Regions of ryanodine receptors that influence activation by the dihydropyridine receptor β1a subunit
© Rebbeck et al. 2015
Received: 5 April 2015
Accepted: 2 July 2015
Published: 22 July 2015
Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR β1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of β1a activation of RyRs and the β1a binding site on RyR1.
We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes.
We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10–100 nM of the β1a subunit. Activation of RyR2 by 10 nM β1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for β1a. A reduction in activation was also observed when 10 nM β1a was added to the alternatively spliced (ASI(−)) isoform of RyR1, which lacks ASI residues (A3481-Q3485). It is notable that the equivalent region of RyR2 also lacks four of five ASI residues, suggesting that the absence of these residues may contribute to the reduced 10 nM β1a activation observed for both RyR2 and ASI(−)RyR1 compared to ASI(+)RyR1. We also investigated the influence of a polybasic motif (PBM) of RyR1 (K3495KKRRDGR3502) that is located immediately downstream from the ASI residues and has been implicated in EC coupling. We confirmed that neutralizing the basic residues in the PBM (RyR1 K-Q) results in an ~50 % reduction in Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for β1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of β1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca2+ release generated during skeletal EC coupling.
We conclude that the β1a subunit likely binds to a region that is largely conserved in RyR1 and RyR2 and that this region is influenced by the presence of the ASI residues and the PBM in RyR1.
KeywordsExcitation-contraction coupling Dihydropyridine receptor β1a subunit Ryanodine receptor isoforms Skeletal muscle Cardiac muscle
Contraction in skeletal and cardiac muscle depends on Ca2+ release from the intracellular sarcoplasmic reticulum (SR) Ca2+ store through ryanodine receptor (RyR) Ca2+ release channels embedded in the SR membrane. This Ca2+ release is crucial to excitation-contraction (EC) coupling. During EC coupling, cardiac RyRs (RyR2) are activated by an influx of extracellular Ca2+ through depolarization-activated dihydropyridine receptor (DHPR) L-type channels located in the surface and transverse-tubule membranes. In contrast, EC coupling in skeletal muscle is independent of extracellular Ca2+, apparently requiring a physical interaction between skeletal isoforms of the RyR (RyR1) and DHPR [1, 2]. However, despite exhaustive investigation, the physical components of this interaction still remain unclear [3, 4] and are investigated in this manuscript.
It is well established that the skeletal isoforms of both the membrane spanning α1S subunit and the cytoplasmic β1a subunit of the DHPR heteropentamer are essential for skeletal EC coupling [5, 6]. The α1S subunit contains the voltage sensor for EC coupling [7, 8] and the “critical” region for skeletal EC coupling (residues L720-764/5) in its intracellular II-III loop [9–11]. The β1a subunit is responsible for the targeting of the DHPR to the triad and assembly into tetrads that are closely aligned with RyR1 in the SR [12–14]. There is also evidence that the β1a subunit also plays an active role in the EC coupling process. The β1a subunit directly activates RyR1 channels incorporated into lipid bilayers and enhances voltage-activated Ca2+ release in skeletal muscle fibers [5, 15, 16]. The C-terminal region of β1a (V490-M524) supports β1a binding to RyR1 in vitro and influences voltage-induced Ca2+ release in mouse myotubes [15, 17, 18]. A peptide corresponding to the same residues mimics full length β1a subunit activation of RyR1 channels in lipid bilayers  and a truncated peptide of the same region enhances voltage-induced Ca2+ release to the same degree as the full length β1a subunit in intact adult mouse muscle fibers [16, 19]. Furthermore, overexpression of a β subunit interacting protein, Rem, in adult mouse skeletal muscle fibers was recently shown to reduce voltage-induced Ca2+ transients by ~65 % without substantially altering α1S subunit membrane targeting or intramembrane gating charge movement or SR Ca2+ store content . This suggests that the DHPR-RyR1 interaction may be uncoupled by virtue of direct interference of β1a subunit. Residues in RyR1 that influence binding to the β1a subunit have also been identified. The M3201-W3661 fragment of RyR1 binds to β1a and the strength of binding is substantially reduced by replacing the six basic residues in a polybasic motif (PBM; K3495KKRRDGR3502) with glutamines . Replacement of the same six residues with glutamines in the full-length RyR protein substantially reduces depolarization-dependent Ca2+ release . The in vitro studies indicate a high-affinity interaction between the isolated RyR1 and the β1a subunit that is influenced by the PBM. However, the basic residues unlikely bind directly to the hydrophobic residues in the β1a C-terminus, although they could contribute to the overall conformation of the binding domain . Similarly, it is unlikely that basic residue binding to the hydrophobic residues could contribute to EC coupling, although both basic residues and hydrophobic residues in the β1a C-terminus influence EC coupling [16, 19].
The fact that skeletal DHPR and RyR isoforms are critical for skeletal-type EC coupling [22–26] suggests that isoform-specific regions of these proteins enable unique interactions in skeletal muscle. Also, in the context of isoform dependence, we reported that an alternatively spliced region of RyR1 (A3481-Q3485), located close to the PBM, is significant in setting the gain of EC coupling . It is notable that RyR2 lacks the equivalent sequence to the ASI residues in ASI(+)RyR1 and, in this respect, more closely resembles the ASI(−)RyR1 isoform. Therefore, here we examined the RyR isoform dependence of the in vitro interaction with the β1a subunit. We use the RyR isoforms as tools to explore regions of the RyR1 that influence its interaction with the C-tail of the β1a subunit. Interactions between RyR2 and the cardiac β subunit were not examined as they have no physiological significance, and there is little sequence homology between the C-terminal tails of the cardiac and skeletal β isoforms [12–14].
The results indicate that while β1a activates RyR1 and RyR2 isolated from the skeletal muscle and the heart and activates recombinant ASI(−)RyR1 and ASI(+)RyR1, β1a activation of RyR2 and AS1(−)RyR1 requires higher β1a concentrations than that required to activate RyR1 or ASI(+)RyR1. In addition, we show that neutralization of the basic residues in the RyR1 PBM abolishes β1a activation of RyR1 in lipid bilayers and confirm that this also markedly reduces voltage-dependent Ca2+ release in skeletal myotubes. Together, the results reinforce the conclusion that β1a binding to RyR1 contributes to EC coupling and suggest that the region encompassing the adjacent ASI residues and PBM is a determinant of β1a binding to and regulation of RyR1.
The work was approved by The Australian National University Animal Experimentation Ethics Committee (Australian Capital Territory, Australia) and by the University Committee on Animal Resources at the University of Rochester (New York, USA).
Preparation of RyR1 ASI (−) and K-Q cDNA
The ASI(−)RyR1 variant was introduced into rabbit RyR1 cDNA (accession #X15750) using two-step site-directed mutagenesis as described previously . The K-Q mutant (K3495KKRRGDR3502) was similarly introduced into a rabbit RyR1 cDNA by two-step site-directed mutagenesis in the following manner: using a BsiWI/BamHI subclone of RyR1, residues R3498Q and R3499Q were introduced via mutagenesis to create a double mutation (R3498Q/R3499Q). This mutant was used as a template to introduce a third mutation R3502Q. Finally, glutamine substitutions for residues K3495, K3496, and K3497 were introduced into the triple mutated plasmid to generate the PBM mutant Q3495QQQQGDQ3502 (K-Q mutant). The entire PCR-modified cDNA portion of the BswiWI/BamHI mutant subclone was confirmed by sequence analysis and then cloned back into full-length RyR1.
Preparation of SR vesicles
Skeletal muscle SR vesicles were prepared from back and leg muscles (fast twitch skeletal muscle) from New Zealand white rabbits [29–31] and cardiac SR vesicles collected from sheep hearts [32, 33]. Vesicles were stored at −70 °C.
Transfection and preparation of microsomal protein
Microsomal vesicles were collected from HEK293 transfected with recombinant rabbit RyR1 ASI(+), ASI(−), or K-Q RyR1 mutant cDNAs in mammalian expression vector (pCIneo) as described previously  with minor modifications. HEK cells were grown in 175-mm2 flasks at 37 °C, 5 % CO2 in 10 % fetal calf serum in MEM. At 50–60 % confluence, cells were transfected with 80 μg cDNA in a phosphate buffer solution (125 mM CaCl2, 70 mM NaH2PO4, 140 mM NaCl, 76 mM HEPES, 7 mM Na2HPO4, pH 7.2) using a calcium phosphate precipitation method. Cells were maintained for 48 h and then harvested in phosphate buffer (137 mM NaCl; 7 mM Na2HPO4; 2.5 mM NaH2PO4.H2O; and, 2 mM EGTA, pH 7.4). The pellet was resuspended in homogenizing buffer (300 mM sucrose, 5 mM imidazole, 1× complete EDTA-free protease inhibitor cocktail, pH 7.4), homogenized and centrifuged at 11,600 × g for 20 min. The resulting pellet was resuspended in homogenizing buffer, further homogenized and centrifuged at 91,943 × g for 2 h. The pellet was resuspended in homogenizing buffer, homogenized, and then briefly sonicated. The microsomal mixture was separated into 15 μL aliquots and stored at −70 °C.
Preparation and injection of dyspedic myotubes
Primary cultures of myotubes were obtained from skeletal myoblasts isolated from newborn RyR1-null (dyspedic) mice as previously described [34, 35]. Four to 6 days after initial plating of myoblasts, nuclei of dyspedic myotubes were microinjected with cDNAs encoding CD8 (0.1 μg/μl) and the appropriate RyR1 expression plasmid (0.5 μg/μl) . Expressing myotubes were identified 2–4 days after cDNA microinjection by incubation with CD8 antibody beads (Dynabeads, Dynal USA). All animals were housed in a pathogen-free area at the University of Rochester and experiments performed in accordance with procedures reviewed and approved by the local University Committees on Animal Resources.
Preparation of β1a subunit
The β1a protein was expressed in transformed Escherichia coli BL21(DE3) and purified as described previously . The proteins were dialyzed against a phosphate buffer (50 mM Na3PO4, 300 mM NaCl, pH 8) and stored at −70 °C.
Single-channel recording and analysis
Channels from cardiac, skeletal, or HEK293 microsomal vesicles were incorporated into lipid bilayers with solutions containing (mM): cis (20 CsCl, 230 CsCH3O3S, 10 TES, and 1 CaCl2) and trans (20 CsCl, 30 CsCH3O3S, 10 mM TES, and 1 CaCl2), pH 7.2. After RyR incorporation, 200 mM CsMS was added to the trans solution for symmetrical [Cs+]. BAPTA was added to the cis solution as determined with a Ca2+ electrode to achieve 10 μM Ca2+, and 2 mM ATP was added. Bilayer potential, Vcis-Vtrans, was switched between −40 and +40 mV. Channel activity under each condition was analyzed over 180 s using the program Channel 2 (developed by P. W. Gage and M. Smith). Threshold levels for channel opening were set to exclude baseline noise at ~20 % of the maximum single-channel conductance and open probability (P o ), mean open time (T o ), and closed open time (T c ) measured. Dwell-time distributions for each channel were obtained using the log-bin method [37–39]. Event frequency (probability) was plotted against equally spaced bins (on a logarithmic scale) for open or closed durations (seven bins per decade). The time constants are indicated by the frequency peaks. The area under each peak indicates the fraction of single-channel open or closed events falling into each time constant component.
Simultaneous measurements of macroscopic Ca2+ currents and transients in myotubes
Parameters of fitted I-V and [ΔF/F]-V curves
G max (nS/nF)
V half (mV)
V half (mV)
WT RyR1 (n = 12)
264 ± 16
5.4 ± 0.4
10.5 ± 1.8
71 ± 1.8
3.4 ± 0.7
3.9 ± 0.5
-4.7 ± 1.6
RyR1 K-Q (n = 10)
201 ± 17*
5.7 ± 0.3
11.5 ± 1.7
70 ± 2.1
1.6 ± 0.3*
4.1 ± 04.
-2.5 ± 1.7
RyR-null (dyspedic) myotubes expressing either WT RyR or RyR K-Q mutant that were plated on glass coverslips were fixed and immunostained with a mouse monoclonal anti-RyR antibody (34C, 1:10; Developmental Studies Hybridoma Bank) and a sheep polyclonal anti-DHPR antibody (1:200; Upstate Biotechnology) overnight at 4 °C as previously described . On the following day, coverslips were washed with PBS three times each for 5 min and then incubated for 1 h at room temperature in blocking buffer containing a 1:500 dilution of Alexa Fluor 488–labeled donkey anti-mouse IgG (Molecular Probes) and 1:500 dilution of rhodamine-labeled donkey anti-sheep IgG (Jackson ImmunoResearch Laboratories Inc.) and washed with PBS (three times for 5 min each). Coverslips were mounted on glass slides and images obtained using a Nikon Eclipse-C1 confocal microscope (Nikon Instruments Inc.) and a 40× oil objective. All confocal images were sampled at a spatial resolution (pixel diameter) of 100 nm.
Average data are given as the mean ± SEM. Statistical significance was evaluated by a paired or unpaired two-way Student’s t-test or analysis of variance (ANOVA) with Fisher’s post hoc test, as appropriate. The numbers of observations (N) are given in the figure legends. To reduce the effects of variability in control single-channel activity parameters (P oC , T cC , T oC ) and to evaluate parameters after β1a subunit (P oB , T cB , T oB ) addition, data were expressed as the difference between the logarithmic values, i.e., log10 rel P o = log10 P oB –log10 P oC . The difference from control was assessed with a paired t-test applied to log10 P oC and log10 P oB . Variance in P o parameter values was assessed with an unpaired t-test. A p value of <0.05 was considered significant.
Ability of the β1a subunit to activate different RyR isoforms
The β1a subunit activates RyR1 and RyR2 channels
The action of β1a on single-channel gating parameters (Fig. 2b, c) reflected the changes in P o (Fig. 2a, left and right). Both RyR1 and RyR2 activity increased with 10 and 100 nM β1a as a result of increases in mean channel open time and an abbreviation of mean channel closed time (Fig. 2b, c). There was also a trend towards a greater increase in mean open time in RyR2 with the higher β1a concentration that is consistent with the greater RyR2 open probability in the presence of 100 nM β1a. In contrast, RyR1 mean open time was similar at both β1a concentrations. Mean closed times were similarly reduced for both RyR isoforms by 10 and 100 nM β1a.
Both 10 and 100 nM concentrations of β1a subunit decreased the fraction of RyR2 openings in τ o1 by 18.7 ± 1.8 % (p = 0.003) and 16.3 ± 2.0 % (p = 0.012), respectively (Fig. 4b). There was a corresponding increase in the fraction of events for the longer open time constant components at both β1a concentrations (Fig. 4b). In contrast to RyR2, the maximal increases in RyR1 activity after exposure to 10 or 100 nM β1a subunit were reflected in a reduction in the fraction of RyR1 open events in τ o1 and increases in events in the longer time constant group (τ o2) at both β1a concentrations (Fig. 4a). The closed time constant distributions in RyR2 and RyR1 were also altered by both 10 and 100 nM β1a, albeit in slightly different ways. There was an apparent transfer of 14.9 ± 3.1 % of closed events in RyR2 from τ c2 to τ c1 with 10 nM β1a and 13.8 ± 4.7 % with 100 nM β1a (Fig. 4b). In contrast, for RyR1, there were fewer long closed events in τ c3 and more short closed events in τ c1 with both 10 and 100 nM β1a than in control (Fig. 4a).
Overall, the results indicate that 10 and 100 nM β1a increase both RyR1 and RyR2 activity but with a reduced activation of RyR2 by 10 nM β1a. The dwell-time distributions indicate subtle differences between RyR1 and RyR2 in the effects of β1a in redistribution between the different time constant components. In particular, β1a induced a significant increase in events in the longest open time constant component in RyR2 but not RyR1 activity, while significantly reducing the number of events in the longest closed time constant component of RyR1 but not RyR2 activity.
The alternatively spliced ASI residues impact the functional interaction between β1a and RyR1
Subconductance activity has been associated with full or partial depletion of FKBP12 from RyRs [29, 30]. Densitometry measurements of immunoprobed RyR and FKBP following co-immunoprecipitation of the RyR1 complex indicate a 65 % reduction (p = 0.014) in FKBP bound to RyR1 in recombinant ASI(+) RyR1 when compared to native RyR1 isolated from muscle. Thus, the sub-conductance activity observed for the recombinant channels is consistent with reduced FKBP12 expression in HEK293 cells and reduced amounts associated with the recombinant RyR1 channels.
Cytoplasmic addition of 10 nM β1a to ASI(+)RyR1 channels produced a significant ~4.4-fold increase in relative P o and a significantly smaller ~2.3-fold increase in relative P o of ASI(-)RyR1 (Fig. 5c). There was no significant difference between the degree of activation of the two RyR1 splice variants following application of 50 nM β1a (Fig. 5c), so that the efficacy of 10 nM β1a on ASI(−)RyR1 isoform appears to be less than that on ASI(+)RyR1. Therefore, the responses of both ASI(-)RyR1 and RyR2 that lack the ASI sequence to application of 10 nM β1a are significantly reduced compared with RyR proteins that contain the ASI sequence, i.e., ASI(+)RyR1 or adult RyR1 isolated from rabbit skeletal muscle.
The impact of the polybasic K3495-R3502 residues on EC coupling and β1a activation of RyR1
The RyR1 polybasic motif facilitates EC coupling in expressing dyspedic myotubes
The PMB (residues K3495-R3502) in RyR1, located immediately downstream from the ASI region (A3481-Q3485), has been implicated in β1a binding to RyR1 and EC coupling . To assess the effect of the PMB on the interaction between β1a and RyR1 channels in bilayers, a mutant of RyR1 in which all six polybasic residues were substituted with glutamines (RyR1 K-Q) was constructed. The functional effects of the RyR1 K-Q mutant on voltage-gated SR Ca2+ release and DHPR L-type currents were confirmed following expression in dyspedic myotubes .
The small reduction in G max is unlikely to fully account for the large reduction in voltage-induced SR Ca2+ release observed in RyR1 K-Q-expressing myotubes (Fig. 6d). This is supported by the sigmoidal voltage dependence of peak Ca2+ release, a feature of skeletal-type EC coupling demonstrating that Ca2+ release is independent of Ca2+ influx. The reduction in depolarization-induced DHPR currents and SR Ca2+ release could have resulted from poor targeting of the RyR1 K-Q mutant to the triad junction. However, double immunofluorescence labeling of RyR1 and the DHPR α1 subunit in expressing myotubes indicates that the DHPR and RyR1 proteins similarly co-localized as indicated by the yellow puncta in the overlays shown in Additional file 1. Therefore, compared to WT RyR1, the efficacy of voltage-induced SR Ca2+ release is reduced in RyR1 K-Q-expressing myotubes.
The polybasic motif in RyR1 is required for β1a activation of RyR1
We explored the possibility that the reduction in efficiency of depolarization-induced Ca2+ release was due to an effect of the K-Q substitution on gating properties of the RyR channel or to its response to cytoplasmic [Ca2+] or ATP. RyR1 K-Q mutant channels exhibited unitary conductance of 222.3 ± 18.5 pS at +40 and 247.3 ± 33.1 pS at −40 mV, similar (p = 0.07–0.78) to that of WT RyR1 (311.9 ± 25.2 pS at +40 and 236.5 ± 12.5 pS at −40 mV), or ~220 pS as previously reported for WT RyR1 expressed in HEK293 cells under the recording conditions used in this study .
The results presented here provide novel insight into the regions of RyR1 that influence the action of the β1a subunit on RyR1 activity and have implications for the role of the β1a subunit in skeletal muscle EC coupling. Our results demonstrate that the functional effect of 100 nM β1a subunit is conserved between RyR1 and RyR2, although the activation by 10 nM β1a was lower in RyR2 than in RyR1. Interestingly, a difference was also observed for the activation of ASI(−)RyR1 and ASI(+)RyR1 isoforms by 10 nM β1a, in that the lower concentration of β1a was also less effective in activating ASI(−)RyR1 than ASI(+)RyR1. In contrast to the maintained, although different, activation of the two RyR isoforms by the β1a subunit, neutralization of the PBM in RyR1 abolished β1a activation of RyR1. One interpretation of this finding is that the ~50 % reduction in depolarization-dependent Ca2+ release results from disruption of direct β1a activation of RyR1 during EC coupling.
The action of β1a subunit on RyR1 and RyR2 channel activity is largely conserved
The activation of RyR1 and RyR2 by β1a suggests that the β1a binding site is conserved across these RyR isoforms. The small concentration-dependent differences between effects on RyR1 and RyR2 suggest minor differences in either the binding residues or the binding pocket that reduces the affinity of β1a for RyR2 (and ASI(−)RyR1). It is difficult to identify specific sequences that could account for the different affinities for β1a as there is a 13.2 % (>600 residues) sequence disparity between RyR1 and RyR2 isoforms, according to a CLUSTALW multiple alignment  of rabbit RyR1 [Swiss-Prot: P11716.1] and rabbit RyR2 [Swiss-Prot: P30957.3]. Given that the string of positive residues is reduced from six to five, this variation is unlikely to account for the observed concentration-dependent difference between β1a modulation of the two isoforms, although such a possibility cannot be fully excluded. Interestingly, except for one additional positive charge in the RyR1, the PBM is conserved in RyR2 (RyR1: K3495KKRR_ _ R3502 and RyR2: K3452MKRK_ _R3459) and thus is unlikely to account for the observed concentration-dependent difference between β1a modulation of the two isoforms. However, just upstream from the PBM, four of the five ASI residues in RyR1 (A3481-Q3485) are missing from the rabbit (and predicted pig) RyR2 sequence. It may be significant that the lack of ASI residues in full-length ASI(−)RyR1 reduces the efficacy of 10 nM β1a-mediated activation. Thus, it is plausible that the difference between β1a modulation of RyR1 and RyR2 is partially due to the presence or absence of the ASI residues, respectively. The conservation of the modulatory effect of β1a on RyR1 and RyR2 does not reflect the in vivo studies showing that RyR2 is unable to replace RyR1 in skeletal muscle EC coupling [25, 46]. However, the lack of skeletal muscle EC coupling in RyR2-expressing dyspedic myotubes is most likely due to the fact that DHPR tetrads are not restored in RyR2-expressing dyspedic myotubes , indicating that β1a is unable to correctly align DHPRs with RyR2 in order to ensure a direct interaction between the two proteins. It is also possible that the II-III loop critical region is unable to engage with RyR2 through β1a.
The importance of the RyR1 polybasic motif for β1a subunit increase in RyR1 channel activity
The role of the RyR1 PBM in the β1a-mediated increase in channel activity was assessed from the response of recombinant RyR1 K-Q channels in bilayers to the addition of the β1a subunit. RyR1 K-Q and WT RyR1 channel conductance and regulation by cytoplasmic modulators were similar, indicating that RyR1 K-Q channels function normally. However, RyR1 K-Q channel activity was unaltered by the β1a subunit. Therefore, the reduction in voltage-gated Ca2+ release observed in RyR1 K-Q-expressing myotubes is likely to reflect a specific effect of the polybasic residues on β1a subunit regulation of RyR1 channel activity during EC coupling rather than a general effect on RyR1 channel function. However, we cannot rule out the possibility that modest differences in RyR1 expression contribute to the reduced L-channel conductance and voltage-gated Ca2+ release in K-Q-expressing myotubes, although this seems unlikely given previous reports of 4-chloro-m-cresol stimulated SR Ca2+ release in myotubes RyR1 K-Q  and the data in Additional file 1.
Given that the PBM in the larger M3201-W3661 fragment of RyR1 is required for pull down of the β1a subunit , it is likely that the lack of an effect of β1a on RyR1 K-Q channels is due to the inability of β1a to bind to the PBM mutant channel. Alternatively, the PBM may be important for maintaining RyR1 in a conformation permissive for β1a binding, rather than directly contributing to binding, as the RyR1 basic residues would be unlikely to interact with the hydrophobic residues in the β1a C-terminal domain (L496, L500, and W503) previously shown to bind RyR1 . In addition, although the PBM is implicated in ASI-mediated inter-domain inhibition of RyR1 [27, 43], the structure of this motif is not altered by substituting three of the six basic residues with alanine residues . Thus, neutralization of the PBM more likely disrupts the inter-domain interaction rather than changes the intrinsic structure of the ASI-polybasic region. In this case, disruption of the RyR1 PBM inter-domain interaction may alter an essential conformation of the β1a binding site or prevent β1a access to its binding site on RyR1.
The β1a subunit is unlikely to be the sole signaling conduit between the DHPR and RyR1 during EC coupling. Consistent with this, expression of the RyR1 K-Q mutant in dyspedic myotubes partially restored sigmoidal, depolarization-dependent Ca2+ release even though β1a modulation of RyR1 in bilayers was abolished. In addition, previous studies have also shown that truncation of β1a C-terminal residues, essential for β1a modulation of RyR1, also reduces but does not abolish depolarization-induced Ca2+ release [17, 40], an outcome that was also observed in adult skeletal muscle fibers that overexpressed a β1a subunit interacting protein, Rem . Finally, alanine substitution of β1a subunit hydrophobic triplet residues (L496, L500, and W503) only partially reduces depolarization-induced Ca2+ release in β1a null myotubes , despite this mutation fully abolishing β1a modulation of RyR1 activity in vitro .
The role of the RyR1 ASI residues in β1a subunit increase in RyR1 channel activity
It is curious that 10 nM β1a subunit increased ASI(−)RyR1 activity less than ASI(+)RyR1 given that EC coupling is enhanced in dyspedic myotubes that express ASI(−)RyR1 relative to ASI(+)RyR1 . The greater activation of ASI(+)RyR1 by 10 nM β1a is consistent with effects reported previously of agonists of RyR1, including caffeine and 4-chloro-m-cresol [27, 28]. Thus, the increased gain of EC coupling observed for ASI(-)RyR1 may not reflect a contribution of the β1a subunit to EC coupling. However, it is possible that activation of RyR1 by agonist binding includes a common mechanism for activation by agonists that differs from that involved in EC coupling. As it is likely that more than one interaction between RyR1 and the DHPR is involved in EC coupling, the combined result of these interactions may produce different effects on the two alternatively spliced variants such that ASI(−)RyR1 channels are activated more strongly by depolarization than ASI(+)RyR1 channels.
The possibility that the ASI region is involved in an inhibitory inter-domain interaction was previously investigated using peptides corresponding to the ASI region from T3471–G3500 . The peptide corresponding to the ASI(−) sequence was more effective in activating ASI(-)RyR1 than ASI(+)RyR1. Together with the finding that ASI(−)RyR1 channels were generally less active than ASI(+)RyR1 channels, these findings suggest that stronger inhibitory inter-domain interactions may exist in ASI(−)RyR1. It is possible then that the triggering mechanism activated during EC coupling disrupts this inhibitory inter-domain interaction giving rise to greater activation of ASI(-)RyR1. This disruption may not occur with RyR1 agonist binding and indeed a stronger inhibitory inter-domain interaction in ASI(-)RyR1 may even oppose activation by β1a and other agonists, allowing for these triggers to more strongly activate ASI(+)RyR1 channels.
The results presented in this study suggest that a functional β1a interaction is conserved between RyR1 and RyR2 and that β1a activation of RyRs is regulated by the presence of the ASI residues. Importantly, we also show that the PBM residues are essential for direct activation of RyR1 by β1a subunit in vitro. This suggests that the ~50 % reduction in Ca2+ release during EC coupling in dyspedic myotubes expressing RyR1 with a neutralized PBM is due to removal of β1a activation of RyR1, and hence, that other DHPR-RyR1 coupling elements (e.g., II-III loop critical domain) contribute to transmission of the remaining Ca2+ release during EC coupling.
alternatively splicing region I
skeletal ryanodine receptor
cardiac ryanodine receptor
The authors are grateful to Suzy Pace and to Joan Stivala for assistance with the preparation of skeletal and cardiac muscle SR vesicles. We also thank Dr. PD. Allen for providing access to the dyspedic mice used in this study. The work was supported by grants from the Australian National Health and Medical Research Council, APP1020589 and APP APP1002589 to AFD, MGC, and PGB, Muscular Dystrophy Association (MDA275574) and National Institutes of Health (AR059646) to RTD, a Career development award (APP1003985) to NAB, an Australian Postgraduate Award to RTR, and an Australia National University postgraduate award to HW.
- Dirksen RT. Bi-directional coupling between dihydropyridine receptors and ryanodine receptors. Front Biosci. 2002;7:d659–670.PubMedView ArticleGoogle Scholar
- Dulhunty AF, Haarmann CS, Green D, Laver DR, Board PG, Casarotto MG. Interactions between dihydropyridine receptors and ryanodine receptors in striated muscle. Prog Biophys Mol Biol. 2002;79:45–75.PubMedView ArticleGoogle Scholar
- Rebbeck RT, Karunasekara Y, Board PG, Beard NA, Casarotto MG, Dulhunty AF. Skeletal muscle excitation-contraction coupling: who are the dancing partners? Int J Biochem Cell Biol. 2014;48:28–38.PubMedView ArticleGoogle Scholar
- Beam KG, Bannister RA. Looking for answers to EC coupling’s persistent questions. J Gen Physiol. 2010;136:7–12.PubMed CentralPubMedView ArticleGoogle Scholar
- Gregg RG, Messing A, Strube C, Beurg M, Moss R, Behan M, et al. Absence of the beta subunit (cchb1) of the skeletal muscle dihydropyridine receptor alters expression of the alpha 1 subunit and eliminates excitation-contraction coupling. Proc Natl Acad Sci U S A. 1996;93:13961–6.
- Tanabe T, Beam KG, Powell JA, Numa S. Restoration of excitation-contraction coupling and slow calcium current in dysgenic muscle by dihydropyridine receptor complementary DNA. Nature. 1988;336:134–9.PubMedView ArticleGoogle Scholar
- Rios E, Brum G. Involvement of dihydropyridine receptors in excitation-contraction coupling in skeletal muscle. Nature. 1987;325:717–20.PubMedView ArticleGoogle Scholar
- Schneider MF, Chandler WK. Voltage dependent charge movement of skeletal muscle: a possible step in excitation-contraction coupling. Nature. 1973;242:244–6.PubMedView ArticleGoogle Scholar
- Nakai J, Tanabe T, Konno T, Adams B, Beam KG. Localization in the II-III loop of the dihydropyridine receptor of a sequence critical for excitation-contraction coupling. J Biol Chem. 1998;273:24983–6.PubMedView ArticleGoogle Scholar
- Takekura H, Paolini C, Franzini-Armstrong C, Kugler G, Grabner M, Flucher BE. Differential contribution of skeletal and cardiac II-III loop sequences to the assembly of dihydropyridine-receptor arrays in skeletal muscle. Mol Biol Cell. 2004;15:5408–19.PubMed CentralPubMedView ArticleGoogle Scholar
- Wilkens CM, Kasielke N, Flucher BE, Beam KG, Grabner M. Excitation-contraction coupling is unaffected by drastic alteration of the sequence surrounding residues L720-L764 of the alpha 1S II-III loop. Proc Natl Acad Sci U S A. 2001;98:5892–7.PubMed CentralPubMedView ArticleGoogle Scholar
- Schredelseker J, Dayal A, Schwerte T, Franzini-Armstrong C, Grabner M. Proper restoration of excitation-contraction coupling in the dihydropyridine receptor beta1-null zebrafish relaxed is an exclusive function of the beta1a subunit. J Biol Chem. 2009;284:1242–51.PubMed CentralPubMedView ArticleGoogle Scholar
- Dayal A, Bhat V, Franzini-Armstrong C, Grabner M. Domain cooperativity in the beta1a subunit is essential for dihydropyridine receptor voltage sensing in skeletal muscle. Proc Natl Acad Sci U S A. 2013;110:7488–93.PubMed CentralPubMedView ArticleGoogle Scholar
- Eltit JM, Franzini-Armstrong C, Perez CF. Amino acid residues 489–503 of dihydropyridine receptor (DHPR) beta1a subunit are critical for structural communication between the skeletal muscle DHPR complex and type 1 ryanodine receptor. J Biol Chem. 2014;289:36116–24.PubMedView ArticleGoogle Scholar
- Rebbeck RT, Karunasekara Y, Gallant EM, Board PG, Beard NA, Casarotto MG, et al. The beta(1a) subunit of the skeletal DHPR binds to skeletal RyR1 and activates the channel via its 35-residue C-terminal tail. Biophys J. 2011;100:922–30.
- Garcia MC, Carrillo E, Galindo JM, Hernandez A, Copello JA, Fill M, et al. Short-term regulation of excitation-contraction coupling by the beta1a subunit in adult mouse skeletal muscle. Biophys J. 2005;89:3976–84.
- Beurg M, Ahern CA, Vallejo P, Conklin MW, Powers PA, Gregg RG, et al. Involvement of the carboxy-terminus region of the dihydropyridine receptor beta1a subunit in excitation-contraction coupling of skeletal muscle. Biophys J. 1999;77:2953–67.
- Cheng W, Altafaj X, Ronjat M, Coronado R. Interaction between the dihydropyridine receptor Ca2+ channel beta-subunit and ryanodine receptor type 1 strengthens excitation-contraction coupling. Proc Natl Acad Sci U S A. 2005;102:19225–30.PubMed CentralPubMedView ArticleGoogle Scholar
- Hernandez-Ochoa EO, Olojo RO, Rebbeck RT, Dulhunty AF, Schneider MF. beta1a490-508, a 19-residue peptide from C-terminal tail of Cav1.1 beta1a subunit, potentiates voltage-dependent calcium release in adult skeletal muscle fibers. Biophys J. 2014;106:535–47.PubMed CentralPubMedView ArticleGoogle Scholar
- Beqollari D, Romberg CF, Filipova D, Meza U, Papadopoulos S, Bannister RA. Rem uncouples excitation-contraction coupling in adult skeletal muscle fibers. J Gen Physiol. 2015;46(1):97–108.View ArticleGoogle Scholar
- Karunasekara Y, Rebbeck RT, Weaver LM, Board PG, Dulhunty AF, Casarotto MG. An alpha-helical C-terminal tail segment of the skeletal L-type Ca2+ channel beta1a subunit activates ryanodine receptor type 1 via a hydrophobic surface. FASEB J. 2012;26:5049–59.PubMedView ArticleGoogle Scholar
- Beurg M, Sukhareva M, Ahern CA, Conklin MW, Perez-Reyes E, Powers PA, et al. Differential regulation of skeletal muscle L-type Ca2+ current and excitation-contraction coupling by the dihydropyridine receptor beta subunit. Biophys J. 1999;76:1744–56.
- Tanabe T, Mikami A, Numa S, Beam KG. Cardiac-type excitation-contraction coupling in dysgenic skeletal muscle injected with cardiac dihydropyridine receptor cDNA. Nature. 1990;344:451–3.PubMedView ArticleGoogle Scholar
- Protasi F, Takekura H, Wang Y, Chen SR, Meissner G, Allen PD, et al. RYR1 and RYR3 have different roles in the assembly of calcium release units of skeletal muscle. Biophys J. 2000;79:2494–508.
- Nakai J, Ogura T, Protasi F, Franzini-Armstrong C, Allen PD, Beam KG. Functional nonequality of the cardiac and skeletal ryanodine receptors. Proc Natl Acad Sci U S A. 1997;94:1019–22.PubMed CentralPubMedView ArticleGoogle Scholar
- Fessenden JD, Wang Y, Moore RA, Chen SR, Allen PD, Pessah IN. Divergent functional properties of ryanodine receptor types 1 and 3 expressed in a myogenic cell line. Biophys J. 2000;79:2509–25.PubMed CentralPubMedView ArticleGoogle Scholar
- Kimura T, Lueck JD, Harvey PJ, Pace SM, Ikemoto N, Casarotto MG, et al. Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling. Cell Calcium. 2009;45:264–74.
- Kimura T, Nakamori M, Lueck JD, Pouliquin P, Aoike F, Fujimura H, et al. Altered mRNA splicing of the skeletal muscle ryanodine receptor and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase in myotonic dystrophy type 1. Hum Mol Genet. 2005;14:2189–200.
- Ahern GP, Junankar PR, Dulhunty AF. Single channel activity of the ryanodine receptor calcium release channel is modulated by FK-506. FEBS Lett. 1994;352:369–74.PubMedView ArticleGoogle Scholar
- Ahern GP, Junankar PR, Dulhunty AF. Subconductance states in single-channel activity of skeletal muscle ryanodine receptors after removal of FKBP12. Biophys J. 1997;72:146–62.PubMed CentralPubMedView ArticleGoogle Scholar
- Saito A, Seiler S, Chu A, Fleischer S. Preparation and morphology of sarcoplasmic reticulum terminal cisternae from rabbit skeletal muscle. J Cell Biol. 1984;99:875–85.PubMedView ArticleGoogle Scholar
- Chamberlain BK, Fleischer S. Isolation of canine cardiac sarcoplasmic reticulum. Methods Enzymol. 1988;157:91–9.PubMedView ArticleGoogle Scholar
- Laver DR, Roden LD, Ahern GP, Eager KR, Junankar PR, Dulhunty AF. Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle. J Membr Biol. 1995;147:7–22.PubMedView ArticleGoogle Scholar
- Nakai J, Dirksen RT, Nguyen HT, Pessah IN, Beam KG, Allen PD. Enhanced dihydropyridine receptor channel activity in the presence of ryanodine receptor. Nature. 1996;380:72–5.PubMedView ArticleGoogle Scholar
- Avila G, Dirksen RT. Functional impact of the ryanodine receptor on the skeletal muscle L-type Ca(2+) channel. J Gen Physiol. 2000;115:467–80.PubMed CentralPubMedView ArticleGoogle Scholar
- Avila G, O'Connell KM, Groom LA, Dirksen RT. Ca2+ release through ryanodine receptors regulates skeletal muscle L-type Ca2+ channel expression. J Biol Chem. 2001;276:17732–8.PubMedView ArticleGoogle Scholar
- Laver DR, van Helden DF. Three independent mechanisms contribute to tetracaine inhibition of cardiac calcium release channels. J Mol Cell Cardiol. 2011;51:357–69.PubMedView ArticleGoogle Scholar
- Sigworth FJ, Sine SM. Data transformations for improved display and fitting of single-channel dwell time histograms. Biophys J. 1987;52:1047–54.PubMed CentralPubMedView ArticleGoogle Scholar
- Tae HS, Cui Y, Karunasekara Y, Board PG, Dulhunty AF, Casarotto MG. Cyclization of the intrinsically disordered alpha1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function. J Biol Chem. 2011;286:22589–99.PubMed CentralPubMedView ArticleGoogle Scholar
- Sheridan DC, Cheng W, Ahern CA, Mortenson L, Alsammarae D, Vallejo P, et al. Truncation of the carboxyl terminus of the dihydropyridine receptor beta1a subunit promotes Ca2+ dependent excitation-contraction coupling in skeletal myotubes. Biophys J. 2003;84:220–37.
- Goonasekera SA, Chen SR, Dirksen RT. Reconstitution of local Ca2+ signaling between cardiac L-type Ca2+ channels and ryanodine receptors: insights into regulation by FKBP12.6. Am J Physiol Cell Physiol. 2005;289:C1476–1484.PubMedView ArticleGoogle Scholar
- Copello JA, Barg S, Onoue H, Fleischer S. Heterogeneity of Ca2+ gating of skeletal muscle and cardiac ryanodine receptors. Biophys J. 1997;73:141–56.PubMed CentralPubMedView ArticleGoogle Scholar
- Kimura T, Pace SM, Wei L, Beard NA, Dirksen RT, Dulhunty AF. A variably spliced region in the type 1 ryanodine receptor may participate in an inter-domain interaction. Biochem J. 2007;401:317–24.PubMed CentralPubMedView ArticleGoogle Scholar
- Goonasekera SA, Beard NA, Groom L, Kimura T, Lyfenko AD, Rosenfeld A, et al. Triadin binding to the C-terminal luminal loop of the ryanodine receptor is important for skeletal muscle excitation contraction coupling. J Gen Physiol. 2007;130:365–78.
- Combet C, Blanchet C, Geourjon C, Deleage G. NPS@: network protein sequence analysis. Trends Biochem Sci. 2000;25:147–50.PubMedView ArticleGoogle Scholar
- Protasi F, Paolini C, Nakai J, Beam KG, Franzini-Armstrong C, Allen PD. Multiple regions of RyR1 mediate functional and structural interactions with alpha(1S)-dihydropyridine receptors in skeletal mudarw1scle. Biophys J. 2002;83:3230–44.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.