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CACNA1D de novo mutations in autism spectrum disorders activate Cav1.3 L-type calcium channels.

Pinggera A, Lieb A, Benedetti B, Lampert M, Monteleone S, Liedl KR, Tuluc P, Striessnig J - Biol. Psychiatry (2014)

Bottom Line: In both cases, these changes are compatible with a gain-of-function phenotype.Our findings have immediate clinical relevance because blockers of LTCCs are available for therapeutic attempts in affected individuals.Patients should also be explored for other symptoms likely resulting from Cav1.3 hyperactivity, in particular, primary aldosteronism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria.

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Biophysical properties of A749G expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and A749G and A749G mutants coexpressed together with wild-type (WT + A749G, equal amounts of complementary DNA transfected for both constructs) in tsA-201 cells as described in Methods and Materials. Sample traces of inward calcium currents measured during depolarizations to maximum voltage are also shown. Current-voltage curves include only data for wild-type channels pooled from parallel recordings with mutants in the same transfections (six independent transfections) to account for differences in expression levels between transfections. A749G cotransfected with wild-type (WT + A749G) resulted in significantly increased peak current amplitudes (for statistics and numbers, see Results). Statistics for gating parameters are summarized in Table 1. (B) Steady-state activation (circles) and inactivation (squares) curves for wild-type and A749G were obtained as described in Methods and Materials. Means ± SEM are illustrated. Wild-type, n = 29 (nine transfections); A749G, n = 27 (six transfections). Steady-state activation parameters for WT + A749G are given in Table 1.
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f0005: Biophysical properties of A749G expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and A749G and A749G mutants coexpressed together with wild-type (WT + A749G, equal amounts of complementary DNA transfected for both constructs) in tsA-201 cells as described in Methods and Materials. Sample traces of inward calcium currents measured during depolarizations to maximum voltage are also shown. Current-voltage curves include only data for wild-type channels pooled from parallel recordings with mutants in the same transfections (six independent transfections) to account for differences in expression levels between transfections. A749G cotransfected with wild-type (WT + A749G) resulted in significantly increased peak current amplitudes (for statistics and numbers, see Results). Statistics for gating parameters are summarized in Table 1. (B) Steady-state activation (circles) and inactivation (squares) curves for wild-type and A749G were obtained as described in Methods and Materials. Means ± SEM are illustrated. Wild-type, n = 29 (nine transfections); A749G, n = 27 (six transfections). Steady-state activation parameters for WT + A749G are given in Table 1.

Mentions: Data from previous mutational studies in Cav1 α1-subunits (33,34) strongly suggested interference of both mutations with Cav1.3 LTCC function. G407R (present in exon 8a, one of two alternative exons) is identical to a Timothy syndrome mutation in Cav1.2 α1-subunits (17). In addition, A749G is located adjacent to p.I750M (I750M), a Cav1.3 mutation for which we reported a pronounced gain-of-function (22). Analysis of the two mutations in a homology model of the Cav1.3 α1-subunit also predicted pronounced changes of the interaction of the affected distal S6 helices with adjacent S6 helices of the activation gate (G407R, A749G) and of the S4–S5 linker with the voltage sensor (G407R) (Figure S1 in Supplement 1). This analysis prompted us to introduce both mutations into human Cav1.3 α1-subunits and express them in tsA-201 cells together with α2δ1 and β3 accessory subunits, which form most LTCC complexes in the brain (35). Western blots revealed robust expression of intact α1-subunits, although slightly higher and lower expression of the G407R and A749G mutant proteins were observed, respectively (Figure S2 in Supplement 1). Both mutations strongly affected channel gating (Figures 1 and 2). A749G significantly enhanced peak current amplitudes (wild-type, −11.6 ± 2.2 pA/pF, n = 17; A749G, −30.5 ± 5.8 pA/pF, n = 27; p < .0001, Mann-Whitney test; wild-type controls from same transfection experiments) (Figure 1A); this was not due to an increased surface expression estimated by quantification of the QON, which was significantly decreased in the mutant (QON [pA*ms] wild-type, 170 ± 26.8, n = 28; A749G, 80.5 ± 11.4, n = 20; p < .009). A749G also shifted steady-state activation and inactivation voltage dependence (Figure 1B) of inward calcium currents to more negative potentials (Table 1).


CACNA1D de novo mutations in autism spectrum disorders activate Cav1.3 L-type calcium channels.

Pinggera A, Lieb A, Benedetti B, Lampert M, Monteleone S, Liedl KR, Tuluc P, Striessnig J - Biol. Psychiatry (2014)

Biophysical properties of A749G expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and A749G and A749G mutants coexpressed together with wild-type (WT + A749G, equal amounts of complementary DNA transfected for both constructs) in tsA-201 cells as described in Methods and Materials. Sample traces of inward calcium currents measured during depolarizations to maximum voltage are also shown. Current-voltage curves include only data for wild-type channels pooled from parallel recordings with mutants in the same transfections (six independent transfections) to account for differences in expression levels between transfections. A749G cotransfected with wild-type (WT + A749G) resulted in significantly increased peak current amplitudes (for statistics and numbers, see Results). Statistics for gating parameters are summarized in Table 1. (B) Steady-state activation (circles) and inactivation (squares) curves for wild-type and A749G were obtained as described in Methods and Materials. Means ± SEM are illustrated. Wild-type, n = 29 (nine transfections); A749G, n = 27 (six transfections). Steady-state activation parameters for WT + A749G are given in Table 1.
© Copyright Policy - CC BY
Related In: Results  -  Collection

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f0005: Biophysical properties of A749G expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and A749G and A749G mutants coexpressed together with wild-type (WT + A749G, equal amounts of complementary DNA transfected for both constructs) in tsA-201 cells as described in Methods and Materials. Sample traces of inward calcium currents measured during depolarizations to maximum voltage are also shown. Current-voltage curves include only data for wild-type channels pooled from parallel recordings with mutants in the same transfections (six independent transfections) to account for differences in expression levels between transfections. A749G cotransfected with wild-type (WT + A749G) resulted in significantly increased peak current amplitudes (for statistics and numbers, see Results). Statistics for gating parameters are summarized in Table 1. (B) Steady-state activation (circles) and inactivation (squares) curves for wild-type and A749G were obtained as described in Methods and Materials. Means ± SEM are illustrated. Wild-type, n = 29 (nine transfections); A749G, n = 27 (six transfections). Steady-state activation parameters for WT + A749G are given in Table 1.
Mentions: Data from previous mutational studies in Cav1 α1-subunits (33,34) strongly suggested interference of both mutations with Cav1.3 LTCC function. G407R (present in exon 8a, one of two alternative exons) is identical to a Timothy syndrome mutation in Cav1.2 α1-subunits (17). In addition, A749G is located adjacent to p.I750M (I750M), a Cav1.3 mutation for which we reported a pronounced gain-of-function (22). Analysis of the two mutations in a homology model of the Cav1.3 α1-subunit also predicted pronounced changes of the interaction of the affected distal S6 helices with adjacent S6 helices of the activation gate (G407R, A749G) and of the S4–S5 linker with the voltage sensor (G407R) (Figure S1 in Supplement 1). This analysis prompted us to introduce both mutations into human Cav1.3 α1-subunits and express them in tsA-201 cells together with α2δ1 and β3 accessory subunits, which form most LTCC complexes in the brain (35). Western blots revealed robust expression of intact α1-subunits, although slightly higher and lower expression of the G407R and A749G mutant proteins were observed, respectively (Figure S2 in Supplement 1). Both mutations strongly affected channel gating (Figures 1 and 2). A749G significantly enhanced peak current amplitudes (wild-type, −11.6 ± 2.2 pA/pF, n = 17; A749G, −30.5 ± 5.8 pA/pF, n = 27; p < .0001, Mann-Whitney test; wild-type controls from same transfection experiments) (Figure 1A); this was not due to an increased surface expression estimated by quantification of the QON, which was significantly decreased in the mutant (QON [pA*ms] wild-type, 170 ± 26.8, n = 28; A749G, 80.5 ± 11.4, n = 20; p < .009). A749G also shifted steady-state activation and inactivation voltage dependence (Figure 1B) of inward calcium currents to more negative potentials (Table 1).

Bottom Line: In both cases, these changes are compatible with a gain-of-function phenotype.Our findings have immediate clinical relevance because blockers of LTCCs are available for therapeutic attempts in affected individuals.Patients should also be explored for other symptoms likely resulting from Cav1.3 hyperactivity, in particular, primary aldosteronism.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria.

Show MeSH
Related in: MedlinePlus