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

Figures & data

Figure 1. Transmembrane topology of the Ca_v1.3 α1-subunit. The pore-forming subunit of voltage gated Ca²⁺ channels consist of four transmembrane repeats each comprising six membrane spanning helices connected by intra- and extracellular linkers. Segments (S) 1–4 of each repeat comprise the voltage- sensing domain, whereas segments 5–6 line the pore. The positions of missense mutations identified in ASD (G407R, A749G) and APAs (F747L, R990H, M1354I) are indicated. Mutations G407R (IS6), F747L (IIS6) and A749G (IIS6) reside within the so-called activation gate at the distal part of the S6 segment. Mutation R990H affects the third of the five gating charges within the IIIS4 helix of the voltage sensing domain and M1354I is localized at the distal part of segment IVS5 facing the extracellular side. For references see text.

Table 1. Activation and inactivation parameters for ASD mutations A749G and G407R.

	activation				inactivation
	Vrev [mV]	Slope [mV]	V0.5 [mV]	n	V0.5 [mV]	Slope [mV]	Remaining current [%]	n
WTS	64.9 ± 1.04	7.71 ± 0.11	−9.49 ± 0.42	66	−31.7 ± 0.88	4.31 ± 0.12	11.5 ± 2.18	25
AGS	54.7 ± 1.46***	6.61 ± 0.11***	−18.3 ± 1.34***	13	−48.6 ± 1.58***	4.27 ± 0.16	6.01 ± 1.50	9
GRS	61.6 ± 1.77	7.68 ± 0.28	−15.3 ± 2.20***	14	nd
WTL	67.7 ± 1.14	8.92 ± 0.20	−2.55 ± 1.05	29	25.7 ± 2.08	5.56 ± 0.23	14.4 ± 3.12	18
AGL	60.3 ± 0.83^###	7.14 ± 0.20^###	−12.3 ± 0.87^###	27	−41.1 ± 1.07^###	5.82 ± 0.18	9.43 ± 1.49	14
GRL	54.7 ± 2.32^###	7.90 ± 0.30	−6.58 ± 1.41	13	nd

All values are presented as mean ± S.E.M. Number of independent transfections >3. Parameters were obtained after fitting normalized (I/I_max) I-V relationships or normalized (I/I_max) steady-state inactivation curves, as described in Methods. Statistical analysis was performed using one-way ANOVA and Bonferroni post-hoc test (activation) or unpaired Student’s t-test (steady-state inactivation). Significance **^{, ##} p < 0.01, ***^{, ###} p < 0.001 in comparison to WT_S (*) or WT_L (^#) (statistical analysis has been performed separately for long and short splice variants). n, number of experiments; nd, not determined; V_0.5, half maximal activation/inactivation voltage; V_rev, reversal potential; WT, wild-type. Note small differences among WT recordings within different sets of experiments (Tables 1, 3 and 5) because WT control experiments were always carried out in parallel with mutated channels. Reference data, which were already published previously, are indicated in italics (taken from reference 10).

Table 2. Remaining current upon 5-s depolarization for ASD mutations A749G and G407R.

	R50 [%]	R500 [%]	R5000 [%]	n
WTS	41.1 ± 1.84	20.7 ± 2.05	13.5 ± 2.09	35
AGS	46.2 ± 3.92	11.8 ± 1.87	8.16 ± 1.84	9
GRS	73.6 ± 4.52***	62.6 ± 5.39***	59.9 ± 5.62***	8
WTL	69.5 ± 3.2	22.8 ± 2.42	6.12 ± 1.13	15
AGL	60.0 ± 4.31	7.22 ± 0.76^###	3.12 ± 0.56^###	6
GRL	99.2 ± 0.65^###	98.1 ± 1.33^###	80.2 ± 3.52^###	13

All values are presented as mean ± S.E.M. Number of independent transfections ≥2. I_Ca inactivation normalized to I_max was obtained 50, 500 and 5000 ms after depolarization to V_max. Statistical analysis was performed using one-way ANOVA and Bonferroni post-hoc test. Significance ***^{, ###} p < 0.001 in comparison to WT_S (*) or WT_L (^#). n, number of experiments; R, remaining current; WT, wild-type. Reference data, which were already published previously, are indicated in italics (taken from reference 10).

Table 3. Activation and inactivation parameters for APA mutations F747L and M1354I.

	activation				inactivation
	Vrev [mV]	Slope [mV]	V0.5 [mV]	n	V0.5 [mV]	Slope [mV]	Remaining current [%]	n
WTL	70.4 ± 0.67	9.06 ± 0.12	1.37 ± 0.60	63	−27.0 ± 0.69	5.54 ± 0.18	14.4 ± 2.02	28
FLL	53.8 ± 0.91***	6.62 ± 0.22***	−15.6 ± 0.71***	30	−29.3 ± 0.98*	4.45 ± 0.32**	32.2 ± 2.50***	19
MIL	69.5 ± 0.69	9.01 ± 0.13	2.48 ± 0.79	40	−25.0 ± 0.82	5.57 ± 0.22	19.6 ± 2.26	25
WTS	64.9 ± 1.04	7.71 ± 0.11	−9.49 ± 0.42	66	−31.7 ± 0.88	4.31 ± 0.12	11.5 ± 2.18	25
MIS	62.5 ± 1.36	7.36 ± 0.18	−9.01 ± 0.64	19	−30.8 ± 0.86	3.92 ± 0.18	13.8 ± 2.49	13

All values are presented as mean ± S.E.M. Number of independent transfections >3. Parameters were obtained after fitting normalized (I/I_max) I-V relationships or normalized (I/I_max) steady-state inactivation curves, as described in Methods. Statistical analysis was performed by one-way ANOVA and Bonferroni post-hoc test (long splice variants) or unpaired Student’s t-test (short splice variants). Significance * p < 0.05, ** p < 0.01, *** p < 0.001 in comparison to WT_L. n, number of experiments; V_0.5, half maximal activation/inactivation voltage; V_rev, reversal potential; WT, wild-type.

Table 4. Remaining current upon 5-s depolarization for APA mutations F747L and M1354I.

	R50 [%]	R500 [%]	R5000 [%]	n
WTL	71.7 ± 2.38	29.4 ± 2.11	9.83 ± 1.86	28
FLL	99.3 ± 0.83^###	63.4 ± 4.78^###	32.2 ± 3.52^###	10
MIL	69.9 ± 3.64	29.7 ± 3.81	16.1 ± 3.84	12
WTS	41.1 ± 1.84	20.7 ± 2.05	13.5 ± 2.09	35
MIS	44.2 ± 3.70	23.9 ± 3.99	18.4 ± 4.34	9

All values are presented as mean ± S.E.M. Number of independent transfections >3. I_Ca inactivation normalized to I_max was obtained 50, 500 and 5000 ms after depolarization to V_max. Statistical analysis was performed using one-way ANOVA and Bonferroni post-hoc test (long splice variants or unpaired Student’s t-test (short splice variants). Significance ^### p < 0.001 in comparison to WT_L. n, number of experiments; R, remaining current; WT, wild-type.

Table 5. Activation and inactivation parameters for APA mutation R990H.

	activation				inactivation
	Vrev [mV]	Slope [mV]	V0.5 [mV]	n	V0.5 [mV]	Slope [mV]	Remaining current [%]	n
WTS	64.9 ± 1.04	7.71 ± 0.11	−9.49 ± 0.42	66	−31.7 ± 0.88	4.31 ± 0.12	11.5 ± 2.18	25
RHS	60.0 ± 1.73	7.55 ± 0.19	−5.37 ± 0.63***	11	−37.3 ± 0.57**	5.26 ± 0.53*	3.92 ± 2.42	8
WTL	66.2 ± 1.10	9.02 ± 0.23	−0.65 ± 1.27	29	−30.8 ± 1.14	5.38 ± 0.30	11.3 ± 2.35	16
RHL	69.7 ± 1.57	8.57 ± 0.14	2.61 ± 0.69^#	33	−32.5 ± 1.12	6.76 ± 0.39^#	11.3 ± 2.05	18

All values are presented as mean ± S.E.M. Number of independent transfections >3. Parameters were obtained after fitting normalized (I/I_max) I-V relationships or normalized (I/I_max) steady-state inactivation curves, as described in Methods. Statistical analysis was performed by unpaired Student’s t-test. Significance *^{, #} p < 0.05, **^{, ##} p < 0.01, ***^{, ###} p < 0.001 in comparison to WT_S (*) or WT_L (^#). n, number of experiments; V_0.5, half maximal activation/inactivation voltage; V_rev, reversal potential; WT, wild-type. Reference data, which were already published previously, are indicated in italics (taken from reference 21).

Figure 2. Biophysical properties of Ca_v1.3 mutant A749G_S. (a) Current-voltage relationships (I_Ca, mean ± S.E.M.; 50-ms depolarizations to indicated voltages) of WT_S and A749G_S. Only WT data recorded in parallel on the same days (> 3- different transfections) were included. Inset (upper): Box plots of WT_S and A749G_S peak current densities. In contrast to the long splice variant, no significant difference was observed in the current amplitudes (median, 5/95 percentile, [pA/pF]): WT_S: −8.74 (−22.4/-5.54) n = 10; A749G_S: −6.18 (−26.4/-4.94), n = 12; p = 0.339, Mann Whitney test). Inset (lower): Representative I_Ca traces from WT_S and A749G_S during depolarizations to V_max. (b) Steady-state activation (circles, solid lines) and inactivation (squares, dashed lines) of WT_S (black symbols) and A749G_S (orange symbols). For statistics and n-numbers see Table 1. Lines without symbols represent the corresponding voltage-dependence of activation and inactivation for full length WT_L and A749G_L recorded under identical conditions (taken from reference 10). (c) Inactivation time course of WT_S (n = 35) and A749G_S (n = 9) during depolarization to V_max for 5 s. Normalized traces are shown as mean ± S.E.M. Inactivation was best described by a bi-exponential decay. The faster inactivation of the mutant resulted from a slightly larger contribution of the fast inactivating component (WT_S: 79.1 ± 1.11 %, n = 26, A749G_S: 84.3 ± 2.21 %, p = 0.036, n = 8) and faster inactivation of the slow component (WT_S: τ_slow = 602 ± 21.8 ms, A749G_S: 420 ± 59.6 ms, p = 0.0012), despite a moderately slower fast inactivation (WT_S: τ_fast = 36.5 ± 1.8 ms, A749G_S: 57.9 ± 7.99 ms, p = 0.0004). Additionally, inactivation was more complete in the mutant (remaining current: WT_S: 14.6 ± 2.5 %, A749G_S: 5.65 ± 1.19 %, p = 0.061, for statistics see Table 2). Statistical significance has been determined by unpaired Student’s t-test.

Figure 3. Biophysical properties of Ca_v1.3 mutant G407R_S. (a) Current-voltage relationships (I_Ca, mean ± S.E.M.) from WT_S and G407R_S recorded in parallel on the same days (>3-different transfections). Inset (upper): Box plots showing significantly reduced peak current densities for G407R_S (median (5/95 percentile) [pA/pF]: WT_S: −6.68 (−17.1/-3.09), n = 17; G407R_S: −2.82 (−4.36/-1.46), n = 13; *** p < 0.0001, Mann Whitney test). Inset (lower): Representative I_Ca traces of WT_S and G407R_S upon depolarization to V_max. (b) Voltage dependence of WT (black circles) and G407R_S (red circles) activation. Lines without symbols depict the corresponding voltage-dependence of activation for full length WT_L (black) and G407R_L (red) recorded under identical conditions (taken from reference 10). Statistics for gating parameters are given in Table 1. (c) Normalized inactivation time course of I_Ca after 5-s depolarization to V_max (mean ± S.E.M.). The fast inactivating component is reduced in G407R_S (n = 8) as compared to WT (n = 35) leading to slower inactivation of the mutant (for statistics see Table 2). (d) Although G407R_S maximal current density is significantly smaller than WT_S, absolute G407R_S current is larger than WT_S after about 200 ms.

Figure 4. Biophysical properties of Ca_v1.3 mutant F747L_L. (a) Current-voltage relationships (I_Ca, mean ± S.E.M.) of WT_L and F747L_L recorded in parallel on the same days (>3-different transfections). Inset (upper): Box plots of peak current densities (median, 5/95 percentile, [pA/pF]): WT_L: −12.7 (−66.1/-5.96), n = 47; F747L_L: −13.3 (−40.3/-3.97), n = 27). Black circles represent extreme values. Peak values were not statistically different (Mann Whitney test, p = 0.97). Inset (lower): Representative I_Ca traces of WT_L and F747L_L upon depolarization to V_max. Note the slower activation time course and the absence of Q_ON gating charge in F747L_L  (arrows). (b) Steady-state activation (circles, solid lines) and inactivation (squares, dashed lines) of WT_L (black symbols) and F747L_L (gray symbols). Gating parameters and statistics are given in Table 3. (c) Normalized inactivation time course of I_Ca after 5-s depolarization to V_max (mean ± S.E.M.) illustrating pronounced slowing of inactivation by F747_L.(n = 10) as compared to WT_L (n = 28). For statistics, see Table 4.

Table 6. Remaining current upon 5-s depolarization for APA mutation R990H.

	R50 [%]	R500 [%]	R5000 [%]	n
WTS	41.1 ± 1.84	20.7 ± 2.05	13.5 ± 2.09	35
RHS	46.6 ± 3.45	19.5 ± 3.73	15.1 ± 3.54	9
WTL	69.7 ± 3.94	24.8 ± 3.50	11.1 ± 2.94	10
RHL	67.1 ± 3.59	21.6 ± 3.75	13.6 ± 3.62	12

All values are presented as mean ± S.E.M. Number of independent transfections >3. I_Ca inactivation normalized to I_max was obtained 50, 500 and 5000 ms after depolarization to V_max. Statistical analysis was performed using unpaired Student’s t-test in comparison to WT_S or WT_L. n, number of experiments; R, remaining current; WT, wild-type.

Figure 5. Biophysical properties of Ca_v1.3 mutant R990H_S. (a) Current-voltage relationships (I_Ca, mean ± S.E.M.) from WT_S and R990H_S recorded in parallel on the same days (>3-different transfections). Inset (upper): Box plots of peak current densities (median (5/95 percentile, [pA/pF]): WT_S: −7.96 (−39.2/-3.03), n = 10; R990H_S: −10.8 (−29.8/-4.65), n = 11; p = 0.549, Mann Whitney test). Inset (lower): Representative I_Ca traces of WT_S and R990H_S upon depolarization to V_max. (b) Steady-state activation (circles, solid lines) and inactivation (squares, dashed lines) of WT_S and R990H_S. For statistics and n-numbers see Table 5. Lines without symbols depict the corresponding voltage-dependence of activation and inactivation for full length WT_L and R990H_L for comparison [21] (c) Normalized inactivation time course of I_Ca after 5-s depolarization to V_max (mean ± S.E.M.) for WT_S (n = 35) and R990H_S (n = 9). For statistics, see Table 6.

Figure 6. Biophysical properties of Ca_v1.3 mutant M1354I. (a) Current-voltage relationships (I_Ca, mean ± S.E.M.) of WT_L and M1354I_L recorded in parallel on the same days (>3-different transfections). Inset (upper): Box plots of peak current densities (median, 5/95 percentile, [pA/pF]): WT_L: −10.9 (−72.3/-5.86) n = 38; M1354I_L: −8.33 (−25.3/-4.44), n = 40) were significantly smaller for the mutant (Mann Whitney test, p = 0.029). Inset (lower): Representative I_Ca traces of WT_L and M1354I_L upon depolarization to V_max. (b) Current-voltage relationships (I_Ca, mean ± S.E.M.) of WT_S and M1354I_S recorded in parallel on the same days (>3-different transfections). Inset (upper): peak current densities (median, 5/95 percentile, [pA/pF]): WT_S: −7.50 (−28.5/-3.63), n = 30; M1354I_S: −5.89 (−12.6/-3.00), n = 19) were significantly smaller for the mutant (Mann Whitney test, p = 0.037). Inset (lower): Representative I_Ca traces of WT_S and M1354I_S upon depolarization to V_max. (c) M1354I steady-state activation (circles, solid lines) and inactivation (squares, dashed lines) of WT_L (filled black symbols), M1354I_L (filled blue symbols), WT_S (open black symbols), and M1354I_S (open blue symbols). No significant changes were observed in mutant channels. Gating parameters and statistics are given in Table 3. (d) Recovery from I_Ca inactivation (mean ± S.E.M.) of WT_L and M1345I_L. First 300 ms are magnified in the inset. Curves were fitted using a mono-exponential function; there was no difference in the time constants (τ as mean ± S.E.M [ms]: WT_L: 166.1 ± 20.6, n = 5; M1354I_L: 154.5 ± 42.5, n = 5; unpaired Student’s t-test). E: Fraction of I_max remaining after repetitive depolarizations to V_max at 3 Hz (mean ± S.E.M.) of WT_L (n = 10) versus M1345I_L (n = 5). (f) Normalized inactivation time course of I_Ca after 5-s depolarization to V_max (mean ± S.E.M.) of WT_L (n = 35) and M1354I_S (n = 9). For statistics, see Table 4.

Table 7. Classification of CACNA1D missense mutations by characteristic functional changes.

Type	Mutation	Disease phenotype	Characteristic functional changes (channel complexes of mutant α1-subunits with α2δ1 and β3)	References
1	G403D	APAs; hyperaldosteronism or hyperinsulinism with seizures and developmental delay	Inactivation almost abolished (voltage-dependence of inactivation not measurable) Voltage-dependence of activation shifted to hyperpolarized voltages or unchanged	[15]
	G403R	APAs		[15]
	G407R	ASD (and ID)		[10]
2	V259D	APAs	Voltage-dependence of activation strongly shifted to hyperpolarized voltages Inactivation not abolished* with voltage-dependence of inactivation strongly shifted to hyperpolarized voltages or unchanged * may be faster, slower, more or less complete after 5 s depolarizations to Vmax	[11]
	V401L	ASD, ID and seizures		[9]
	F747L	APAs		[11]
	A749G	ASD (and ID)		[9,16]
	I750M	APAs; hyperaldosteronism with seizures and developmental delay		[11,15]
	V1153G	APAs		[12]
3	Q558H	ID, seizures, hearing impairment	Slower and less complete inactivation during 3–5s depolarizations to Vmax and no changes in voltage-dependence of gating	[14]
	P1336R	APAs		[11]
4	R990H	APAs	Mutation-induced (depolarizing) ω-current	[11,21]

Pinggera A, Lieb A, Benedetti B, et al. CACNA1D De Novo Mutations in Autism Spectrum Disorders Activate Cav1.3 L-Type Calcium Channels. Biol Psychiatry. 2015;77:816–822.

 PubMed Web of Science ®Google Scholar

Monteleone S, Lieb A, Pinggera A, et al. Mechanisms Responsible for ω-Pore Currents in Cav Calcium Channel Voltage-Sensing Domains. Biophys J. 2017;113:1485–1495.

 PubMed Web of Science ®Google Scholar

Scholl UI, Goh G, Stolting G, et al. Somatic and germline CACNA1D calcium channel mutations in aldosterone-producing adenomas and primary aldosteronism. Nat Genet. 2013;45:1050–1054.

 PubMed Web of Science ®Google Scholar

Azizan EAB, Poulsen H, Tuluc P, et al. Somatic mutations in ATP1A1 and CACNA1D underlie a common subtype of adrenal hypertension. Nat Genet. 2013;45:1055–1060.

 PubMed Web of Science ®Google Scholar

Pinggera A, Mackenroth L, Rump A, et al. New gain-of-function mutation shows CACNA1D as recurrently mutated gene in autism spectrum disorders and epilepsy. Hum Mol Genet. 2017;26:2923–2932.

 PubMed Web of Science ®Google Scholar

Limpitikul WB, Dick IE, Ben-Johny M, et al. An autism-associated mutation in Cav1.3 channels has opposing effects on voltage- and Ca2+-dependent regulation. Sci. Rep. 2016;6:27235.

 PubMed Web of Science ®Google Scholar

Tan GC, Negro G, Pinggera A, et al. Aldosterone-Producing Adenomas: histopathology-Genotype Correlation and Identification of a Novel CACNA1D Mutation. Hypertension. 2017;70:129–136.

 PubMed Web of Science ®Google Scholar

Garza-Lopez E, Lopez JA, Hagen J, et al. Role of a conserved glutamine in the function of voltage-gated Ca(2+) channels revealed by a mutation in human CACNA1D. J Biol Chem. 2018;293:14444–14454.

 PubMed Web of Science ®Google Scholar