Excitation–contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720–L764 of the α(1S) II-III loop

The II-III loop of the skeletal muscle dihydropyridine receptor (DHPR) α(1S) subunit is responsible for bidirectional-signaling interactions with the ryanodine receptor (RyR1): transmitting an orthograde, excitation–contraction (EC) coupling signal to RyR1 and receiving a retrograde, current-enhancing signal from RyR1. Previously, several reports argued for the importance of two distinct regions of the skeletal II-III loop (residues R681–L690 and residues L720–Q765, respectively), claiming for each a key function in DHPR–RyR1 communication. To address whether residues 720–765 of the II-III loop are sufficient to enable skeletal-type (Ca(2+) entry-independent) EC coupling and retrograde interaction with RyR1, we constructed a green fluorescent protein (GFP)-tagged chimera (GFP-SkLM) having rabbit skeletal (Sk) DHPR sequence except for a II-III loop (L) from the DHPR of the house fly, Musca domestica (M). The Musca II-III loop (75% dissimilarity to α(1S)) has no similarity to α(1S) in the regions R681–L690 and L720–Q765. GFP-SkLM expressed in dysgenic myotubes (which lack endogenous α(1S) subunits) was unable to restore EC coupling and displayed strongly reduced Ca(2+) current densities despite normal surface expression levels and correct triad targeting (colocalization with RyR1). Introducing rabbit α(1S) residues L720–L764 into the Musca II-III loop of GFP-SkLM (substitution for Musca DHPR residues E724–T755) completely restored bidirectional coupling, indicating its dependence on α(1S) loop residues 720–764 but its independence from other regions of the loop. Thus, 45 α(1S)-residues embedded in a very dissimilar background are sufficient to restore bidirectional coupling, indicating that these residues may be a site of a protein–protein interaction required for bidirectional coupling.