Stac3 has a direct role in skeletal muscle-type excitation-contraction coupling that is disrupted by a myopathy-causing mutation

Abstract

In skeletal muscle, conformational coupling between CaV1.1 in the plasma membrane and type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR) is thought to underlie both excitation-contraction (EC) coupling Ca(2+) release from the SR and retrograde coupling by which RyR1 increases the magnitude of the Ca(2+) current via CaV1.1. Recent work has shown that EC coupling fails in muscle from mice and fish null for the protein Stac3 (SH3 and cysteine-rich domain 3) but did not establish the functional role of Stac3 in the CaV1.1-RyR1 interaction. We investigated this using both tsA201 cells and Stac3 KO myotubes. While confirming in tsA201 cells that Stac3 could support surface expression of CaV1.1 (coexpressed with its auxiliary β1a and α2-δ1 subunits) and the generation of large Ca(2+) currents, we found that without Stac3 the auxiliary γ1 subunit also supported membrane expression of CaV1.1/β1a/α2-δ1, but that this combination generated only tiny Ca(2+) currents. In Stac3 KO myotubes, there was reduced, but still substantial CaV1.1 in the plasma membrane. However, the CaV1.1 remaining in Stac3 KO myotubes did not generate appreciable Ca(2+) currents or EC coupling Ca(2+) release. Expression of WT Stac3 in Stac3 KO myotubes fully restored Ca(2+) currents and EC coupling Ca(2+) release, whereas expression of Stac3W280S (containing the Native American myopathy mutation) partially restored Ca(2+) currents but only marginally restored EC coupling. We conclude that membrane trafficking of CaV1.1 is facilitated by, but does not require, Stac3, and that Stac3 is directly involved in conformational coupling between CaV1.1 and RyR1.

Keywords: L-type Ca2+ channels; Stac3 protein; excitation–contraction coupling.

Fig. 1.

The γ₁ subunit alters the kinetics of Ca²⁺ current via Ca_V1.1 in tsA201 cells. (A) Representative peak calcium currents (Left) and peak I-V relationships (Right) in tsA201 cells transfected with YFP-Ca_V1.1, β_1a, and α₂-δ₁ together with Stac3 alone, or with both Stac3 and γ₁. Test potentials: +40 mV; calibrations: 5 pA/pF (vertical), 50 ms (horizontal). (B) Representative charge movements (Left, V_test of −20, 0, +20, and +40 mV) and average Q-V relationships (Right) in tsA201 cells transfected with YFP-Ca_V1.1, β_1a, and α₂-δ₁ together with Stac3 alone, or with both Stac3 and γ₁. Calibrations: 2 pA/pF (vertical), 10 ms (horizontal).

Fig. 2.

(A) G_max/Q_max for the indicated combinations of cell type and construct. Values of G_max and Q_max (and thus G_max/Q_max) were obtained only from cells in which both peak I-V and Q-V relationships were measured. ** indicates a statistically significant difference (P < 0.05) between the center two combinations and the indicated combinations flanking them to the left and right. The G_max/Q_max ratios were not corrected for any background charge (unrelated to Ca_V1.1), which was small in dysgenic myotubes in the current experiments (0.83 ± 0.16 nC/μF, n = 7, measured at +40 mV). (B and C) Time constants and relative amplitudes, respectively, derived from a double-exponential function (Eq. 3) fitted to Ca²⁺ currents at +40 mV. There was a statistically significant difference (***P < 0.001; **P < 0.05) between the center three combinations and the indicated combinations flanking them to the left and right. Numbers of cells (from left to right): 9 [dysgenic (Ca_V1.1-null) myotubes expressing YFP-Ca_V1.1], 10 (tsA201 cells expressing YFP-Ca_V1.1, α₂-δ₁, β_1a + Stac3), 9 (tsA201 cells expressing YFP-Ca_V1.1, α₂-δ₁, β_1a + Stac3 + γ₁), 7 [dyspedic (RyR1-null) myotubes], 7 (Stac3 KO myotubes), 9 (Stac3 heterozygous myotubes), 10 (Stac3 KO myotubes expressing WT Stac3), and 6 (Stac3 KO myotubes expressing Stac3_W280S).

Fig. 3.

The γ₁ subunit supports membrane expression of Ca_V1.1 in tsA201 cells. (A) Representative peak calcium current (test potential: +30 mV) and peak I-V relationships in tsA201 cells transfected with YFP-Ca_V1.1, β_1a, and α₂-δ₁ and γ₁. Calibrations: 1 pA/pF (vertical), 50 ms (horizontal). (B) Representative charge movements (V_test of −20, 0, +20, and +40 mV) and average Q-V relationships in tsA201 cells transfected with YFP-Ca_V1.1, β_1a, α₂-δ₁ and γ₁. Calibrations: 2 pA/pF (vertical), 10 ms (horizontal). For comparison, the smooth curves fitted to the peak I-V and Q-V data for tsA201 cells transfected with YFP-Ca_V1.1, β_1a, α₂-δ₁ and WT Stac3 (Fig. 1 A and B) are replotted in blue.

Fig. S1.

Subcellular distribution in tsA201 cells of transiently expressed γ₁-BFP alone (Left) or YFP-Ca_V1.1 coexpressed with β_1a, α₂-δ₁, and γ₁ (Right). (Scale bars: 5 μm.)

Fig. 4.

The absence of Stac3 in myotubes partially reduces Ca_V1.1 membrane expression, significantly alters L-type Ca²⁺ current, and abolishes EC coupling Ca²⁺ release. (A) Representative charge movements (V_test of −20, 0, +20, and +40 mV, Left) and average Q-V relationships (Right) in myotubes homo- or heterozygous for a null mutation of Stac3. Calibrations: 1 pA/pF (vertical), 10 ms (horizontal). (B) Representative Ca²⁺ currents (V_test of −10, +10 and +30 mV, Left), and average peak I-V relationships (Right) in myotubes homo- or heterozygous for Stac3 KO. Calibrations: 5 pA/pF (vertical), 50 ms (horizontal). (C) Representative whole-cell, voltage-clamp measurements of Ca²⁺ transients with Fluo-3 (200 ms depolarizations from −80 mV to −40, −20, 0, and +20 mV, Left) and average peak change in fluorescence normalized by baseline (ΔF/F) as a function of test potential (Right) for heterozygous or homozygous Stac3 KO myotubes. Calibrations: 1 ΔF/F (vertical), 5 s (horizontal).

Fig. 5.

Ca_V1.1 immunostaining in myotubes heterozygous (Left) or homozygous (Right) for Stac3 KO. (Scale bars: 5 μm.)

Fig. 6.

In myotubes, the NAM mutation of Stac3 has modest effects on membrane expression of Ca_V1.1 and L-type current properties but causes a very large reduction in EC coupling Ca²⁺ release. (A) Representative charge movements (V_test of −20, 0, +20, and +40 mV, Left) and average Q-V relationships (Right) in Stac3 KO myotubes expressing WT Stac3 or Stac3_W280S. Calibrations: 2 pA/pF (vertical), 10 ms (horizontal). (B) Representative ionic peak currents at +30 mV (Left), and average peak I-V relationships (Right) for Stac3 KO myotubes expressing WT Stac3 or Stac3_W280S. Calibrations: 5 pA/pF (vertical), 50 ms (horizontal). (C) Representative whole-cell, voltage-clamp measurements of Ca²⁺ transients with Fluo-3 (200-ms depolarizations from −80 mV to −40, −20, 0, and +20 mV, Left) and average peak change in fluorescence normalized by baseline (ΔF/F) as a function of test potential (Right) for Stac3 KO myotubes expressing WT Stac3 or Stac3_W280S. Calibrations: 1 ΔF/F (vertical), 5 s (horizontal). The smooth curves for data from myotubes heterozygous (red) and homozygous (black) for Stac3 KO are replotted from Fig. 4 A–C.

Fig. S2.

In tsA201 cells, the NAM mutation introduced into mouse Stac3 (Stac3_W280S) depresses membrane expression of Ca_V1.1 and accelerates L-type current activation kinetics. (A) Representative charge movements (V_test of −20, 0, +20, and +40 mV, Left) and average Q-V relationship (Right) for YFP-Ca_V1.1 coexpressed in tsA201 cells with β_1a, α₂-δ₁, and Stac3_W280S. Calibrations: 2 pA/pF (vertical), 10 ms (horizontal). (B) Representative peak currents at +40 mV (Left) and average peak I-V relationships (Right) in tsA201 cells expressing YFP-Ca_V1.1, β_1a, and α₂-δ₁ together with Stac3_W280S (green) or with WT Stac3 (blue). The gray trace (Left) represents a vertically scaled (3.75×) version of the peak current for Stac3_W280S. Calibrations: 5 pA/pF (vertical), 50 ms (horizontal). For comparison, the smooth curves fitted to the peak I-V and Q-V data for YFP-Ca_V1.1, β_1a, α₂-δ₁ and WT Stac3 expressed in tsA201 cells (Fig. 1B) are replotted in blue.