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The phosphoprotein AHNAK is a large, ubiquitously expressed scaffolding protein involved in mediating a host of protein-protein interactions. This enables AHNAK to participate in various multi-protein complexes thereby orchestrating a range of diverse biological processes, including tumour suppression, immune regulation and cell architecture maintenance. A less studied but nonetheless equally important function occurs in calcium homeostasis. It does so by largely interacting with the L-type voltage-gated calcium channel (LVGCC) present in the plasma membrane of excitable cells such as muscles and neurons. Several studies have characterized the underlying basis of AHNAK's functional role in calcium channel modulation, which has led to a greater understanding of this cellular process and its associated pathologies. In this article we review and examine recent advances relating to the physiological aspects of AHNAK in calcium regulation. Specifically, we will provide a broad overview of AHNAK including its structural makeup and its interaction with several isoforms of LVGCC, and how these molecular interactions regulate calcium modulation across various tissues and their implication in muscle and neuronal function., (Copyright © 2021. Published by Elsevier Ltd.)

Aims: Our aim was to characterise the actions of novel BIT compounds with structures based on peptides and toxins that bind to significant regulatory sites on ryanodine receptor (RyR) Ca 2+ release channels. RyRs, located in sarcoplasmic reticulum (SR) Ca 2+ store membranes of striated muscle, are essential for muscle contraction. Although severe sometimes-deadly myopathies occur when the channels become hyperactive following genetic or acquired changes, specific inhibitors of RyRs are rare., Main Methods: The effect of BIT compounds was determined by spectrophotometric analysis of Ca 2+ release from isolated SR vesicles, analysis of single RyR channel activity in artificial lipid bilayers and contraction of intact and skinned skeletal muscle fibres., Key Findings: The inhibitory compounds reduced: (a) Ca 2+ release from SR vesicles with IC50s of 1.1-2.5 μM, competing with activation by parent peptides and toxins; (b) single RyR ion channel activity with IC50s of 0.5-1.5 μM; (c) skinned fibre contraction. In contrast, activating BIT compounds increased Ca 2+ release with an IC50 of 5.0 μM and channel activity with AC50s of 2 to 12 nM and enhanced skinned fibre contraction. Sub-conductance activity dominated channel activity with both inhibitors and activators. Effects of all compounds on skeletal and cardiac RyRs were similar and reversible. Competition experiments suggest that the BIT compounds bind to the regulatory helical domains of the RyRs that impact on channel gating mechanisms through long-range allosteric interactions., Significance: The BIT compounds are strong modulators of RyR activity and provide structural templates for novel research tools and drugs to combat muscle disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

There is an overwhelming body of work supporting the idea that excitation-contraction coupling in skeletal muscle depends on a physical interaction between the skeletal muscle isoform of the dihydropyridine receptor L-type Ca(2+) channel and the skeletal isoform of the ryanodine receptor Ca(2+) release channel. A general assumption is that this physical interaction is between "critical" residues that have been identified in the II-III loop of the dihydropyridine receptor alpha subunit and the ryanodine receptor. However, despite extensive searches, the complementary "critical" residues in the ryanodine receptor have not been identified. This raises the possibility that the coupling proceeds either through other subunits of the dihydropyridine receptor and/or other co-proteins within the large RyR1 protein complex. There have been some remarkable advances in recent years in identifying proteins in the RyR complex that impact on the coupling process, and these are considered in this review. A major candidate for a role in the coupling mechanism is the beta subunit of the dihydropyridine receptor, because specific residues in both the beta subunit and ryanodine receptor have been identified that facilitate an interaction between the two proteins and these also impact on excitation-contraction coupling. This role of beta subunit remains to be fully investigated as well as the degree to which it may complement any other direct or indirect voltage-dependent coupling interactions between the DHPR alpha II-III loop and the ryanodine receptor., (Copyright © 2014. Published by Elsevier Ltd.)

The adamantanes are a class of anti-influenza drugs that inhibit the M2 ion channel of the influenza A virus. However recently, the clinical effectiveness of these drugs has been called into question due to the emergence of adamantane-insensitive A/M2 mutants. Although we previously reported (1R,2R,3R,5S)-3-pinanamine 3 as a novel inhibitor of the wild type influenza A virus M2 protein (WT A/M2), limited inhibition was found for adamantane-resistant M2 mutants. In this study, we explored whether newly synthesized pinanamine derivatives were capable of inhibiting WT A/M2 and selected adamantane-resistant M2 mutants. Several imidazole and guanazole derivatives of pinanamine were found to inhibit WT A/M2 to a comparable degree as amantadine and one of these compounds 12 exhibits weak inhibition of A/M2-S31N mutant and it is marginally more effective in inhibiting S31NM2 than amantadine. This study provides a new insight into the structural nature of drugs required to inhibit WT A/M2 and its mutants., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Diazepam modulation of native γ2-containing GABA(A) (γGABA(A)) receptors increases channel conductance by facilitating protein interactions involving the γ2-subunit amphipathic (MA) region, which is found in the cytoplasmic loop between transmembrane domains 3 and 4 (Everitt et al., 2009). However, many drugs, predicted to act on different GABA(A) receptor subtypes, increase channel conductance leading us to hypothesize that conductance variation in GABA(A) receptors may be a general property, mediated by protein interactions involving the cytoplasmic MA stretch of amino acids. In this study we have tested this hypothesis by potentiating extrasynaptic GABA(A) currents with etomidate and examining the ability of peptides mimicking either the γ2- or δ-subunit MA region to affect conductance. In inside-out hippocampal patches from newborn rats the general anesthetic etomidate potentiated GABA currents, producing either an increase in open probability and single-channel conductance or an increase in open probability, as described previously (Seymour et al., 2009). In patches displaying high conductance channels application of a δ((392-422)) MA peptide, but not a scrambled version or the equivalent γ2((381-403)) MA peptide, reduced the potentiating effects of etomidate, significantly reducing single-channel conductance. In contrast, when GABA currents were potentiated by the γ2-specific drug diazepam the δ MA peptide had no effect. These data reveal that diazepam and etomidate potentiate different extrasynaptic GABA(A) receptor subtypes but both drugs modulate conductance similarly. One interpretation of the data is that these drugs elicit potentiation through protein interactions and that the MA peptides compete with these interactions to disrupt this process., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Alternative splicing of ASI residues (Ala(3481)-Gln(3485)) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(-)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr(3471)-Gly(3500)) in the splice variants. Depolarisation-dependent Ca(2+) release was enhanced by >50% in myotubes expressing ASI(-)RyR1 compared with ASI(+)RyR1, although DHPR L-type currents and SR Ca(2+) content were unaltered, while ASI(-)RyR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RyR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RyR1. The action of the peptide depended on a sequence of basic residues and their alignment in an alpha-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RyR1 that influences EC coupling. Implications for development and DM1 are discussed.

The II-III loop of the dihydropyridine receptor (DHPR) alpha(1s) subunit is a modulator of the ryanodine receptor (RyR1) Ca(2+) release channel in vitro and is essential for skeletal muscle contraction in vivo. Despite its importance, the structure of this loop has not been reported. We have investigated its structure using a suite of NMR techniques which revealed that the DHPR II-III loop is an intrinsically unstructured protein (IUP) and as such belongs to a burgeoning structural class of functionally important proteins. The loop does not possess a stable tertiary fold: it is highly flexible, with a strong N-terminal helix followed by nascent helical/turn elements and unstructured segments. Its residual structure is loosely globular with the N and C termini in close proximity. The unstructured nature of the II-III loop may allow it to easily modify its interaction with RyR1 following a surface action potential and thus initiate rapid Ca(2+) release and contraction. The in vitro binding partner for the II-III was investigated. The II-III loop interacts with the second of three structurally distinct SPRY domains in RyR1, whose function is unknown. This interaction occurs through two preformed N-terminal alpha-helical regions and a C-terminal hydrophobic element. The A peptide corresponding to the helical N-terminal region is a common probe of RyR function and binds to the same SPRY domain as the full II-III loop. Thus the second SPRY domain is an in vitro binding site for the II-III loop. The possible in vivo role of this region is discussed.

We have determined the structure of a domain peptide corresponding to the extreme 19 C-terminal residues of the ryanodine receptor Ca2+ release channel. We examined functional interactions between the peptide and the channel, in the absence and in the presence of the regulatory protein Homer. The peptide was partly alpha-helical and structurally homologous to the C-terminal end of the T1 domain of voltage-gated K+ channels. The peptide (0.1-10 microM) inhibited skeletal ryanodine receptor channels when the cytoplasmic Ca2+ concentration was 1 microM; but with 10 microM cytoplasmic Ca2+, skeletal ryanodine receptors were activated by < or = 1.0 microM peptide and inhibited by 10 microM peptide. Cardiac ryanodine receptors on the other hand were inhibited by all peptide concentrations, at both Ca2+ concentrations. When channels did open in the presence of the peptide, they were more likely to open to substate levels. The inhibition and increased fraction of openings to subconductance levels suggested that the domain peptide might destabilise inter-domain interactions that involve the C-terminal tail. We found that Homer 1b not only interacts with the channels, but reduces the inhibitory action of the C-terminal tail peptide, perhaps by stabilizing inter-domain interactions and preventing their disruption.