Coordinated Movement of Cytoplasmic and Transmembrane Domains of RyR1 upon Gating

Abstract

Ryanodine receptor type 1 (RyR1) produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 Å resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic “inner branches” and the transmembrane “inner helices”). Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 Å diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right-handed bundle structures along a common 4-fold axis.

How to open a Ryanodine Receptor

“…The socalled central domain plays the role of 'relay', linking these movements to the U-motif and thus the TM region. Right around the 4-fold symmetry axis, the region of the cap containing the Nterminal domains moves upward and outward, confirming previous studies [5,9]. Bai et al already see similar movements of the cap in distinct closed states.…”

“…Again, cryo-EM has proven to be invaluable in solving this puzzle. In 2009, Samso et al [5] compared open and closed RyR1 at ~10 Å resolution, and this work has now been further refined at higher resolution by Wei et al [6] and Bai et al [7] in Cell Research. Both groups come to similar conclusions on the movements within the transmembrane region, and offer distinct viewpoints about the role of the cytosolic cap.…”

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…Most cryo-EM studies on RyRs, (Radermacher et al 1992;Radermacher et al 1994;Serysheva et al 1995;Orlova et al 1996;Sharma et al 1998;Serysheva et al 1999;Benacquista et al 2000;Sharma et al 2000;Ludtke et al 2005;Samsó et al 2005;Serysheva et al 2005;Serysheva et al 2008;Samsó et al 2009) and all the subnanometer resolution analysis (Serysheva et al 2008;Samsó et al 2009) have focused on the RyR1, however, some progress has been made with RyR2 (Sharma et al 1998;Liu et al 2002) and RyR3 Liu et al 2001). Overall, the structures of all three isoforms are similar, consistent with the high sequence homology ( 65%).…”

“…The clamps (Fig. 3, subdomains 5, 6, 7, 8, 9, 10) undergo major conformational changes during the opening and closing of the channel (Serysheva et al 2008;Samsó et al 2009), are likely to participate in intermolecular interactions with neighboring RyRs, and are the sites of interactions with modulators Wagenknecht et al 1996;Wagenknecht et al 1997;Samsó and Wagenknecht 2002;Samsó et al 2006;Sharma et al 2006;Meng et al 2009). Two of the areas of high divergence in the primary sequence of the RyR isoforms were mapped in the clamps Liu et al 2004).…”

“…Although these studies Samsó et al 2005) were performed at similar conditions, they differ in interpretations, one study suggesting conformational similarity to the open K þ channel ) and the other suggesting a structure more similar to the closed K þ channel (Samsó et al 2005;Samsó et al 2009). The conformation of the open RyR has so far only been proposed to resemble the conformation of the open K þ channel (Samsó et al 2009). The pore region has been predicted to consist of between 4 and 12 transmembrane segments (Takeshima et al 1989;Zorzato et al 1990;Tunwell et al 1996;Du et al 2002).…”