Regulation of excitation-contraction coupling and L-type Ca(2+) channel activation in skeletal muscle by calmodulin and prolonged depolarization

In skeletal muscle, the dihydropyridine receptor (DHPR) serves a dual function: a slowly activating L-type Ca2+ channel and voltage-sensor for ryanodine receptor (RyR1) activation during excitation-contraction coupling (EC coupling). The DHPR and RyR1 display bi-directional coupling, whereby the DHPR regulates activation of RyR1 (orthograde coupling), and RyR1 interacts with the DHPR to increase Ca2+ permeation (retrograde coupling). In amphibian skeletal muscle, it has been established that depolarizing conditioning prepulses induce rapid activation of the L-channel. Similar protocols failed to alter activation of L-current in mammalian skeletal muscle. We described a novel paradigm of prolonged depolarization (60 s to +40 mV) followed by a brief repriming interval (10 s to −80 mV) that accelerated the activation of the slowly activating L-type current in mammalian skeletal muscle. Rapid gating of the channel is an intrinsic property of the channel, as L-currents in RyR1-null myotubes and adult muscle fibers were also accelerated by prolonged depolarization. These results suggest that the DHPR is capable of the rapid gating transitions associated with the voltage sensor of EC coupling.; In vitro, calmodulin has been shown to strongly modulate RyR1 activity, but a functional role for CaM in EC coupling has not yet been demonstrated. To examine the role of CaM on EC coupling in skeletal muscle, mutant calmodulins with altered Ca2+ sensitivity were overexpressed in myotubes. CaM_1–4, a Ca2+ insensitive mutant of CaM, significantly increased both L-type Ca2+ currents and voltage-gated SR Ca2+ release in cultured myotubes while CaM-CC, a chimeric CaM with only high-affinity Ca2+ binding sites, significantly decreased L-type Ca2+ currents and SR Ca 2+ release. Neither mutant altered RyR1 caffeine sensitivity. To specifically examine the role of CaM modulation of RyR1, mutations in RyR1 that prevented CaM binding (L3624D) or permitted only apoCaM binding (W3620A) were expressed in RyR1-null myotubes. Neither L3624D nor W3620A altered orthograde coupling. However, W3620A selectively increased retrograde coupling, while L3624D altered the caffeine sensitivity of RyR1. These results are the first to examine CaM modulation of EC coupling proteins in intact muscle cells and suggest that CaM does not strongly influence RyR1 activation during EC coupling, but does affect voltage sensor function.