Germline de novo variant F747S extends the phenotypic spectrum ofCACNA1DCa2+ channelopathies

Figures

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  Figure 1. Transmembrane topology of the pore-forming α1-subunit of the Cav1.3 channel. (A) In each of the four homologous repeats (I–IV) the segments 1–4 comprise the voltage-sensing domain and segments 5–6 together with their connecting loop form the ion conducting pore (see also Fig. 6 below for a homology model of the channel). All four cytoplasmic ends of S6 together form the activation gate. The position of de novo variant F747S (grey circle) is indicated together with other pathogenic germline missense mutations functionally characterized so far (open circles, see main text for details). (B) Sequence alignment of parts of the IIS6 segment of all human α1-subunits (gene names are given). The box indicates the position of F747. Amino acid conservation in all (dark grey) or some (light grey) α1-subunits is indicated.

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  Figure 2. Voltage-dependence of WT and F747S channel gating. WT or mutant channels were coexpressed with β3 (A–C) or β2a (D–F) together with α2δ1-subunits. Current–voltage relationships (A, D) were generated with 25-ms depolarizing square test pulses from a holding potential of −89 mV in 5 mV increments to the indicated test potentials. Current densities are shown (current normalized to the cell size, pA/pF). Representative traces upon depolarization to Vmax are shown in the insets. (B, E) Normalized steady-state activation (circles; obtained from the I–V-relationships) and inactivation curves (squares) were obtained as described in methods. For detailed parameters, n-numbers and statistics see Table 1. (C, F) Window currents calculated from the current densities in I-V-curves and steady-state inactivation parameters as described in methods. Statistics: one-way ANOVA with Bonferroni post hoc test. Data are presented as mean ± SEM from at least 3–5 independent transfections.

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  Figure 3. Inactivation time course of Cav1.3 WT and F747S channels. Inactivation kinetics of normalized Cav1.3 WT (black traces) and F747S (grey traces) channel currents during 5-s depolarizing pulses to Vmax from a HP of −89 mV. Channels were co-expressed with β3- (A) or β2a- (B) together with α2δ1-subunits. Curves represent the means ± SEM from at least 3–5 independent transfections. For statistical significance of inactivation after pre-specified time points see Table 2. Note the slowing of the inactivation time course induced by β2a-subunits (B). Parameters obtained by fitting individual inactivation curves to exponential decay are given in Table 3. (C) Time courses of recovery from inactivation of Cav1.3-WT (black) and F747S (grey) co-expressed with β3 examined using a two-pulse protocol (top). The current amplitude during the 25 ms control pulse was normalized to peak current evoked by the 2 s prepulse and plotted as a function of recovery time. Data are present as mean ± SEM, n numbers are indicated in parenthesis. Data were fit to a double exponential function (solid lines). The following parameters (means ± SEM) were obtained: WT: Afast = 0.72 ± 0.02, τfast = 67.6 ± 5.72 ms, Aslow…

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  Figure 4. Deactivation kinetics and tail currents of Cav1.3 WT and F747S channels. (A, D). Representative traces of tail currents recorded in Cav1.3 WT (black) or F747S channels (grey) co-expressed with β3- (A) or β2a- (D) together with α2δ1-subunits during 20-ms repolarizations to −69 mV or − 49 mV after 10-ms test pulses to the reversal potential. To quantify the mutation-induced relative change in Ca2+ charge during deactivation, normalized tail currents at both test voltages were integrated over 20 ms and are shown for both −69 mV (B, E) and −49 mV (C, F) for WT (dark grey bars) and F747S (light grey bars) co-expressed with β3- (B, C) or β2a -subunits (E, F). Statistics: one-way ANOVA with Bonferroni post-hoc test. Data are presented as mean ± SEM (>3 independent transfections).

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  Figure 5. Inhibition of β3- and β2a-containing Cav1.3 WT and F747S channels by isradipine. (A, B) Representative experiment for inhibition of peak inward ICa (with co-expressed β2a) through Cav1.3 WT (A, black open circles) or F747S channels (B, grey open circles) by 30 and 300 nm isradipine (arrows indicate steady-state inhibition) during continuous 100-ms depolarizations (0.1 Hz) from the HP to Vmax. Insets: Representative current traces obtained before (bath solution, BS) and after maximal inhibition by the indicated isradipine concentrations (3 μm were used for full block at the end of each experiment). The mean time course of current decay in the absence of drug (linear current run-down; BS only) is also shown as the mean (±SEM) from all control experiments measured in parallel with the WT and mutant channels on different days (A, B, filled circles). Run-down-corrected concentration-inhibition curves are shown for both β3- (C) and β2a-containing channel complexes (D) for WT (black) and F747S channels (grey). Data are presented as means ± SEM. Curves were computer-fitted as described in Methods using a Hill slope of 1 with top and bottom fixed (bottom = 0; top = 100). Concentrations…

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  Figure 6. Molecular modeling of the F747S mutation in homology models of different conformational states of the Cav1.3 α1-subunit. (A) Top (top panel) or side view (bottom panel) of the activated-open channel conformation of the Cav1.3 α1-subunit. The box highlights the region of interaction between the newly formed hydrogen bond between F747S (IIS6, orange) and N1145 (IIIS6, green). VSD, voltage-sensing domains. (B) Detailed view of the F747S—N1154 interaction in the predicted activated-open channel conformation of mutant channels. (C) This novel hydrogen bond also forms in a recently published Cav1.3 α1-subunit structure (11) in which the activation gate assumes a conformation with structural features of both the open and inactivated-closed conformation and may represent a "pre-open" channel state (Supplementary Material, Fig. S1). (D) The F747S—N1154 is absent in the inactivated (and resting, not illustrated) channel conformation. For further details for modeling of the different states, see section Methods.