A Component of Excitation-Contraction Coupling Triggered in the Absence of the T671-L690 and L720-Q765 Regions of the II-III Loop of the Dihydropyridine Receptor α1s Pore Subunit

Abstract

We conducted a deletion analysis of two regions identified in the II-III loop of alpha(1S), residues 671-690, which were shown to bind to ryanodine receptor type 1 (RyR1) and stimulate RyR1 channels in vitro, and residues 720-765 or the narrower 724-743 region, which confer excitation-contraction (EC) coupling function to chimeric dihydropyridine receptors (DHPRs). Deletion mutants were expressed in dysgenic alpha(1S)-null myotubes and analyzed by voltage-clamp and confocal fluo-4 fluorescence. Immunostaining of the mutant subunits using an N-terminus tag revealed abundant protein expression in all cases. Furthermore, the maximum recovered charge movement density was >80% of that recovered by full-length alpha(1S) in all cases. Delta671-690 had no effect on the magnitude of voltage-evoked Ca(2+) transients or the L-type Ca(2+) current density. In contrast, Delta720-765 or Delta724-743 abolished Ca(2+) transients entirely, and L-type Ca(2+) current was reduced or absent. Surprisingly, Ca(2+) transients and Ca(2+) currents of a moderate magnitude were recovered by the double deletion mutant Delta671-690/Delta720-765. A simple explanation for this result is that Delta720-765 induces a conformation change that disrupts EC coupling, and this conformational change is partially reverted by Delta671-690. To test for Ca(2+)-entry independent EC coupling, a pore mutation (E1014K) known to entirely abolish the inward Ca(2+) current was introduced. alpha(1S) Delta671-690/Delta720-765/E1014K expressed Ca(2+) transients with Boltzmann parameters identical to those of the Ca(2+)-conducting double deletion construct. The data strongly suggest that skeletal-type EC coupling is not uniquely controlled by alpha(1S) 720-765. Other regions of alpha(1S) or other DHPR subunits must therefore directly contribute to the activation of RyR1 during EC coupling.

Structural Requirements of the Dihydropyridine Receptor α1S II-III Loop for Skeletal-type Excitation-Contraction Coupling

“…The II-III loop is not only important for transmitting this orthograde EC coupling signal to the RyR1, it also receives a retrograde, current-enhancing signal from the RyR1 to the DHPR (10,11). Beside unequivocally strong indications for an essential role of the II-III loop for this bidirectional coupling mechanism, accumulating evidence suggests an additional influence of other regions of the DHPR ␣ 1S subunit and/or of the accessory ␤ subunit (12,13).…”

“…Are Required for the Bidirectional Signaling Interaction with RyR1-In previous studies, residues Leu 720 -Leu 764 of the rabbit skeletal muscle DHPR ␣ 1S II-III loop were demonstrated to be essential for the full restoration of skeletal-type EC coupling (11,12,14,15) as well as for retrograde coupling, which is the RyR1-mediated enhancement of Ca 2ϩ influx through the DHPR (11,12,15). To better understand the molecular mechanism underlying the direct skeletal DHPR-RyR1 interaction, we identified the minimum sequence within ␣ 1S residues Leu 720 -Leu 764 required for the bidirectional coupling.…”

The recombinant dihydropyridine receptor II–III loop and partly structured ‘C’ region peptides modify cardiac ryanodine receptor activity

“…The II-III loop and its 'C' region have been studied because they are required for skeletal EC coupling and are thought to contain the parts of the DHPR that physically interact with RyR1 in this process [15][16][17]. In addition, the corresponding fragments of the DHPR interact with RyR1 in vitro.…”

Functional implications of modifying RyR‐activating peptides for membrane permeability

“…A strict alignment (Block et al, 1988) allows physical coupling between the II-III loop of the DHPR and the RyR to initiate skeletal-type excitation-contraction (EC) coupling (which is independent of external Ca 2 þ ). Although the A region of the II-III loop is not required for EC coupling (Proenza et al, 2000;Wilkens et al, 2001), it has been implicated in the overall physical interaction between the DHPR and RyR (Ahern et al, 2001).…”