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. 2018 Sep 10;7:e35222. doi: 10.7554/eLife.35222

Figure 4. Stac enhances the PO of CaV1.3.

(A) A general four-state scheme for stac and CaM modulation. (1) CaV1.3S devoid of CaM and stac possess a low baseline PO (PO/E). (2) Without stac, apoCaM binding to CaV1.3S upregulates baseline PO (PO/A). Baseline PO of CaV1.3S bound to stac in the absence (configuration 3, PO/E*) and the presence of apoCaM (configuration 4, PO/A*) are unknown. (B) Schematic outlines three mechanistic possibilities for stac binding to CaV1 and their functional outcomes. Scenario 1, stac uniformly suppresses PO of CaV1 (PO/E) and abolishes CDI. Scenario 2, apoCaM tunes baseline PO of CaV1 despite concurrent stac binding. Stac, nonetheless, abrogates CDI. Scenario 3, stac uniformly upregulates the baseline PO of CaV1 and abolishes CDI (PO/A). (C) Top, cartoon shows the canonical CaV1.3S variant with a high apoCaM binding affinity. Single-channel analysis of recombinant CaV1.3S in the absence (middle) and presence of stac (bottom). In both panels, the unitary Ba2+ currents during voltage-ramp are shown between −50 mV and +40 mV (slanted gray lines, GHK fit). Robust CaV1.3 openings are detected in the absence and presence of stac. (D) Average single-channel PO-voltage relationship for CaV1.3S obtained from multiple patches in the absence (gray) and presence of stac2 (blue). Error bars indicate ±S.E.M. for specified number of patches and 80–150 stochastic records per patch. (E) Histogram shows distribution of single-trial average PO (P-O) for the voltage range -30 mV ≤ V ≤ +25 mV under control (top), stac-bound (middle), and CaM-bound (bottom) conditions. P-O-distribution is bimodal in the absence of stac corresponding to high PO (gray) and low PO (rose) gating modes. With stac, P-O-distribution is largely restricted to the high PO mode. (F–H) Single-channel analysis of a recombinant CaV1.3RNA-edited variant reveals a marked upregulation in the baseline PO in the presence of stac compared with control conditions in which apoCaM preassociation is weak. Absent stac or CaM, single-trial P-O-distribution is restricted to the low PO limits, With either stac or CaM, the high PO gating mode re-emerges. Format as in (C–E). (I–K) Stac also upregulates the baseline PO of CaV1.3L, an alternatively spliced variant, by stabilizing the high PO gating configuration. Format as in (C–E).

Figure 4.

Figure 4—figure supplement 1. Extended data show that stac2 preferentially biases a high PO gating mode for CaV1.3.

Figure 4—figure supplement 1.

(A) Cartoon schematizes CaV1.3S. (B) Left, 10 sequential single-channel trials of CaV1.3S under endogenous levels of CaM illustrate high PO gating mode with rare sojourns into a low PO gating mode. Here, traces show Ba2+ currents elicited every 12 s in response to a voltage ramp shown between -40 mV and +40 mV. Right, diary plot displays single trial average PO computed for the voltage-range -30 mV ≤ V ≤ +25 mV (P-O (-30 ≤ V ≤ 25)). Dashed line discriminates traces to low PO (red shaded area) or high PO (gray shaded area) categories as previously established. (C) Average PO at each voltage calculated for all traces within the high PO range (gray region in (B)) and the low PO range (red shaded region in (B)). These relationships estimate the PO-V relationship for high PO and low PO gating modes. (D–E) In the presence of stac2, CaV1.3S preferentially adopts the high PO gating configuration. Format as in (B–C).
Figure 4—figure supplement 2. Extended data show that stac2 and CaM enhance the PO of CaV1.3RNA-edited variant via discreet transitions into a high PO gating mode.

Figure 4—figure supplement 2.

(A) Cartoon schematizes CaV1.3RNA-edited. RNA-editing results in a methionine substitution of the central isoleuicine residue, resulting in sharply diminished CaM binding. Functionally, CaM binding enhances the PO of CaV1.3RNA-edited. (B–C) Under endogenous levels of CaM, CaV1.3RNA-edited exhibits a uniformly low PO as evident from individual trials. The average PO – V relation is consistent with that for the low PO gating mode. Format as in Figure 4—figure supplement 1B–C. (D–E) CaM overexpression enhances the PO of CaV1.3RNA-edited variant. Exemplar traces depict channels switching between discrete high and low PO gating modes. A PO-V relationship for high PO and low PO gating modes is evident. Format as in Figure 4—figure supplement 1B–C. (F–G) Similar to CaM, stac2 overexpression also enhances the PO of CaV1.3RNA-edited variant by stabilizing the high PO gating mode. Exemplar traces depict channels largely adopting the high gating mode. Format as in Figure 4—figure supplement 1B–C.
Figure 4—figure supplement 3. Extended data show that both stac2 and CaM enhance the PO of CaV1.3L variant.

Figure 4—figure supplement 3.

(A) Cartoon schematizes CaV1.3L. The long-splice variant includes a distal carboxy-tail (DCT) that interacts with the IQ domain and competitively displaces CaM. (B–C) Under endogenous levels of CaM, CaV1.3L exhibits a uniformly low PO as evident from individual trials. The average PO – V relation is consistent with that for the low PO gating mode. Format as in Figure 4—figure supplement 1B–C. (D–E) CaM overexpression enhances the PO of CaV1.3L variant. Exemplar traces depict channels switching between discrete high and low PO gating modes. Format as in Figure 4—figure supplement 1B–C. (F–G) Similar to CaM, stac2 overexpression also enhances the PO of CaV1.3L variant by stabilizing the high PO gating mode. Exemplar traces depict channels largely adopting the high gating mode. Format as in Figure 4—figure supplement 1B–C.