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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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Functional consequences of A749G and G407R expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and G407R and G407R mutants coexpressed together with wild-type (WT + G407R, 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 (n = 4) to account for differences in expression levels between experiments. Transfections with high expression levels of wild-type were selected for analysis to provide sufficient current amplitudes for G407R channels. Peak current amplitudes of G407R mutants and G407R cotransfected with wild-type were significantly reduced (mean ± SEM [pA/pF]; wild-type, −24.6 ± 4.18, n = 15; G407R, −8.12 ± .70, n = 13; WT + G407R, −7.63 ± .87, n = 9; both p < .0001 vs. wild-type, Mann-Whitney test). Statistics for gating parameters obtained from all transfections are summarized in Table 1.(B) Inactivation of wild-type, A749G, and G407R mutants during 5-sec depolarizations from a holding potential of −80 mV to maximum voltage. Peak inward calcium currents for overlapping wild-type and A749G traces are indicated by arrows for clarity. Traces represent the means ± SEM (only ± SEM is illustrated) from parallel recordings of wild-type and mutant channels performed on the same day in four (G407R) or two (A749G) independent transfections. The pronounced slowing of inactivation is evident from the respective normalized current traces shown in the inset. See text for numbers and % current persisting after 5-sec depolarizations. WT, wild-type.
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f0010: Functional consequences of A749G and G407R expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and G407R and G407R mutants coexpressed together with wild-type (WT + G407R, 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 (n = 4) to account for differences in expression levels between experiments. Transfections with high expression levels of wild-type were selected for analysis to provide sufficient current amplitudes for G407R channels. Peak current amplitudes of G407R mutants and G407R cotransfected with wild-type were significantly reduced (mean ± SEM [pA/pF]; wild-type, −24.6 ± 4.18, n = 15; G407R, −8.12 ± .70, n = 13; WT + G407R, −7.63 ± .87, n = 9; both p < .0001 vs. wild-type, Mann-Whitney test). Statistics for gating parameters obtained from all transfections are summarized in Table 1.(B) Inactivation of wild-type, A749G, and G407R mutants during 5-sec depolarizations from a holding potential of −80 mV to maximum voltage. Peak inward calcium currents for overlapping wild-type and A749G traces are indicated by arrows for clarity. Traces represent the means ± SEM (only ± SEM is illustrated) from parallel recordings of wild-type and mutant channels performed on the same day in four (G407R) or two (A749G) independent transfections. The pronounced slowing of inactivation is evident from the respective normalized current traces shown in the inset. See text for numbers and % current persisting after 5-sec depolarizations. WT, wild-type.

Mentions: Depolarizations to the reversal potential revealed an increased ratio of maximal tail current amplitude to integrated QON (Itail/QON [msec−1]: wild-type, 11.1 ± 1.1, n = 28; A749G, 34.1 ± 2.25, n = 20; p < .0001 vs. wild-type, unpaired Student t test). This finding is compatible with a higher channel open probability or conductance or both, a feature previously observed by us also for other Cav1.3 gain-of-function mutations (22). In contrast to A749G, mutation G407R reduced maximal current amplitudes (Figure 2A) and caused no change in activation voltage dependence (Figure 2A and Table 1). However, in contrast to A749G, G407R dramatically slowed the inactivation time course during 5-sec depolarizations. At the end of the 5-sec pulse to Vmax, only 5.12% ± .98% (n = 15) of maximal wild-type and 3.07% ± .37% (n = 6) of A749G current remained, whereas 82.8% ± .04% (n = 13) of G407R current persisted (p < .001 vs. wild-type, Mann-Whitney test) (Figure 2B, inset). The failure of G407R currents to inactivate prevented the measurement of steady-state inactivation parameters. Despite reduced maximal current amplitudes (Figure 2A), the slow inactivation resulted in larger absolute current amplitudes during prolonged depolarization than in wild-type Cav1.3 channels (Figure 2B). The absence of a fast inactivating component (mediated by calcium-dependent inactivation in Cav1.3) (9,36) suggests that calcium-dependent and voltage-dependent inactivation were strongly weakened by the mutation. The smaller current and QON amplitudes were unlikely because of a lower expression of mutant channel protein as demonstrated by Western blots (Figure S2 in Supplement 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)

Functional consequences of A749G and G407R expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and G407R and G407R mutants coexpressed together with wild-type (WT + G407R, 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 (n = 4) to account for differences in expression levels between experiments. Transfections with high expression levels of wild-type were selected for analysis to provide sufficient current amplitudes for G407R channels. Peak current amplitudes of G407R mutants and G407R cotransfected with wild-type were significantly reduced (mean ± SEM [pA/pF]; wild-type, −24.6 ± 4.18, n = 15; G407R, −8.12 ± .70, n = 13; WT + G407R, −7.63 ± .87, n = 9; both p < .0001 vs. wild-type, Mann-Whitney test). Statistics for gating parameters obtained from all transfections are summarized in Table 1.(B) Inactivation of wild-type, A749G, and G407R mutants during 5-sec depolarizations from a holding potential of −80 mV to maximum voltage. Peak inward calcium currents for overlapping wild-type and A749G traces are indicated by arrows for clarity. Traces represent the means ± SEM (only ± SEM is illustrated) from parallel recordings of wild-type and mutant channels performed on the same day in four (G407R) or two (A749G) independent transfections. The pronounced slowing of inactivation is evident from the respective normalized current traces shown in the inset. See text for numbers and % current persisting after 5-sec depolarizations. WT, wild-type.
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Related In: Results  -  Collection

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f0010: Functional consequences of A749G and G407R expressed in tsA-201 cells. (A) Calcium current voltage relationships for human wild-type and G407R and G407R mutants coexpressed together with wild-type (WT + G407R, 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 (n = 4) to account for differences in expression levels between experiments. Transfections with high expression levels of wild-type were selected for analysis to provide sufficient current amplitudes for G407R channels. Peak current amplitudes of G407R mutants and G407R cotransfected with wild-type were significantly reduced (mean ± SEM [pA/pF]; wild-type, −24.6 ± 4.18, n = 15; G407R, −8.12 ± .70, n = 13; WT + G407R, −7.63 ± .87, n = 9; both p < .0001 vs. wild-type, Mann-Whitney test). Statistics for gating parameters obtained from all transfections are summarized in Table 1.(B) Inactivation of wild-type, A749G, and G407R mutants during 5-sec depolarizations from a holding potential of −80 mV to maximum voltage. Peak inward calcium currents for overlapping wild-type and A749G traces are indicated by arrows for clarity. Traces represent the means ± SEM (only ± SEM is illustrated) from parallel recordings of wild-type and mutant channels performed on the same day in four (G407R) or two (A749G) independent transfections. The pronounced slowing of inactivation is evident from the respective normalized current traces shown in the inset. See text for numbers and % current persisting after 5-sec depolarizations. WT, wild-type.
Mentions: Depolarizations to the reversal potential revealed an increased ratio of maximal tail current amplitude to integrated QON (Itail/QON [msec−1]: wild-type, 11.1 ± 1.1, n = 28; A749G, 34.1 ± 2.25, n = 20; p < .0001 vs. wild-type, unpaired Student t test). This finding is compatible with a higher channel open probability or conductance or both, a feature previously observed by us also for other Cav1.3 gain-of-function mutations (22). In contrast to A749G, mutation G407R reduced maximal current amplitudes (Figure 2A) and caused no change in activation voltage dependence (Figure 2A and Table 1). However, in contrast to A749G, G407R dramatically slowed the inactivation time course during 5-sec depolarizations. At the end of the 5-sec pulse to Vmax, only 5.12% ± .98% (n = 15) of maximal wild-type and 3.07% ± .37% (n = 6) of A749G current remained, whereas 82.8% ± .04% (n = 13) of G407R current persisted (p < .001 vs. wild-type, Mann-Whitney test) (Figure 2B, inset). The failure of G407R currents to inactivate prevented the measurement of steady-state inactivation parameters. Despite reduced maximal current amplitudes (Figure 2A), the slow inactivation resulted in larger absolute current amplitudes during prolonged depolarization than in wild-type Cav1.3 channels (Figure 2B). The absence of a fast inactivating component (mediated by calcium-dependent inactivation in Cav1.3) (9,36) suggests that calcium-dependent and voltage-dependent inactivation were strongly weakened by the mutation. The smaller current and QON amplitudes were unlikely because of a lower expression of mutant channel protein as demonstrated by Western blots (Figure S2 in Supplement 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