A Destructive Interaction Mechanism Accounts for Dominant-Negative Effects of Misfolded Mutants of Voltage-Gated Calcium Channels

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.

Dramatically different levels of cacna1a gene expression between pre-weaning wild type and leaner mice

“…A dominant negative effect of EA2 mutant Ca V 2.1 α-subunit on normal protein has been demonstrated by double transfection methods for mutations producing PTCs as well [6,[11][12][13][14]. However, the present results on leaner mice and the similar phenotype of cacna1a null, knockout and knockdown mice [20][21][22] would indicate that also a haploinsufficiency mechanism can cause ataxia.…”

“…EA2 is an early onset, autosomal dominant disorder characterised by attacks of vertigo/ataxia, visual disturbance, dysarthria, interictal cerebellar deficit of extremely variable severity, and cerebellar atrophy; epilepsy has occasionally been described [9,10]. A dominant negative effect of the Ca V 2.1 mutated protein affecting the wild type product, rather than a haploinsufficiency, has been hypothesised [6,[11][12][13][14]. Loss of function mutations of the mouse orthologue of the CACNA1A gene cause recessive neurological phenotypes, in tottering (cacna1a tg ; tg), leaner (cacna1a tg-la ; tg-la), rocker (cacna1a rkr ) and rolling Nagoya (cacna1a tg-rol ) mice [15,16].…”

Mutations in potassium channel kcnd3 cause spinocerebellar ataxia type 19

“…27 We therefore assessed whether the SCA19 missense mutations (T352P, M373I, and S390N) would also affect the subcellular localization/maturation of Kv4.3. Confocal microscopic analysis of transiently transfected HeLa cells revealed that, unlike wild-type Kv4.3 that was mainly located on the plasma membrane (74.5 6 3.5%; Supplementary Fig 2), all 3 mutant proteins showed almost no or markedly reduced cell surface expression (T352P, 4.9 6 0.6%; M373I, 40.4 6 6.7%; S390N, 5.0 6 2.7%; Supplementary Fig 2), but rather accumulated in perinuclear foci (Fig 3A).…”

The Physiology, Pathology, and Pharmacology of Voltage-Gated Calcium Channels and Their Future Therapeutic Potential

“…These mutations include missense mutations, splice site mutations, and frame shifts the lead to the truncation of the Ca V 2.1 protein junctions and typically lead loss of channel function (Wappl et al, 2002;Kipfer et al, 2013). In addition, dominant negative effects of mutated channels on normal Ca V 2.1 channels have been reported (Jouvenceau et al, 2001;Jeng et al, 2008;Mezghrani et al, 2008;Page et al, 2010). Consistent with what has been observed with Ca V 2.1 null mice, loss of function of Ca V 2.1 due to a premature truncation of the protein has been shown to give rise to absence seizures in one patient with episodic ataxia (Jouvenceau et al, 2001).…”