Rem inhibits EC coupling in FDB fibers without affecting intramembrane charge movement. Representative recordings of intramembrane charge movements elicited by 25-ms depolarizations from −80 to −40, −20, 0, and 20 mV shown for transfected FDB fibers expressing either V-Rem (A) or YFP (B). (C) The Q-V relationships for fibers expressing either V-Rem (n = 7; ○) or YFP (n = 5; ●) are shown. Charge movements were evoked at 0.1 Hz by test potentials ranging from −70 through 50 mV in 10-mV increments. The smooth curves for V-Rem– or YFP-expressing fibers are plotted according to , with the respective fit parameters shown in . Representative recordings of myoplasmic Ca²⁺ transients elicited by 25-ms depolarizations from −80 to −40, −20, 0, 20, and 40 mV are shown for FDB fibers overexpressing V-Rem (D) or YFP (E). (F) The peak ΔF/F-V relationships for V-Rem (n = 8; ○)– and YFP (n = 6; ●)-expressing fibers are shown. Ca²⁺ transients were evoked at 0.1 Hz by test potentials ranging from −70 through 60 mV in 10-mV increments. The smooth curves for V-Rem– and YFP-expressing fibers are plotted according to with the respective fit parameters shown in . Error bars represent ±SEM.

SR Ca²⁺ store content is not significantly affected by overexpression of Rem. SR Ca²⁺ store content as assessed by changes in Fluo-3 AM fluorescence (ΔF/F) in response to the application of 1 mM 4-CmC to fibers expressing either YFP (A) or V-Rem (B). Insets show images of loaded fibers before 4-CmC application (left) and at the peak of fluorescence (right). Bars, 100 µm. (C) A comparison of the average peak ΔF/F values for YFP- and V-Rem–expressing fibers is shown. Error bars represent ±SEM.

Rem overexpression does not alter targeting of Ca_V1.1 α_1S or β_1a subunits. Confocal images of FDB fibers expressing CFP-α_1S alone (A), CFP-α_1S coexpressed with V-Rem (B), CFP-β_1a alone (C), or CFP-β_1a coexpressed with V-Rem (D). For each panel, the left, left-middle, and right-middle images show CFP fluorescence (red), Venus fluorescence (green), and an overlay, respectively. Bars, 10 µm. The right images are blowups of the area indicated by the yellow boxes in the adjacent overlays; average image profile analyses are shown below. The green lines indicate Venus fluorescence and the red lines represent the fluorescence emitted by either CFP-α_1S or CFP-β_1a in arbitrary units. Note that the transverse-tubular targeting of CFP-α_1S or β_1a is intact both in the absence and in the presence of coexpressed V-Rem. For experiments with each channel subunit clone, images were acquired with nearly identical microscope settings.

Introduction of alanines at Rem positions R200, L227, and H229 disrupts the interaction with β_1a. (A) In the duplicate representative experiments shown, a monoclonal antibody directed to Rem was used to immunoprecipitate V-Rem–YFP-β_1a complexes from tsA201 cells expressing YFP-β_1a (lanes 2 and 6), YFP-β_1a and V-Rem (lanes 3 and 7), and YFP-β_1a and V-Rem AAA (lanes 4 and 8). Blots were probed with an antibody directed to XFP (see Materials and methods). (B) Similar affinity of the Rem antibody for V-Rem and V-Rem AAA is presented, where the immunoprecipitated Rem and Rem AAA are detected by the Rem antibody (lanes 3–4 and 7–8). (C) Comparable expression of YFP-β_1a, V-Rem, and V-Rem AAA in harvested tsA201 cells is confirmed in total lysates before coimmunoprecipitation. Lanes 1 and 5 are loaded with protein markers (molecular weights indicated). Results shown are representative of five separate experiments.

Introduction of alanines at Rem positions R200, L227, and H229 ablates the ability of V-Rem to inhibit L-type Ca²⁺ current conducted by Ca_V1.3/β_1a/α₂δ-1 channels expressed in tsA201 cells. Representative L-type currents are shown for tsA201 cells coexpressing Ca_V1.3/β_1a/α₂δ-1 and V-Rem (A), V-Rem AAA (B), or YFP (C). Current families shown were evoked by 50-ms steps from −80 to −40 through 60 mV in 10-mV increments. Current amplitudes were normalized by linear cell capacitance (pA/pF). (D) I-V relationships are shown. (E) Confocal images confirming successful heterologous expression of V-Rem and V-Rem AAA in tsA201 cells are shown. Bars, 10 µm. Error bars represent ±SEM.

Expression of V-Rem AAA in FDB fibers has very little effect on native Ca_V1.1 function. Representative recordings of skeletal muscle L-type Ca²⁺ currents elicited by 500-ms depolarizations from −50 to −20, 0, 20, and 40 mV are shown for FDB fibers expressing V-Rem AAA (A; left). The peak I-V relationship for fibers expressing V-Rem AAA (n = 6; gray circles) is shown with the peak I-V relationship for fibers expressing unfused YFP (n = 5; black circles) and V-Rem (n = 5; white circles) in the right panel. L-type currents were evoked at 0.1 Hz by test potentials ranging from −40 through 80 mV in 10-mV increments. The smooth curves are plotted according to with the following respective parameters for V-Rem AAA–, V-Rem–, and YFP-expressing fibers: G_max = 212 ± 26, 128 ± 19, and 205 ± 12 nS/nF; V_1/2 = 4.3 ± 3.1, 8.3 ± 3.2, and 4.0 ± 2.0 mV; k_G = 5.0 ± 0.4, 5.4 ± 0.5, and 5.1 ± 0.5 mV; V_rev = 70.0 ± 1.7, 70.0 ± 3.4, and 67.0 ± 1.4 mV. Representative recordings of intramembrane charge movements elicited by 25-ms depolarizations from −80 to −40, −20, 0, and 20 mV are shown for transfected FDB fibers expressing V-Rem AAA (B; left). The Q-V relationships for fibers expressing V-Rem (n = 7; white circles), V-Rem AAA (n = 5; gray circles), or YFP (n = 5; black circles) are shown in the right panel. Charge movements were evoked at 0.1 Hz by test potentials ranging from −70 through 50 mV in 10-mV increments. The smooth curves for V-Rem–, V-Rem AAA–, or YFP-expressing fibers are plotted according to with the respective fit parameters shown in . Representative recordings of myoplasmic Ca²⁺ transients elicited by 25-ms depolarizations from −80 to −40, −20, 0, 20, and 40 mV are shown for FDB fibers overexpressing V-Rem AAA (C; left). The peak ΔF/F-V relationships for V-Rem AAA (n = 6; gray circles)–, V-Rem (n = 8; white circles)–, and YFP (n = 6; black circles)-expressing fibers are presented in the right panel. Ca²⁺ transients were evoked at 0.1 Hz by test potentials ranging from −70 through 60 mV in 10-mV increments. The smooth curves for V-Rem–, V-Rem AAA–, and YFP-expressing fibers are plotted according to with the respective fit parameters shown in . The Q-V and ΔF/F-V relationships for YFP- and V-Rem–expressing fibers are reproduced from . Error bars represent ±SEM.

Schematic depicting potential mechanisms for Rem-mediated EC uncoupling. (A) The diagram represents the intact Ca_V1.1–RYR1 ultrastructure requisite for skeletal-type EC coupling. Four Ca_V1.1 α_1S (red circles)–β_1a (white ovals) channel complexes are shown coupled to each subunit of a single RYR1 (gray tetramer) from a transverse-tubular vantage point. For clarity, the β_1a subunits are superimposed on the α_1S subunits, and the α₂δ-1 subunits, γ₁ subunits, and other nonessential components of the junction have been omitted. The orientation of β_1a within the tetrad follows on previous work (; ). In the right panels (B and C), we present two potential mechanisms by which Rem (black ovals) may disrupt EC coupling. In B, Rem displaces the Ca_V1.1 channel complex from RYR1 sufficiently to disrupt the tetradic ultrastructure that is required for Ca_V1.1–RYR1 communication by interacting with the conserved guanylate kinase–like domain of β_1a (; ) on the periphery of the tetrad (). If ultrastructure is preserved in Rem-overexpressing fibers (as depicted in C), the binding of Rem to β_1a within the intact CRU would most likely induce conformational rearrangements within β_1a that deter transmission of the EC coupling signal from the membrane-bound, voltage-sensing regions of Ca_V1.1 to RYR1.