The Timothy syndrome mutation differentially affects voltage- and calcium-dependent inactivation of CaV1.2 L-type calcium channels

ArticleinProceedings of the National Academy of Sciences 105(6):2157-62 · March 2008with10 Reads
DOI: 10.1073/pnas.0710501105 · Source: PubMed
Calcium entry into excitable cells is an important physiological signal, supported by and highly sensitive to the activity of voltage-gated Ca2+ channels. After membrane depolarization, Ca2+ channels first open but then undergo various forms of negative feedback regulation including voltage- and calcium-dependent inactivation (VDI and CDI, respectively). Inactivation of Ca2+ channel activity is perturbed in a rare yet devastating disorder known as Timothy syndrome (TS), whose features include autism or autism spectrum disorder along with severe cardiac arrhythmia and developmental abnormalities. Most cases of TS arise from a sporadic single nucleotide change that generates a mutation (G406R) in the pore-forming subunit of the L-type Ca2+ channel Ca(V)1.2. We found that the TS mutation powerfully and selectively slows VDI while sparing or possibly speeding the kinetics of CDI. The deceleration of VDI was observed when the L-type channels were expressed with beta1 subunits prominent in brain, as well as beta2 subunits of importance for the heart. Dissociation of VDI and CDI was further substantiated by measurements of Ca2+ channel gating currents and by analysis of another channel mutation (I1624A) that hastens VDI, acting upstream of the step involving Gly406. As highlighted by the TS mutation, CDI does not proceed to completeness but levels off at approximately 50%, consistent with a change in gating modes and not an absorbing inactivation process. Thus, the TS mutation offers a unique perspective on mechanisms of inactivation as well as a promising starting point for exploring the underlying pathophysiology of autism.
    • "TS is a multi-organ disorder caused by a single mutation, G406R (TS mutation ), of an alternatively spliced human Ca V 1.2 calcium channel containing exon 8a [14]. This TS mutation can lead to lethal arrhythmias, which are thought to be caused by impaired VDI [14, 15]. Our results showed that 6869DD and DDDD could enhance PLM-induced increases in VDI, so we speculate that it is possible that PLM phosphorylation might restore TS-impaired VDI. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Phospholemman (PLM) is an important phosphorylation substrate for protein kinases A and C in the heart. Until now, the association between PLM phosphorylation status and L-type calcium channels (LTCCs) gating has not been fully understood. We investigated the kinetics of LTCCs in HEK 293T cells expressing phosphomimetic or nonphosphorylatable PLM mutants. Methods: The LTCCs gating was measured in HEK 293T cells transfected with LTCC and wild-type (WT) PLM, phosphomimetic or nonphosphorylatable PLM mutants: 6263AA, 6869AA, AAAA, 6263DD, 6869DD or DDDD. Results: WT PLM significantly slowed LTCCs activation and deactivation while enhanced voltage-dependent inactivation (VDI). PLM mutants 6869DD and DDDD significantly increased the peak of the currents. 6263DD accelerated channel activation, while 6263AA slowed it more than WT PLM. 6869DD significantly enhanced PLM-induced increase of VDI. AAAA slowed the channel activation more than 6263AA, and DDDD accelerated the channel VDI more than 6869DD. Conclusions: Our results demonstrate that phosphomimetic PLM could stimulate LTCCs and alter their dynamics, while PLM nonphosphorylatable mutant produced the opposite effects. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.
    Full-text · Article · Feb 2015
    • "The consequences are prolonged action potentials and delayed after- depolarizations [6]. Single channel analysis of both kinds of TS mutants revealed, that they opened longer and more frequently [24,25], mainly resulting from slowed and decreased voltage-dependent inactivation [26]. Gain-of-function mutations in Cav1.2 were for a long time exclusively linked to TS [6,14]. "
    [Show abstract] [Hide abstract] ABSTRACT: Gain-of-function mutations in CACNA1C, encoding the L-type Ca(2+) channel Cav1.2, cause Timothy syndrome (TS), a multi-systemic disorder with dysmorphic features, long-QT syndrome (LQTS) and autism spectrum disorders. TS patients have heterozygous mutations (G402S and G406R) located in the alternatively spliced exon 8, causing a gain-of-function by reduced voltage-dependence of inactivation. Screening 540 unrelated patients with non-syndromic forms of LQTS, we identified six functional relevant CACNA1C mutations in different regions of the channel. All these mutations caused a gain-of-function combining different mechanisms, including changes in current amplitude, rate of inactivation and voltage-dependence of activation or inactivation, similar as in TS. Computer simulations support the theory that the novel CACNA1C mutations prolong action potential duration. We conclude that genotype-negative LQTS patients should be investigated for mutations in CACNA1C, as a gain-of-function in Cav1.2 is likely to cause LQTS and only specific and rare mutations, i.e. in exon 8, cause the multi-systemic TS. Copyright © 2015 International Society for Heart Research. Published by Elsevier Ltd. All rights reserved.
    Full-text · Article · Jan 2015
    • "402; Splawski et al., 2004, 2005). These mutations profoundly impair voltage-dependent inactivation of I CaL (VDI), and this is generally accepted to be the proximal molecular dysfunction associated with the disease (Barrett and Tsien, 2008). The cardiac signatures of TS include a range of cardiac structural defects and markedly prolonged ventricular repolarization (Splawski et al., 2004, 2005). "
    [Show abstract] [Hide abstract] ABSTRACT: Calcium/calmodulin-dependent protein kinase II (CaMKII) activity has been shown to contribute to arrhythmogenesis in a remarkably broad range of cardiac pathologies. Several of these involve significant structural and electrophysiologic remodeling, whereas others are due to specific channelopathies, and are not typically associated with arrhythmogenic changes to protein expression or cellular and tissue structure. The ability of CaMKII to contribute to arrhythmia across such a broad range of phenotypes suggests one of two interpretations regarding the role of CaMKII in cardiac arrhythmia: (1) some CaMKII-dependent mechanism is a common driver of arrhythmia irrespective of the specific etiology of the disease, or (2) these different etiologies expose different mechanisms by which CaMKII is capable of promoting arrhythmia. In this review, we dissect the available mechanistic evidence to explore these two possibilities and discuss how the various molecular actions of CaMKII promote arrhythmia in different pathophysiologic contexts.
    Full-text · Article · May 2014
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