The RyR1 P3528S Substitution Alters Mouse Skeletal Muscle Contractile Properties and RyR1 Ion Channel Gating.

The recessive Ryanodine Receptor Type 1 (RyR1) P3527S mutation causes mild muscle weakness in patients and increased resting cytoplasmic [Ca2+] in transformed lymphoblastoid cells. In the present study, we explored the cellular/molecular effects of this mutation in a mouse model of the mutation (RyR1 P3528S). The results were obtained from 73 wild type (WT/WT), 82 heterozygous (WT/MUT) and 66 homozygous (MUT/MUT) mice with different numbers of observations in individual data sets depending on the experimental protocol. The results showed that WT/MUT and MUT/MUT mouse strength was less than that of WT/WT mice, but there was no difference between genotypes in appearance, weight, mobility or longevity. The force frequency response of extensor digitorum longus (EDL) and soleus (SOL) muscles from WT/MUT and MUT/MUT mice was shifter to higher frequencies. The specific force of EDL muscles was reduced and Ca2+ activation of skinned fibres shifted to a lower [Ca2+], with an increase in type I fibres in EDL muscles and in mixed type I/II fibres in SOL muscles. The relative activity of RyR1 channels exposed to 1 µM cytoplasmic Ca2+ was greater in WT/MUT and MUT/MUT mice than in WT/WT mice. We suggest the altered RyR1 activity due to the P2328S substitution could increase resting [Ca2+] in muscle fibres, leading to changes in fibre type and contractile properties.

Ion channel gating in cardiac ryanodine receptors from the arrhythmic RyR2-P2328S mouse.

Mutations in the cardiac ryanodine receptor Ca2+ release channel (RyR2) can cause deadly ventricular arrhythmias and atrial fibrillation (AF). The RyR2-P2328S mutation produces catecholaminergic polymorphic ventricular tachycardia (CPVT) and AF in hearts from homozygous RyR2P2328S/P2328S (denoted RyR2S/S) mice. We have now examined P2328S RyR2 channels from RyR2S/S hearts. The activity of wild-type (WT) and P2328S RyR2 channels was similar at a cytoplasmic [Ca2+] of 1 mM, but P2328S RyR2 was significantly more active than WT at a cytoplasmic [Ca2+] of 1 µM. This was associated with a >10-fold shift in the half maximal activation concentration (AC50) for Ca2+ activation, from ∼3.5 µM Ca2+ in WT RyR2 to ∼320 nM in P2328S channels and an unexpected >1000-fold shift in the half maximal inhibitory concentration (IC50) for inactivation from ∼50 mM in WT channels to ≤7 µM in P2328S channels, which is into systolic [Ca2+] levels. Unexpectedly, the shift in Ca2+ activation was not associated with changes in sub-conductance activity, S2806 or S2814 phosphorylation or the level of FKBP12 (also known as FKBP1A) bound to the channels. The changes in channel activity seen with the P2328S mutation correlate with altered Ca2+ homeostasis in myocytes from RyR2S/S mice and the CPVT and AF phenotypes.This article has an associated First Person interview with the first author of the paper.

Association of FK506 binding proteins with RyR channels - effect of CLIC2 binding on sub-conductance opening and FKBP binding.

Ryanodine receptor (RyR) Ca2+ channels are central to striated muscle function and influence signalling in neurons and other cell types. Beneficially low RyR activity and maximum conductance opening may be stabilised when RyRs bind to FK506 binding proteins (FKBPs) and destabilised by FKBP dissociation, with submaximal opening during RyR hyperactivity associated with myopathies and neurological disorders. However, the correlation with submaximal opening is debated and quantitative evidence is lacking. Here, we have measured altered FKBP binding to RyRs and submaximal activity with addition of wild-type (WT) CLIC2, an inhibitory RyR ligand, or its H101Q mutant that hyperactivates RyRs, which probably causes cardiac and intellectual abnormalities. The proportion of sub-conductance opening increases with WT and H101Q CLIC2 and is correlated with reduced FKBP-RyR association. The sub-conductance opening reduces RyR currents in the presence of WT CLIC2. In contrast, sub-conductance openings contribute to excess RyR 'leak' with H101Q CLIC2. There are significant FKBP and RyR isoform-specific actions of CLIC2, rapamycin and FK506 on FKBP-RyR association. The results show that FKBPs do influence RyR gating and would contribute to excess Ca2+ release in this CLIC2 RyR channelopathy.

Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-ß1a subunit (CACNB1).

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of ß1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca2+ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR ß1a subunit and assess its role on Ca2+ 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 ß1a subunit. Expression of the variant subunit in ß1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of ß1a-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca2+ conductance, current kinetics, or sarcoplasmic reticulum Ca2+ load were observed. Measurement of resting free Ca2+ and Na+ concentrations shows that both cations were significantly elevated in ß1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca2+ entry through Na+/Ca2+ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of ß1a subunit leading to a phenotype of Ca2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR α1S subunit. These data prove that homozygous expression of variant ß1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

Structural and biophysical analyses of the skeletal dihydropyridine receptor ß subunit ß1a reveal critical roles of domain interactions for stability.

Excitation-contraction (EC) coupling in skeletal muscle requires a physical interaction between the voltage-gated calcium channel dihydropyridine receptor (DHPR) and the ryanodine receptor Ca2+ release channel. Although the exact molecular mechanism that initiates skeletal EC coupling is unresolved, it is clear that both the α1 and ß 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 ß subunit ß1a Removal of the intrinsically disordered N and C termini and the hook region of ß1a prevented oligomerization, allowing for its structural determination by X-ray crystallography. The structure had a topology similar to that of previously determined ß 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 ß1a and ß2a constructs, and the addition of the DHPR α1s I-II loop (α-interaction domain) peptide stabilized both ß isoforms by ∼6-8 °C. Similar to other ß isoforms, ß1a 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 ß1a while also providing a conduit for allosteric signaling events.

The Anthracycline Metabolite Doxorubicinol Abolishes RyR2 Sensitivity to Physiological Changes in Luminal Ca2+ through an Interaction with Calsequestrin.

The chemotherapeutic anthracycline metabolite doxorubicinol (doxOL) has been shown to interact with and disrupt the function of the cardiac ryanodine receptor Ca2+ release channel (RyR2) in the sarcoplasmic reticulum (SR) membrane and the SR Ca2+ binding protein calsequestrin 2 (CSQ2). Normal increases in RyR2 activity in response to increasing diastolic SR [Ca2+] are influenced by CSQ2 and are disrupted in arrhythmic conditions. Therefore, we explored the action of doxOL on RyR2's response to changes in luminal [Ca2+] seen during diastole. DoxOL abolished the increase in RyR2 activity when luminal Ca2+ was increased from 0.1 to 1.5 mM. This was not due to RyR2 oxidation, but depended entirely on the presence of CSQ2 in the RyR2 complex. DoxOL binding to CSQ2 reduced both the Ca2+ binding capacity of CSQ2 (by 48%-58%) and its aggregation, and lowered CSQ2 association with the RyR2 complex by 67%-77%. Each of these effects on CSQ2, and the lost RyR2 response to changes in luminal [Ca2+], was duplicated by exposing native RyR2 channels to subphysiologic (≤1.0 µM) luminal [Ca2+]. We suggest that doxOL and low luminal Ca2+ both disrupt the CSQ2 polymer, and that the association of the monomeric protein with the RyR2 complex shifts the increase in RyR2 activity with increasing luminal [Ca2+] away from the physiologic [Ca2+] range. Subsequently, these changes may render the channel insensitive to changes of luminal Ca2+ that occur through the cardiac cycle. The altered interactions between CSQ2, triadin, and/or junctin and RyR2 may produce an arrhythmogenic substrate in anthracycline-induced cardiotoxicity.

A new cytoplasmic interaction between junctin and ryanodine receptor Ca2+ release channels.

Junctin, a non-catalytic splice variant encoded by the aspartate-ß-hydroxylase (Asph) gene, is inserted into the membrane of the sarcoplasmic reticulum (SR) Ca(2+) store where it modifies Ca(2+) signalling in the heart and skeletal muscle through its regulation of ryanodine receptor (RyR) Ca(2+) release channels. Junctin is required for normal muscle function as its knockout leads to abnormal Ca(2+) signalling, muscle dysfunction and cardiac arrhythmia. However, the nature of the molecular interaction between junctin and RyRs is largely unknown and was assumed to occur only in the SR lumen. We find that there is substantial binding of RyRs to full junctin, and the junctin luminal and, unexpectedly, cytoplasmic domains. Binding of these different junctin domains had distinct effects on RyR1 and RyR2 activity: full junctin in the luminal solution increased RyR channel activity by ∼threefold, the C-terminal luminal interaction inhibited RyR channel activity by ∼50%, and the N-terminal cytoplasmic binding produced an ∼fivefold increase in RyR activity. The cytoplasmic interaction between junctin and RyR is required for luminal binding to replicate the influence of full junctin on RyR1 and RyR2 activity. The C-terminal domain of junctin binds to residues including the S1-S2 linker of RyR1 and N-terminal domain of junctin binds between RyR1 residues 1078 and 2156.

Core skeletal muscle ryanodine receptor calcium release complex.

The core skeletal muscle ryanodine receptor (RyR1) calcium release complex extends through three compartments of the muscle fibre, linking the extracellular environment through the cytoplasmic junctional gap to the lumen of the internal sarcoplasmic reticulum (SR) calcium store. The protein complex is essential for skeletal excitation-contraction (EC)-coupling and skeletal muscle function. Its importance is highlighted by perinatal death if any one of the EC-coupling components are missing and by myopathies associated with mutation of any of the proteins. The proteins essential for EC-coupling include the DHPR α1S subunit in the transverse tubule membrane, the DHPR ß1a subunit in the cytosol and the RyR1 ion channel in the SR membrane. The other core proteins are triadin and junctin and calsequestrin, associated mainly with SR. These SR proteins are not essential for survival but exert structural and functional influences that modify the gain of EC-coupling and maintain normal muscle function. This review summarises our current knowledge of the individual protein/protein interactions within the core complex and their overall contribution to EC-coupling. We highlight significant areas that provide a continuing challenge for the field. Additional important components of the Ca2+ release complex, such as FKBP12, calmodulin, S100A1 and Stac3 are identified and reviewed elsewhere.

Unexpected dependence of RyR1 splice variant expression in human lower limb muscles on fiber-type composition.

The skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1), essential for excitation-contraction (EC) coupling, demonstrates a known developmentally regulated alternative splicing in the ASI region. We now find unexpectedly that the expression of the splice variants is closely related to fiber type in adult human lower limb muscles. We examined the distribution of myosin heavy chain isoforms and ASI splice variants in gluteus minimus, gluteus medius and vastus medialis from patients aged 45 to 85 years. There was a strong positive correlation between ASI(+)RyR1 and the percentage of type 2 fibers in the muscles (r = 0.725), and a correspondingly strong negative correlation between the percentages of ASI(+)RyR1 and percentage of type 1 fibers. When the type 2 fiber data were separated into type 2X and type 2A, the correlation with ASI(+)RyR1 was stronger in type 2X fibers (r = 0.781) than in type 2A fibers (r = 0.461). There was no significant correlation between age and either fiber-type composition or ASI(+)RyR1/ASI(-)RyR1 ratio. The results suggest that the reduced expression of ASI(-)RyR1 during development may reflect a reduction in type 1 fibers during development. Preferential expression of ASI(-) RyR1, having a higher gain of in Ca(2+) release during EC coupling than ASI(+)RyR1, may compensate for the reduced terminal cisternae volume, fewer junctional contacts and reduced charge movement in type 1 fibers.

Cardiac ryanodine receptor activation by a high Ca²âº store load is reversed in a reducing cytoplasmic redox environment.

Here, we report the impact of redox potential on isolated cardiac ryanodine receptor (RyR2) channel activity and its response to physiological changes in luminal [Ca(2+)]. Basal leak from the sarcoplasmic reticulum is required for normal Ca(2+) handling, but excess diastolic Ca(2+) leak attributed to oxidative stress is thought to lower the threshold of RyR2 for spontaneous sarcoplasmic reticulum Ca(2+) release, thus inducing arrhythmia in pathological situations. Therefore, we examined the RyR2 response to luminal [Ca(2+)] under reducing or oxidising cytoplasmic redox conditions. Unexpectedly, as luminal [Ca(2+)] increased from 0.1 to 1.5 mM, RyR2 activity declined when pretreated with cytoplasmic 1 mM DTT or buffered with GSH∶GSSG to a normal reduced cytoplasmic redox potential (-220 mV). Conversely, with 20 µM cytoplasmic 4,4'-DTDP or buffering of the redox potential to an oxidising value (-180 mV), RyR2 activity increased with increasing luminal [Ca(2+)]. The luminal redox potential was constant at -180 mV in each case. These responses to luminal [Ca(2+)] were maintained with cytoplasmic 2 mM Na2ATP or 5 mM MgATP (1 mM free Mg(2+)). Overall, the results suggest that the redox potential in the RyR2 junctional microdomain is normally more oxidised than that of the bulk cytoplasm.

Multiple actions of phi-LITX-Lw1a on ryanodine receptors reveal a functional link between scorpion DDH and ICK toxins.

We recently reported the isolation of a scorpion toxin named U1-liotoxin-Lw1a (U1-LITX-Lw1a) that adopts an unusual 3D fold termed the disulfide-directed hairpin (DDH) motif, which is the proposed evolutionary structural precursor of the three-disulfide-containing inhibitor cystine knot (ICK) motif found widely in animals and plants. Here we reveal that U1-LITX-Lw1a targets and activates the mammalian ryanodine receptor intracellular calcium release channel (RyR) with high (fM) potency and provides a functional link between DDH and ICK scorpion toxins. Moreover, U1-LITX-Lw1a, now described as ϕ-liotoxin-Lw1a (ϕ-LITX-Lw1a), has a similar mode of action on RyRs as scorpion calcines, although with significantly greater potency, inducing full channel openings at lower (fM) toxin concentrations whereas at higher pM concentrations increasing the frequency and duration of channel openings to a submaximal state. In addition, we show that the C-terminal residue of ϕ-LITX-Lw1a is crucial for the increase in full receptor openings but not for the increase in receptor subconductance opening, thereby supporting the two-binding-site hypothesis of scorpion toxins on RyRs. ϕ-LITX-Lw1a has potential both as a pharmacological tool and as a lead molecule for the treatment of human diseases that involve RyRs, such as malignant hyperthermia and polymorphic ventricular tachycardia.

ß1a490-508, a 19-residue peptide from C-terminal tail of Cav1.1 ß1a subunit, potentiates voltage-dependent calcium release in adult skeletal muscle fibers.

The α1 and ß1a subunits of the skeletal muscle calcium channel, Cav1.1, as well as the Ca(2+) release channel, ryanodine receptor (RyR1), are essential for excitation-contraction coupling. RyR1 channel activity is modulated by the ß1a subunit and this effect can be mimicked by a peptide (ß1a490-524) corresponding to the 35-residue C-terminal tail of the ß1a subunit. Protein-protein interaction assays confirmed a high-affinity interaction between the C-terminal tail of the ß1a and RyR1. Based on previous results using overlapping peptides tested on isolated RyR1, we hypothesized that a 19-amino-acid residue peptide (ß1a490-508) is sufficient to reproduce activating effects of ß1a490-524. Here we examined the effects of ß1a490-508 on Ca(2+) release and Ca(2+) currents in adult skeletal muscle fibers subjected to voltage-clamp and on RyR1 channel activity after incorporating sarcoplasmic reticulum vesicles into lipid bilayers. ß1a490-508 (25 nM) increased the peak Ca(2+) release flux by 49% in muscle fibers. Considerably fewer activating effects were observed using 6.25, 100, and 400 nM of ß1a490-508 in fibers. ß1a490-508 also increased RyR1 channel activity in bilayers and Cav1.1 currents in fibers. A scrambled form of ß1a490-508 peptide was used as negative control and produced negligible effects on Ca(2+) release flux and RyR1 activity. Our results show that the ß1a490-508 peptide contains molecular components sufficient to modulate excitation-contraction coupling in adult muscle fibers.

Adverse effects of doxorubicin and its metabolic product on cardiac RyR2 and SERCA2A.

The use of anthracycline chemotherapeutic drugs is restricted owing to potentially fatal cardiotoxic side effects. It has been hypothesized that anthracycline metabolites have a primary role in this cardiac dysfunction; however, information on the molecular interactions of these compounds in the heart is scarce. Here we provide novel evidence that doxorubicin and its metabolite, doxorubicinol, bind to the cardiac ryanodine receptor (RyR2) and to the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA2A) and deleteriously alter their activity. Both drugs (0.01 µM-2.5 µM) activated single RyR2 channels, and this was reversed by drug washout. Both drugs caused a secondary inhibition of RyR2 activity that was not reversed by drug washout. Preincubation with the reducing agent dithiothreitol (DTT, 1 mM) prevented drug-induced inhibition of channel activity. Doxorubicin and doxorubicinol reduced the abundance of thiol groups on RyR2, further indicating that oxidation reactions may be involved in the actions of the compounds. Ca(2+) uptake into sarcoplasmic reticulum vesicles by SERCA2A was inhibited by doxorubicinol, but not doxorubicin. Unexpectedly, in the presence of DTT, doxorubicinol enhanced the rate of Ca(2+) uptake by SERCA2A. Together the evidence provided here shows that doxorubicin and doxorubicinol interact with RyR2 and SERCA2A in similar ways, but that the metabolite acts with greater efficacy than the parent compound. Both compounds modify RyR2 and SERCA2A activity by binding to the proteins and also act via thiol oxidation to disrupt SR Ca(2+) handling. These actions would have severe consequences on cardiomyocyte function and contribute to clinical symptoms of acute anthracycline cardiotoxicity.

An X-linked channelopathy with cardiomegaly due to a CLIC2 mutation enhancing ryanodine receptor channel activity.

Chloride intracellular channel 2 (CLIC2) protein is a member of the glutathione transferase class of proteins. Its' only known function is the regulation of ryanodine receptor (RyR) intracellular Ca(2+) release channels. These RyR proteins play a major role in the regulation of Ca(2+) signaling in many cells. Utilizing exome capture and deep sequencing of genes on the X-chromosome, we have identified a mutation in CLIC2 (c.303C>G, p.H101Q) which is associated with X-linked intellectual disability (ID), atrial fibrillation, cardiomegaly, congestive heart failure (CHF), some somatic features and seizures. Functional studies of the H101Q variant indicated that it stimulated rather than inhibited the action of RyR channels, with channels remaining open for longer times and potentially amplifying Ca(2+) signals dependent on RyR channel activity. The overly active RyRs in cardiac and skeletal muscle cells and neuronal cells would result in abnormal cardiac function and trigger post-synaptic pathways and neurotransmitter release. The presence of both cardiomegaly and CHF in the two affected males and atrial fibrillation in one are consistent with abnormal RyR2 channel function. Since the dysfunction of RyR2 channels in the brain via 'leaky mutations' can result in mild developmental delay and seizures, our data also suggest a vital role for the CLIC2 protein in maintaining normal cognitive function via its interaction with RyRs in the brain. Therefore, our patients appear to suffer from a new channelopathy comprised of ID, seizures and cardiac problems because of enhanced Ca(2+) release through RyRs in neuronal cells and cardiac muscle cells.

An α-helical C-terminal tail segment of the skeletal L-type Ca2+ channel ß1a subunit activates ryanodine receptor type 1 via a hydrophobic surface.

Excitation-contraction (EC) coupling in skeletal muscle depends on protein interactions between the transverse tubule dihydropyridine receptor (DHPR) voltage sensor and intracellular ryanodine receptor (RyR1) calcium release channel. We present novel data showing that the C-terminal 35 residues of the ß(1a) subunit adopt a nascent α-helix in which 3 hydrophobic residues align to form a hydrophobic surface that binds to RyR1 isolated from rabbit skeletal muscle. Mutation of the hydrophobic residues (L496, L500, W503) in peptide ß(1a)V490-M524, corresponding to the C-terminal 35 residues of ß(1a), reduced peptide binding to RyR1 to 15.2 ± 7.1% and prevented the 2.9 ± 0.2-fold activation of RyR1 by 10 nM wild-type peptide. An upstream hydrophobic heptad repeat implicated in ß(1a) binding to RyR1 does not contribute to RyR1 activation. Wild-type ß(1a)A474-A508 peptide (10 nM), containing heptad repeat and hydrophobic surface residues, increased RyR1 activity by 2.3 ± 0.2- and 2.2 ± 0.3-fold after mutation of the heptad repeat residues. We conclude that specific hydrophobic surface residues in the 35 residue ß(1a) C-terminus bind to RyR1 and increase channel activity in lipid bilayers and thus may support skeletal EC coupling.

Biophysical reviews top five: voltage-dependent charge movement in nerve and muscle.

The discovery of gating currents and asymmetric charge movement in the early 1970s represented a remarkable leap forward in our understanding of the biophysical basis of voltage-dependent events that underlie electrical signalling that is vital for nerve and muscle function. Gating currents and charge movement reflect a fundamental process in which charged amino acid residues in an ion channel protein move in response to a change in the membrane electrical field and therefore activate the specific voltage-dependent response of that protein. The detection of gating currents and asymmetric charge movement over the past 50 years has been pivotal in unraveling the multiple molecular and intra-molecular processes which lead to action potentials in excitable tissues and excitation-contraction (EC) coupling in skeletal muscle. The recording of gating currents and asymmetric charge movement remains an essential component of investigations into the basic molecular mechanisms of neuronal conduction and muscle contraction.

Feedback contributions to excitation-contraction coupling in native functioning striated muscle.

Skeletal and cardiac muscle excitation-contraction coupling commences with Nav1.4/Nav1.5-mediated, surface and transverse (T-) tubular, action potential generation. This initiates feedforward, allosteric or Ca2+-mediated, T-sarcoplasmic reticular (SR) junctional, voltage sensor-Cav1.1/Cav1.2 and ryanodine receptor-RyR1/RyR2 interaction. We review recent structural, physiological and translational studies on possible feedback actions of the resulting SR Ca2+ release on Nav1.4/Nav1.5 function in native muscle. Finite-element modelling predicted potentially regulatory T-SR junctional [Ca2+]TSR domains. Nav1.4/Nav1.5, III-IV linker and C-terminal domain structures included Ca2+ and/or calmodulin-binding sites whose mutations corresponded to specific clinical conditions. Loose-patch-clamped native murine skeletal muscle fibres and cardiomyocytes showed reduced Na+ currents (INa) following SR Ca2+ release induced by the Epac and direct RyR1/RyR2 activators, 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate and caffeine, abrogated by the RyR inhibitor dantrolene. Conversely, dantrolene and the Ca2+-ATPase inhibitor cyclopiazonic acid increased INa. Experimental, catecholaminergic polymorphic ventricular tachycardic RyR2-P2328S and metabolically deficient Pgc1ß-/- cardiomyocytes also showed reduced INa accompanying [Ca2+]i abnormalities rescued by dantrolene- and flecainide-mediated RyR block. Finally, hydroxychloroquine challenge implicated action potential (AP) prolongation in slowing AP conduction through modifying Ca2+ transients. The corresponding tissue/organ preparations each showed pro-arrhythmic, slowed AP upstrokes and conduction velocities. We finally extend discussion of possible Ca2+-mediated effects to further, Ca2+, K+ and Cl-, channel types. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Cyclization of the intrinsically disordered α1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function.

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α(1S) subunit of the dihydropyridine receptor (DHPR). The II-III loop has been previously examined in vitro using a linear II-III loop with unrestrained N- and C-terminal ends. To better reproduce the loop structure in its native environment (tethered to the DHPR transmembrane domains), we have joined the N and C termini using intein-mediated technology. Circular dichroism and NMR spectroscopy revealed a structural shift in the cyclized loop toward a protein with increased α-helical and ß-strand structure in a region of the loop implicated in its in vitro function and also in a critical region for EC coupling. The affinity of binding of the II-III loop binding to the SPRY2 domain of the skeletal ryanodine receptor (RyR1) increased 4-fold, and its ability to activate RyR1 channels in lipid bilayers was enhanced 3-fold by cyclization. These functional changes were predicted consequences of the structural enhancement. We suggest that tethering the N and C termini stabilized secondary structural elements in the DHPR II-III loop and may reflect structural and dynamic characteristics of the loop that are inherent in EC coupling.