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.
Highlights
Maintaining a precise intracellular Ca2þ concentration that is 10,000-fold lower than the surrounding environment of the cell, and the ability to dramatically increase intracellular calcium to trigger downstream events in response to specific stimulus are key for cell survival [1]
The typical flickering behavior of Ryanodine receptor type 1 (RyR1) under physiologic activating conditions represents a significant limitation, since it produces a mixed population of open and closed states, e.g., under maximum Ca2þ activating conditions (50 lM Ca2þ on the cytoplasmic side), the channel open probability (Po) of reconstituted purified RyR1-FK506-binding protein 12 kDa (FKBP12) channels is less than 30%
The bilayer lipid membrane (BLM) studies of reconstituted purified RyR1-FKBP12 channels indicate that PCB 95 stabilizes the full open state in ten out of ten reconstituted channels, resulting in extremely long-lived openings interspersed with rare short-lived transitions to the closed state
Summary
Maintaining a precise intracellular Ca2þ concentration that is 10,000-fold lower than the surrounding environment of the cell, and the ability to dramatically increase intracellular calcium to trigger downstream events in response to specific stimulus are key for cell survival [1]. Ryanodine receptors (RyRs) are high-conductance intracellular Ca2þ channels regulated by both exogenous and intracellular mediators, which release Ca2þ stored in the endoplasmic reticulum. RyRs are the largest ion channels known, with an average molecular weight of 2.26 MDa, with most of its mass (;4/5) forming the cytoplasmic domain. The skeletal muscle isoform, RyR1, has a bidirectional interaction with the slow voltage-gated calcium channel in the cell membrane, or dihydropyridine receptor (DHPR), which acts as RyR19s voltage sensor for cell membrane depolarization [2]. Two key questions to understand RyR19s function are how are signals transmitted from peripheral cytoplasmic domains to the ion gate, and what is the gating mechanism itself. It has been possible to map the binding sites for several ligands: the FK506-binding protein
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