The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.

JGP modeling study suggests that selectivity filter constriction is a plausible mechanism for C-type inactivation of the Shaker voltage-gated potassium channel.

X-ray diffraction studies of muscle have been tremendously powerful in providing fundamental insights into the structures of, for example, the myosin and actin filaments in a variety of muscles and the physiology of the cross-bridge mechanism during the contractile cycle. However, interpretation of x-ray diffraction patterns is far from trivial, and if modeling of the observed diffraction intensities is required it needs to be performed carefully with full knowledge of the possible pitfalls. Here, we discuss (1) how x-ray diffraction can be used as a tool to monitor various specific muscle properties and (2) how to get the most out of the rest of the observed muscle x-ray diffraction patterns by modeling where the reliability of the modeling conclusions can be objectively tested. In other x-ray diffraction methods, such as protein crystallography, the reliability of every step of the process is estimated and quoted in published papers. In this way, the quality of the structure determination can be properly assessed. To be honest with ourselves in the muscle field, we need to do as near to the same as we can, within the limitations of the techniques that we are using. We discuss how this can be done. We also use test cases to reveal the dos and don’ts of using x-ray diffraction to study muscle physiology.

X-ray diffraction studies of muscle have provided a wealth of information on muscle structure and physiology, and the meridian of the diffraction pattern is particularly informative. Reconditi et al. (2014. J. Physiol. https://doi.org/10.1113/jphysiol.2013.267849 ) performed superb experiments on changes to the M3 meridional peak as a function of sarcomere length (SL). They found that the M3 intensity dropped almost linearly as sarcomere length increased at least to about SL = 3.0 µm, and that it followed the same track as tension, pointing toward zero at the end of overlap at ∼3.6 µm. They concluded that, just as tension could only be generated by overlapped myosin heads, so ordered myosin heads contributing to the M3 intensity could only occur in the overlap region of the A-band, and that nonoverlapped heads must be highly disordered. Here we show that this conclusion is not consistent with x-ray diffraction theory; it would not explain their observations. We discuss one possible reason for the change in M3 intensity with increasing sarcomere length in terms of increasing axial misalignment of the myosin filaments that at longer sarcomere lengths is limited by the elastic stretching of the M-band and titin.

Single-channel recordings reveal that norfluoxetine inhibits the two-pore domain K⁺ channel TREK-2 by a complex array of mechanisms.

JGP study shows that the phosphorylation state of cMyBPC modulates the ability of omecamtiv mecarbil to enhance myocardial force generation.

JGP study shows that the subendocardium is more susceptible to spontaneous Ca²⁺ release events that can initiate arrhythmias, and this may be reduced by local CaMKII inhibition.

JGP study demonstrates how recordings from neuron–HEK cell cocultures provide a clearer picture of the factors involved in synaptic transmission.

Pannexin 1 (Panx1) plays a decisive role in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It is widely accepted that Panx1 exerts its role in the context of purinergic signaling by providing a transmembrane pathway for ATP. However, under certain conditions, Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability. A recent flurry of publications has provided structural information about the Panx1 channel. However, while these structures are consistent with a chloride selective channel, none show a conformation with strong support for the ATP release function of Panx1. In this Viewpoint, we critically assess the existing evidence for the function and structure of the Panx1 channel and conclude that the structure corresponding to the ATP permeation pathway is yet to be determined. We also list a set of additional topics needing attention and propose ways to attain the large-pore, ATP-permeable conformation of the Panx1 channel.

Our review focuses on sarcomere regulatory mechanisms with a discussion of cardiac-specific modifications to the three-state model of thin filament activation from a blocked to closed to open state. We discuss modulation of these thin filament transitions by Ca²⁺, by crossbridge interactions, and by thick filament–associated proteins, cardiac myosin–binding protein C (cMyBP-C), cardiac regulatory light chain (cRLC), and titin. Emerging evidence supports the idea that the cooperative activation of the thin filaments despite a single Ca²⁺ triggering regulatory site on troponin C (cTnC) cannot be considered in isolation of other functional domains of the sarcomere. We discuss long- and short-range interactions among these domains with the regulatory units of thin filaments, including proteins at the barbed end at the Z-disc and the pointed end near the M-band. Important to these discussions is the ever-increasing understanding of the role of cMyBP-C, cRLC, and titin filaments. Detailed knowledge of these control processes is critical to the understanding of mechanisms sustaining physiological cardiac state with varying hemodynamic load, to better defining genetic and acquired cardiac disorders, and to developing targets for therapies at the level of the sarcomeres.