Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α and β subunits of DHPR are essential for this process. Here, we employed a series of techniques, including size-exclusion chromatography-multi-angle light scattering, differential scanning fluorimetry, and isothermal calorimetry, to characterize various biophysical properties of the skeletal DHPR β subunit β Removal of the intrinsically disordered N and C termini and the hook region of β prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined β isoforms, which consist of SH3 and guanylate kinase domains. However, transition melting temperatures derived from the differential scanning fluorimetry experiments indicated a significant difference in stability of ∼2-3 °C between the β and β constructs, and the addition of the DHPR α I-II loop (α-interaction domain) peptide stabilized both β isoforms by ∼6-8 °C. Similar to other β isoforms, β bound with nanomolar affinity to the α-interaction domain, but binding affinities were influenced by amino acid substitutions in the adjacent SH3 domain. These results suggest that intramolecular interactions between the SH3 and guanylate kinase domains play a role in the stability of β while also providing a conduit for allosteric signaling events.

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β subunit and assess its role on Ca metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β subunit. Expression of the variant subunit in β-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of β-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca conductance, current kinetics, or sarcoplasmic reticulum Ca load were observed. Measurement of resting free Ca and Na concentrations shows that both cations were significantly elevated in β-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca entry through Na/Ca exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β subunit leading to a phenotype of Ca dysregulation that partly resembles that of other MH-linked mutations of DHPR α subunit. These data prove that homozygous expression of variant β-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

Excitation‐contraction coupling (ECC) is the physiological process in which an electrical signal originating from the central nervous system is converted into muscle contraction. In skeletal muscle tissue, the key step in the molecular mechanism of ECC initiated by the muscle action potential is the cooperation between two Ca2+ channels, dihydropyridine receptor (DHPR; voltage‐dependent L‐type calcium channel) and ryanodine receptor 1 (RyR1). These two channels were originally postulated to communicate with each other via direct mechanical interactions; however, the molecular details of this cooperation have remained ambiguous. Recently, it has been proposed that one or more supporting proteins are in fact required for communication of DHPR with RyR1 during the ECC process. One such protein that is increasingly believed to play a role in this interaction is the SH3 and cysteine‐rich domain‐containing protein 3 (STAC3), which has been proposed to bind a cytosolic portion of the DHPR α1S subunit known as the II–III loop. In this work, we present direct evidence for an interaction between a small peptide sequence of the II–III loop and several residues within the SH3 domains of STAC3 as well as the neuronal isoform STAC2. Differences in this interaction between STAC3 and STAC2 suggest that STAC3 possesses distinct biophysical features that are potentially important for its physiological interactions with the II–III loop. Therefore, this work demonstrates an isoform‐specific interaction between STAC3 and the II–III loop of DHPR and provides novel insights into a putative molecular mechanism behind this association in the skeletal muscle ECC process.

Ahnak is a ~ 700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca signaling in cardiomyocytes by interacting with the β subunit of the L-type Ca channel (Ca1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete H,C and N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the Ca1.2-Ahnak complex.

The starting point for structure-based drug design (SBDD) efforts is a high quality structural model obtained using X-ray crystallography or NMR spectroscopic techniques. In most instances classical tools are used as structural surrogates in X-ray and NMR refinement protocols in order to improve the parameter to observation ratio realized from these experimental techniques. While classical approaches are useful structural surrogates, they do suffer from a number of issues that affect their performance including: electrostatic modeling, parameter defects and missing parameters. The way in which these issues can be mitigated is to use more robust structural theories like quantum mechanical (QM) methods, which have had a tremendous impact on our understanding of "small" chemical and biological systems. In this talk we will focus on the application of ab initio QM methods to refine protein/ligand complexes for use in SBDD applications using NMR and X-ray methods. We will discuss the computational details and describe several uses of QM in structure refinement efforts using NMR and X-ray datasets. The strengths and weaknesses of a QM approach in structure refinement will be discussed as well as future prospects of this strategy.