The alpha(1S) III-IV loop influences 1,4-dihydropyridine receptor gating but is not directly involved in excitation-contraction coupling interactions with the type 1 ryanodine receptor

Abstract

In skeletal muscle, coupling between the 1,4-dihydropyridine receptor (DHPR) and the type 1 ryanodine receptor (RyR1) underlies excitation-contraction (EC) coupling. The III-IV loop of the DHPR alpha(1S) subunit binds to a segment of RyR1 in vitro, and mutations in the III-IV loop alter the voltage dependence of EC coupling, raising the possibility that this loop is directly involved in signal transmission from the DHPR to RyR1. To clarify the role of the alpha(1S) III-IV loop in EC coupling, we examined the functional properties of a chimera (GFP-alpha(1S)[III-IVa]) in which the III-IV loop of the divergent alpha(1A) isoform replaced that of alpha(1S). Dysgenic myotubes expressing GFP-alpha(1S)[III-IVa] yielded myoplasmic Ca(2+) transients that activated at approximately 10 mV more hyperpolarized potentials and that were approximately 65% smaller than those of GFP-alpha(1S). A similar reduction was observed in voltage-dependent charge movements for GFP-alpha(1S)[III-IVa], indicating that the chimeric channels trafficked less well to the membrane but that those that were in the membrane functioned as efficiently in EC coupling as GFP-alpha(1S). Relative to GFP-alpha(1S), L-type currents mediated by GFP-alpha(1S)[III-IVa] were approximately 40% smaller and activated at approximately 5 mV more hyperpolarized potentials. The altered gating of GFP-alpha(1S)[III-IVa] was accentuated by exposure to +/-Bay K 8644, which caused a much larger hyperpolarizing shift in activation compared with its effect on GFP-alpha(1S). Taken together, our observations indicate that the alpha(1S) III-IV loop is not directly involved in EC coupling but does influence DHPR gating transitions important both for EC coupling and activation of L-type conductance.

FIGURE 1.

Rationale for examination of the functional properties of the GFP-α_1S[III-IVa] chimera. A, sequence comparison of the III-IV loops of rabbit α_1S (GenBank™ accession number X05921), rabbit α_1C (GenBank™ accession number X15539), and rabbitα_1A (GenBank™ accession number X57477). Residues of α_1C or α_1A identical to those of α_1S are shown boxed in black, and residues conserved with those of α_1S are shown boxed in gray. Note that the residue corresponding to α_1S R1086 (indicated with a red arrow) that, if exchanged to H or C is linked to the MH phenotype, is conserved in all three channels. B, schematic representation of GFP-α_1S[III-IVa].

FIGURE 2.

EC coupling is reduced in myotubes expressing GFP-α_1S[III-IVa]. Simultaneous recordings of myoplasmic Ca²⁺ transients (top) and L-type Ca²⁺ currents (bottom), elicited by 50-ms depolarizations from -50 mV to the indicated test potentials are shown for dysgenic myotubes expressing either GFP-α_1S (A) or GFP-α_1S[III-IVa] (B). C, average ΔF/F-V relationships. D, ΔF/F-V relationships normalized to average ΔF/F at +50 mV to allow direct comparison of voltage dependence. The smooth curves are plotted according to Equation 2, with fit parameters presented in Table 1. Throughout, the error bars represent ± S.E.

FIGURE 3.

l-type Ca²⁺ currents are reduced in myotubes expressing GFP-α_1S[III-IVa]. A, recordings of L-type Ca²⁺ currents, elicited by 200-ms depolarizations to the indicated test potentials are shown for dysgenic myotubes expressing GFP-α_1S or GFP-α_1S[III-IVa]. B, comparison of average peak I-V relationships. The dashed curve represents the I-V relationship for GFP-α_1S[III-IVa] scaled to match the peak current density for GFP-α_1S. The currents were evoked at 0.1 Hz by test potentials ranging from -20 mV through +80 mV in 10-mV increments following a prepulse protocol (11). The current amplitudes were normalized by linear cell capacitance (pA/pF). The smooth I-V curves are plotted according to Equation 1. The best fit parameters for each plot are presented in Table 2. C, tail current amplitudes measured 1 ms after repolarization to -50 mV are plotted as a function of the preceding test potential.

FIGURE 4.

GFP-α_1S[III-IVa] releases Ca²⁺ as efficiently as GFP-α_1S but has a higher l-type channel open probability (P_o). A, recordings of immobilization-resistant charge movements elicited by 20-ms depolarizations from -50 mV to the indicated test potentials are shown for dysgenic myotubes expressing GFP-α_1S or GFP-α_1S[III-IVa]. B, comparison of Q-V relationships. Charge movements were evoked at 0.1 Hz by test potentials ranging from -40 mV through +60 mV in 10-mV increments, following a prepulse protocol (11). The smooth curves are plotted according to Equation 3, with best fit parameters presented in Table 2. C, (ΔF/F)_max/Q′ and I_tail(40)/Q′ (left and right, respectively) for GFP-α_1S and GFP-α_1S[III-IVa]. Q′ represents the charge attributable to heterologously expressed DHPRs in the membrane and was calculated as Q_max - Q_dys (Table 2). The values for (ΔF/F)_max are from Table 1. The values for I_tail(40) are given in the text and Table 2.

FIGURE 5.

GFP-α_1S[III-IVa] is more sensitive to ±Bay K 8644 than GFP-α_1S. A, average peak I-V relationships for GFP-α_1S in the presence (▴, n = 8) and absence (•, n = 13) of 10 μm ± Bay K 8644. B, average peak I-V relationships for GFP-α_1S[III-IVa] in the presence (▵, n = 7) and absence (○, n = 20) of 10 μm ±Bay K 8644. In both (A) and (B), peak I-V data obtained in the absence of ±Bay K 8644 are replotted from Fig. 3, and the smooth curves are plotted according to Equation 1 with best fit parameters given in Table 2. The insets illustrate representative currents obtained at a test potential of +30 mV in the presence of ±Bay K 8644. The vertical scale bar equals 5 pA/pF. The horizontal scale bar equals 50 ms. Note that these tail currents decay more slowly than currents in the absence of ±Bay K 8644 (Fig. 3A). C, potentiation ratios (I_{Bay K 8644}/I_untreated) for GFP-α_1S (▴) or GFP-α_1S[III-IVa] (▵). The dashed line represents a potentiation ratio of 1.