Gating defects of disease-causing de novo mutations in Ca_v1.3 Ca²⁺ channels

Abstract

Recently, we and others identified somatic and germline de novo gain-of-function mutations in CACNA1D, the gene encoding the α1-subunit of voltage-gated Cav1.3 Ca2+-channels. While somatic mutations identified in aldosterone producing adenomas (APAs) underlie treatment-resistant hypertension, germline CACNA1D mutations are associated with a neurodevelopmental disorder characterized by a wide symptomatic spectrum, including autism spectrum disorder. The number of newly identified CACNA1D missense mutations is constantly growing, but their pathogenic potential is difficult to predict in silico, making functional studies indispensable to assess their contribution to disease risk.Here we report the functional characterization of previously identified CACNA1D APA mutations F747L and M1354I using whole-cell patch-clamp electrophysiology upon recombinant expression in tsA-201 cells. We also investigated if alternative splicing of Cav1.3 affects the aberrant gating of the previously characterized APA mutation R990H and two mutations associated with autism spectrum disorder (A479G and G407R). Splice-variant dependent gating changes are of particular interest for germline mutations, since the relative expression of Cav1.3 splice variants differs across different tissues and within brain regions and might therefore result in tissue-specific phenotypes. Our data revealed a complex gain-of-function phenotype for APA mutation F747L confirming its pathogenic role. Furthermore, we found splice-variant dependent gating changes in R990H, A749G and G407R. M1354I did not change channel function of Cav1.3 splice variants and should therefore be considered a rare non-pathogenic variant until further proof for its pathogenicity is obtained. Our new findings together with previously published data allow classification of pathogenic CACNA1D mutations into four categories based on prototypical functional changes.

Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder

“…For example, in neurons firing from more depolarized membrane potentials, the negative shift in steady-state inactivation may also reduce the availability of Cav1.3 channels. S652L adds to another six de novo missense mutations functionally characterized so far by us [14][15][16] and Fig. 8 Mutation S652L shows higher isradipine sensitivity.…”

“…In this study, we provide compelling evidence for the CACNA1D S652L variant as a high-risk and likely disease-causing mutation in two individuals of the Deciphering Developmental Disorders cohort (decipher.sanger.ac.uk) of children with severe, undiagnosed developmental disorders [23]. This evidence builds on a detailed biophysical characterization that demonstrates gating changes able to also induce enhanced channel activity as is typical for six other de novo germline missense mutations in patients with ASD with and without other neurodevelopmental symptoms [14][15][16][17][18]20]. Our data adds the CACNA1D gene to the other 12 developmental-disorder-linked genes identified in the Deciphering Developmental Disorders study and thus further increases its diagnostic yield.…”

“…For a detailed biophysical characterization in tsA-201 cells we introduced this mutation into the biophysically distinct C-terminally long (WT L , S652L L ) and short (WT S , S652L S ) Cav1.3 splice variants [26]. Both splice variants are abundantly expressed in the brain and differ considerably with respect to their biophysical properties, with higher voltage-sensitivity and faster Ca 2+dependent inactivation of WT S [3,16]. Mutant α 1 -subunit proteins were expressed as intact proteins with the expected molecular mass (Additional file 1: Figure S1).…”

“…We [14][15][16] and others [17][18][19][20] have recently provided accumulating evidence that de novo missense mutations in the pore-forming α 1 -subunit of Cav1.3 LTCCs (CACNA1D) confer high risk for neurodevelopmental disorders in humans. Symptoms range from ASD with (mutations A749G, Q547H [14,20];) and without (G407R [14]) intellectual disability to more severely affected patients with seizures, muscle hypotonia, and global developmental delay (V401L [15]).…”

“…Moreover, electrophysiological analysis of six mutations (from seven of these patients) after expression in HEK-293 cells revealed a highly consistent pattern of functional changes: they all induce gating changes that can enhance Cav1.3 Ca 2+ -current through these channels, in particular at subthreshold voltages. This gain-of-function is evident from a drastic slowing of channel inactivation and/ or by facilitation of channel opening at more negative voltages [16]. Therefore, the demonstration of such typical gain-of-function gating changes in functional studies may allow to distinguish likely pathogenic from nonpathogenic CACNA1D missense variants and help in the genetic diagnosis of individuals with neurodevelopmental disorders.…”