Giving structure meaning: Modelling the cellular synapse of excitation–contraction coupling in skeletal muscle

Early electron microscopy studies showed large macromolecular entities in the transverse tubular (TT)-SR junction whose main constituents were later identified as the voltage-sensitive calcium channel Ca_V1.1 of the TT membrane and the giant SR calcium release channel (RyR). Both can nowadays be explored with cryogenic electron microscopy (cryo-EM) techniques at near atomic resolution (Shishmarev, 2020; Xu et al, 2024). These two proteins are assembled in regular quasicrystalline complexes, called couplons (Franzini-Armstrong, 2018; Rios, 2026; Block et al., 1988), which in mouse skeletal muscle reach a length of up to about 1 μm, consisting of a double row of RyR tetramers (isoform RyR1) in which every other is associated with a tetrad of Ca_V1.1 channels (Fig. 1 A). Couplons provide the structural substrate for the control of Ca²⁺ release by the TT membrane voltage conveyed by the voltage sensors of Ca_V1.1. Other than in cardiac muscle, it is not the inward current passed by the Ca_V channel that initiates SR Ca²⁺ release (Dayal et al., 2017). Instead, the coupling between the two proteins appears to be merely conformational (Flucher and Campiglio, 2019).

The pattern of the channel arrangement in the couplon suggests a crucial mechanistic role and a tight functional interaction among its individual elements. The interesting fact that every other RyR1 is void of an association with Ca_V1.1 prompted speculations on the nature of this interaction. Because RyRs are Ca²⁺ sensitive (Ríos, 2018; Laver, 2018), one possible scenario suggests that those RyRs (V channels) that are in contact with Ca_V1.1 release a “trigger” Ca²⁺ from the SR, whereas the others (C channels), activated by the local rise in Ca²⁺ concentration, cause further release in a regenerative positive feedback mechanism. One condition that must be met by any valid model of excitation–contraction coupling (EC coupling) is the graded dependence of Ca²⁺ release on voltage, which might be at odds with a regenerative Ca²⁺ response. This is where mathematical modelling and comparison with experimental results can provide important clues. Previous such studies demonstrated that a local regenerative rise of Ca²⁺ release needs not necessarily be in conflict with graded voltage control (Stern et al., 1997; Stephenson, 2024). The orderly arrangement of RyR1 channels would also permit conformational transmission between these proteins. Evidence for neighboring release channels influencing each other came from the statistical analysis of RyR current fluctuations in artificial bilayers, leading to the concept of “coupled gating” (Marx et al., 1998). The study by Rios and Pizarro (2026) explores the voltage response of a model couplon that acts as a continuum by allosteric transmission between individual RyR1 channels.

The quality of a model depends on the precision by which it describes experimental results. In a large number of studies, skeletal muscle Ca²⁺ signals were assessed, globally on the whole-cell level as well as locally in cellular micro-domains. Those measurements were often combined with voltage clamp techniques to maintain control of Ca²⁺ release via the membrane potential (Hernández-Ochoa and Schneider, 2012). To estimate the kinetics of the Ca²⁺ release flux during step depolarizations, optically recorded Ca²⁺ signals were analyzed by us and others by fitting the measurements with models that describe cytoplasmic Ca²⁺ binding and transport (Melzer et al., 1987; Ursu et al., 2005). Fig. 1 B shows the typical shape of the estimated flux of Ca²⁺ release. Characteristic is the rapid decay from an early peak to an almost constant much lower level. Double step experiments with varying time intervals showed that the peak recovers within less than a second, a time much shorter than it would take the SERCA pump to restore SR Ca²⁺ from depletion (Sárközi et al., 2000; Schneider et al., 1987). Therefore, the decay must result from some sort of inactivation. Its molecular origin is still unresolved. Calcium seems to be involved, and potential molecular determinants have been addressed using genetically modified mice (Prosser et al., 2011).

Moreover, Ca²⁺ release events at the level of individual couplons in micro-domains of the mammalian muscle cells have been studied by confocal microscopy and were termed “embers” (Zhou et al., 2003). They respond in a graded fashion to voltage (Csernoch et al., 2004), indicating that basic features of voltage-controlled SR Ca²⁺ permeability are already present on the single couplon level in contrast to other tissues (heart, frog skeletal muscle) that show “sparks” as the elementary Ca²⁺ events (Cserne Szappanos et al., 2026).

Rios and Pizarro (2026) calculated the statistical fluctuations in a cluster with allosterically interacting elements, allowing them to simulate experimental data both at the molecular level and at the cell level (by summing individual runs). Their standard model couplon consists of 60 RyRs (V + C) plus the corresponding Ca_V channels in the arrangement shown in the middle part of Fig. 1 A. The gating schemes of the individual channels contain resting and active states plus two inactive states, the latter exclusively assigned to the C channels (Fig. 1 B). The stochastic state transitions of the cluster entities during voltage activation were computed using Markov chain methods extending an approach applied in a previous publication (Stern et al., 1997). This model defines the couplon as an excitable structure in which channel opening can propagate while Ca²⁺ release remains strictly under voltage control. It is rewarding to see how well it simulates the various known features of voltage-controlled Ca²⁺ release and (with shorter couplons) also the phenomena of coupled gating observed in bilayer work. For simplicity, it is assumed that the C channels are responsible for most of the Ca²⁺ permeability and the peaky part of the macroscopic release flux, whereas the V channels function primarily as recipients of the signal from the voltage sensors of Ca_V1.1 and produce the much smaller non-inactivating component of the flux (Fig. 1 B). Further research will tell if this assumption is true, but it is conceivable that steric hindrance by the large Ca_V1.1 proteins may restrict the Ca²⁺ flux through the V channels. Although abstract and formal, the simulations provide important insights, in particular into the role and necessity of the inactivation process. It could be learned that the inactivation is essential for maintaining voltage control over the Ca²⁺ flux of the couplon. Reducing or removing it would make the couplon respond in an all or none manner. Inactivation, as implemented in the gating scheme, also explains other observations obtained with special pulse paradigms, indicating that only channels that were opened by a voltage step inactivate and do so completely, a behavior called “deterministic inactivation” (Szentesi et al., 2000).

The study by Rios and Pizarro is a successful attempt to link the mysterious structure of the EC coupling signalling machine to a possible function. Model building is always part of the scientific process to understand nature. Its task is to determine whether certain mechanistic ideas are compatible with experimental findings or not. In this sense, the present model works very well. With just a few basic assumptions, it can simulate fundamental features of EC coupling, determined both globally and locally. Further experiments may challenge this model or lead to its refinement. As a new piece in the EC coupling puzzle, it may also be joined to available ideas on how Ca²⁺ is handled outside and inside of the SR (Royer and Ríos, 2009) with the prospect of developing a large scale model to simulate the whole chain of events. Further, this study should encourage the search for the molecular determinants of the proposed allosteric signal transmission within the couplon (Xu et al., 2024). Regions critical for the V–C interaction might be affected in some forms of RyR-based diseases. For instance, malignant hyperthermia mutations cause a lower threshold for Ca²⁺ release activation by chemical effectors like volatile anesthetics and the membrane potential (Zullo et al., 2018). Alterations of the allosteric coupling between V and C channels could result in similar effects.

The road to a full understanding of the intra- and intermolecular rearrangements that lead from voltage sensing in Ca_V1.1 to SR Ca²⁺ release may still be long, but the dramatic progress in the field of structural biology and molecular dynamics gives reason to be confident. The promising final goal is a complete molecular model of the EC coupling mechanism that will also explain the biophysical reasons for the regular pattern of Ca²⁺ channel proteins in the couplon and their different preferences for interaction.

Olaf S. Andersen served as an editor.

Author contributions: Werner Melzer: conceptualization, visualization, and writing—original draft, review, and editing.