease pathogenesis.
    Preview · Article · Mar 2007 · Molecular and Cellular Neuroscience
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