The cardiac alpha(1C) subunit can support excitation-triggered Ca2+ entry in dysgenic and dyspedic myotubes

Abstract

Depolarization-induced entry of divalent ions into skeletal muscle has been attributed to a process termed Excitation-Coupled Ca(2+) Entry (ECCE), which is hypothesized to require the interaction of the ryanodine receptor (RyR1), the L-type Ca(2+) channel (DHPR) and another unidentified cation channel. Thus, ECCE is absent in myotubes lacking either the DHPR (dysgenic) or RyR1 (dyspedic). Furthermore, ECCE, as measured by Mn(2+) quench of Fura-2, is reconstituted by expression of a mutant DHPR alpha(1S) subunit (SkEIIIK) thought to be impermeable to divalent cations. Previously, we showed that the bulk of depolarization-induced Ca(2+) entry could be explained by the skeletal L-type current. Accordingly, one would predict that any Ca(2+) current similar to the endogenous current would restore such entry and that this entry would not require coupling to either the DHPR or RyR1. Here, we show that expression of the cardiac alpha(1C) subunit in either dysgenic or dyspedic myotubes does result in Ca(2+) entry similar to that ascribed to ECCE. We also demonstrate that, when potentiated by strong depolarization and Bay K 8644, SkEIIIK supports entry of Mn(2+). These results strongly support the idea that the L-type channel is the major route of Ca(2+) entry in response to repetitive or prolonged depolarization of skeletal muscle.

Figure 1

Long, weak depolarizations in 2 mM Ca²⁺ elicit slowly-inactivating L-type currents in dysgenic myotubes expressing α_1C-CFP. (A) A family of L-type Ca²⁺ currents elicited by 200-ms depolarizations from −50 mV to the indicated test potentials is shown for an α_1C-CFP expressing dysgenic myotube in 2 mM external Ca²⁺. (B) The average peak I–V relationship is shown for dysgenic myotubes expressing α_1C-CFP (●; n = 6) elicited at 0.1 Hz by test potentials ranging from −20 mV through +80 mV in 10 mV increments, following a prepulse protocol (see Materials and Methods). The smooth curve is plotted according to Eq. 1, where G_max = 452.4 nS/nF, V_rev = 66.5 mV, V_1/2 = 29.2 mV and k_G = 6.42 mV. (C) Representative L-type currents from a dysgenic myotube expressing α_1C-CFP elicited by 9800-ms depolarizations from −80 mV to the indicated test potentials in 2 mM external Ca²⁺.

Figure 2

ECCE-like Ca²⁺ entry is observed in dysgenic myotubes expressing cardiac α_1C subunits. (A) Representative Ca²⁺ transient evoked by application of 60 mM K⁺ Ringer’s solution to a dysgenic myotube expressing α_1C-CFP. (B) Absence of elevated K⁺-evoked Ca²⁺ transient in an untransfected dysgenic myotube. (C) Partial block by 10 μM nifedipine of the elevated K⁺-evoked Ca²⁺ transient in an α_1C-CFP-expressing dysgenic myotube. (D) Complete block by 50 μM nifedipine of the K⁺-evoked Ca²⁺ transient in an α_1C-CFP-expressing dysgenic myotube. (E) Average peak ΔF/F (left) and t_1/2peak (right) for dysgenic myotubes expressing α_1C-CFP in the absence and presence of 10 μM nifedipine. Asterisks indicate significant differences (**, p < 0.005; ***, p < 0.0005; t-test). Myotubes were exposed to 200 μM ryanodine for >1 hr at 37°C prior to experiments in order to block contribution of RyR1 and/or RyR3 to the Ca²⁺ transient.

Figure 3

ECCE-like Ca²⁺ entry is observed in dyspedic myotubes expressing cardiac α_1C subunits. (A) Ca²⁺ transient in a dyspedic myotube expressing α_1C-CFP evoked by 60 mM K⁺ Ringer’s solution. Elevated K⁺ failed to evoke Ca²⁺ transients in a non-transfected dyspedic myotube (B) or in a non-transfected dyspedic myotube exposed to 5 μM ±Bay K 8644 (C). Myotubes were exposed to 200 μM ryanodine for >1 hr at 37°C prior to experiments in order to block any potential contribution of RyR3 to the Ca²⁺ transient.

Figure 4

The SkEIIIK pore mutant allows inward Mn²⁺ current. Representative currents in a dysgenic myotube expressing SkEIIIK before (A) and after exposure to 5 μM ±Bay K 8644 (B). The external solution contained 10 mM Mn²⁺. SkEIIIK currents were elicited by depolarizations from −50 mV to either 0 or +80 mV, following a prepulse protocol (see Materials and Methods). (C) Summary of deactivation time constants of SkEIIIK Mn²⁺ tail currents recorded upon repolarization from 80 mV to −50 mV in the absence (n = 4) and presence (n = 4) of ±Bay K 8644. Tail currents were fit by Eq. 2. The asterisks indicate a significant difference (p < 0.006; t-test).