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  • Research
  • Open Access

Regions of ryanodine receptors that influence activation by the dihydropyridine receptor β1a subunit

  • 1,
  • 2,
  • 3,
  • 2,
  • 2,
  • 4,
  • 3 and
  • 2Email author
Skeletal Muscle20155:23

  • Received: 5 April 2015
  • Accepted: 2 July 2015
  • Published:



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.


  • Excitation-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 [911]. 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 [1214]. 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 [15] 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 [20]. 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 [18]. Replacement of the same six residues with glutamines in the full-length RyR protein substantially reduces depolarization-dependent Ca2+ release [18]. 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 [21]. 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 [2226] 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 [27]. 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 [1214].

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 [28]. 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 [2931] 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 [28] 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) [36]. 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 [15]. 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 [3739]. 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

The whole-cell patch clamp technique was used to simultaneously measure voltage-gated L-type Ca2+ currents (L currents) and Ca2+ transients in expressing myotubes [36]. Patch clamp experiments were conducted using an external solution consisting of (in millimolar): 145 TEA-Cl, 10 CaCl2, and 10 HEPES, pH 7.4 with TEA-OH and an internal pipette solution consisting of (in millimolar): 145 Cs-aspartate, 10 CsCl, 0.1 Cs2-EGTA, 1.2 MgCl2, 5 Mg-ATP, 0.2 K5-fluo-4, and 10 HEPES, pH 7.4 with CsOH. Peak L-current magnitude was normalized to cell capacitance (pA/pF), plotted as a function of the membrane potential (I-V curves in Fig. 6c), and fitted according to:
$$ I = {G}_{\mathbf{max}}*\ \left({V}_m - {V}_{\mathbf{rev}}\right)\ /\ \left(1 + \exp \left[\left({V}_{\mathbf{G1}/\mathbf{2}} - {V}_m\right)\ /\ {k}_G\right]\right) $$
where G max is the maximal L-channel conductance, V m is test potential, V rev is the L-channel reversal potential, V G1/2 is the potential for half-maximal activation of G max, and k G is a slope factor. Relative changes in fluo-4 fluorescence (ΔF/F) were measured at the end of each 200-ms depolarization, plotted as a function of the membrane potential, and fitted according to:
$$ \varDelta F/F = \left(\varDelta F\ /\ {F}_{\mathbf{max}}\right)/\left\{1 + \exp\ \left[\left({V}_{\mathbf{F1}/\mathbf{2}}-{V}_m\right)\ /\ {k}_F\right]\right\} $$
where ΔF/F max is the maximal fluorescence change, V F1/2 is the potential for half-maximal activation of ΔF/F max, and k F is a slope factor. The bell-shaped voltage dependence of ΔF/F measurements obtained in RyR1 K-Q mutant-expressing myotubes were fitted according to the following equation:
$$ \varDelta F/F = \left({\left(\varDelta F\ /\ F\right)}_{\mathbf{max}}\left(\left({V}_m - {V}_{\mathbf{rev}}\right)/k^{\prime}\right)\right)/\left(1 + \exp\ \left(\left({V}_{\mathbf{F1}/\mathbf{2}}-{V}_m\right)\ /\ {k}_F\right)\right) $$
where (ΔF/F)max, V m , V rev, V F1/2, and k F have their usual meanings. The additional variable k′ is a scaling factor that varies with (ΔF/F)max [40, 41]. The maximal rate of voltage-gated SR Ca2+ release was approximated from the peak of the first derivative of the fluo-4 fluorescence trace (dF/dt) elicited during the test depolarization at 30 mV. Pooled current-voltage (I-V) and fluorescence-voltage (ΔF/F-V) data in Table 1 are expressed as mean ± SEM.
Table 1

Parameters of fitted I-V and [ΔF/F]-V curves


I-V data


[∆F/F]-V data


G max (nS/nF)

k (mV)

V half (mV)

Vrev (mV)


k (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

Maximal L-channel conductance (G max), the potential for half-maximal G max (V half), slope factor (k), and reversal potential (V rev). Values presented as mean ± SEM for I-V data presented in Fig. 6c. Maximal Ca2+ transient [(ΔF/F)max], the potential at half maximal fluorescence (V half) and slope factor (k). Values presented as mean ± SE for [ΔF/F]-V data presented in Fig. 6d

*p < 0.05 vs WT RyR1-expressing dyspedic myotubes

Immunofluorescence labeling

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 [41]. 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

As we reported previously [15], when added to the cytoplasmic cis chamber, the full-length β1a subunit increases the activity of native RyR1 channels incorporated into planar lipid bilayers (Fig. 1a). Both 10- or 100-nM concentrations of β1a subunit maximally activate RyR1 channels in the presence of 10 μM Ca2+ and 2 mM Na2 ATP [15]. The records in Fig. 1b show that RyR2 channel activity also increases upon cytoplasmic exposure to 10 nM β1a subunit, but in contrast to RyR1, greater activation of RyR2 is observed with 100 nM β1a. On average, addition of 10 nM or 100 nM β1a to the cis solution significantly increased the relative P o of RyR2 by 1.8-fold and 2.6-fold, respectively (Fig. 2a, left). Data is presented as average relative P o which is the average of the logarithm to the base 10 of P o of each individual channel in the presence of β1a, relative to the logarithm of the P o of its internal control activity measured before application of β1a. Use of relative P o eliminates any effect of the normal variability between individual RyR channels [39, 42]. The logarithm is used to reveal the extent of variation of the effects of β1a. The average of the P o parameter values are also shown to indicate absolute level of each parameter (Fig. 2a–c, right), however, the relative changes should be used as the most accurate indicator of effects of β1a on RyRs. The effects on RyR2 channel activity were similar at +40 and −40 mV (relative P o with 10 nM β1a increasing by ~2-fold at +40 mV and ~1.7-fold at −40 mV), and these values were combined in the average data in Fig. 2a. It has been established that the activation of RyR1 by β1a is maximal at 10 nM and does not increase between 10 and 1000 nM [15]. Therefore, the reduced efficacy of 10 nM β1a on RyR2 suggests that affinity of RyR2 for β1a is lower than that of RyR1.
Fig. 1
Fig. 1

β1a subunit increases RyR1 and RyR2 channel activity in lipid bilayers. a and b Three second (3 s) traces of representative activity from native (a) RyR1 or (b) RyR2 channels recorded at a test potential of +40 mV. Openings are shown as upward inflections from the closed (c) state to the maximum open (o) level. Results are shown before (top panel; control, cis 10 μM [Ca2+] and 2 mM ATP) and after addition of 10 nM β1a subunit (middle panel) and then 100 nM β1a subunit (bottom panel) to the cis chamber. Open probability (P o ) is shown at the right hand corner of each trace

Fig. 2
Fig. 2

β1a subunit increases RyR1 and RyR2 channel activity in lipid bilayers. Single-channel gating parameters of RyR1 and RyR2 in response to 10 or 100 nM β1a subunit. a (left) Average relative P o (log10 rel P o ) is the average of the differences between the logarithm of P o following addition of β1a subunit (log10 P oB ) and the logarithm of the control P o (log10 P oC ), where P oC was measured before β1a subunit addition. b (left) Average relative mean open time (log10 rel T c ). c (left) Average relative mean closed time (log10 rel T o ) were calculated in the same way as the average log10 rel P o (above). ac (right) The average single-channel parameter values are shown right of the corresponding relative values. ac Single-channel parameters were calculated from ~180 s of channel activity (at +40 and −40 mV). Data are shown for 0 nM β1a (black bar), 10 nM β1a subunit (dark shade bar), and 100 nM β1a subunit (light shade bar), when examined. Error bars indicate ± SEM., n = 7−15 experiments/bar. *p < 0.05 vs control determined using paired (left) or un-paired (right) Student’s t-test, # p < 0.05 vs 10 nM β1a subunit with RyR2 determined by ANOVA

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.

The effects of β1a on the open (τ o ) and closed (τ c ) time constant components and the relative distribution of events between time constants is presented in Figs. 3 and 4. Open events in RyR1 and RyR2 channels were well described by the sum of three time constants of ~1 (τ o1), ~10 (τ o2), and ~100 ms (τ o3) (Fig. 3). Closed times were also characterized by three time constants of ~1 (τ c1), ~10 (τ c2), and ~100 ms (τ c3) (Fig. 3). Figure 4 shows plots of the average probability of open (Fig. 4a, b, upper plots) and closed (Fig. 4a, b, lower plots) events as a function of the average time constant in the absence (control) and presence of either 10 or 100 nM β1a. Neither the time constants nor the relative probability of events for each time constant varied significantly (p = 0.12–0.99) between +40 and −40 mV and thus were combined in the average data.
Fig. 3
Fig. 3

Effects of β1a subunit on the distribution of representative RyR channel open and closed dwell times. Exponential open and closed time constants determined for RyR1 (ac) and RyR2 (df). Open and closed times were collected into logged bins and the square root of the relative frequency of events (probability1/2) was plotted against the logarithm of open (open circles) or closed times (filled circles) in milliseconds. Examples are shown for the data from representative individual channels under control (a, d) and after exposure to 10 nM β1a subunit (b, e) and then 100 nM β1a subunit (c, f). The solid lines represent the fit of multiple exponentials to the data. The individual open time constants (τ o1, τ o2, and τ o3) and individual closed time constants (τ c1, τ c2, and τ c3) are indicated by arrows

Fig. 4
Fig. 4

Effects of β1a subunit on the distribution of RyR channel open and closed dwell times. The probability of open and closed events falling into each time constant is plotted against the respective time constant. The open (τ o , top graphs) and closed (τ c , bottom graphs) time constants and the probability of events in each time constant component were calculated from ~180 s of single channel activity (at +40 and −40 mV). Data is shown for a RyR1 and b RyR2 before (open circle) and after addition of 10 nM β1a subunit (open triangle) or 100 nM β1a subunit (open square), n = 6–12 channel traces. Error bars indicate ± SEM. The individual open time constants (τ o1, τ o2, and τ o3) and individual closed time constants (τ c1, τ c2, and τ c3) are indicated on the top and bottom graphs, respectively. *p < 0.05 vs the probability of events in each time constant in control with 10 nM β1a subunit, determined by ANOVA. # p < 0.05 vs the probability of events in each time constant in control with 100 nM β1a subunit, determined by ANOVA

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

There is a curious similarity between the cardiac RyR2 isoform and the ASI(−) splice variant of RyR1 in that both lack ASI residues. This may be relevant to the effect of β1a on RyR1 and its contribution to EC coupling as we have shown that the presence of the alternatively spliced AS1 residues influences the gain of EC coupling in skeletal myotubes [27] and modulates RyR1 activity in vitro [28]. Therefore, we determined the impact of the alternatively spliced ASI residues on the activation of RyR1 by β1a. Recombinant ASI(-)RyR1 and ASI(+)RyR1 constructs [28, 43] were incorporated into lipid bilayers and the actions of the β1a subunit on channel activity examined (Fig. 5). The ASI(+) isoform is the adult isoform of RyR1 and its sequence is equivalent to the adult rabbit RyR1 used in the previous section and to the cloned wild type (WT) rabbit RyR1 sequence described in the following section. It is notable in the single-channel activity, as shown in Fig. 5a, b (and in Fig. 7 below), that the recombinant channels (both ASI(-)RyR1 and ASI(+)RyR1) display strong sub-conductance (or sub-state) activity, with long channel openings to levels at ~50 % of the maximal conductance. Channel activity was measured as usual (“Methods” section) with an open threshold set at ~20 % of the maximum single-channel conductance to exclude baseline noise but to include sub-conductance openings to levels >20 % of the maximum. It is important to note that similar amounts of sub-conductance activity were seen in HEK293-expressed WT and ASI(−) compared in Fig. 5 and in WT and RyR1 K-Q channels compared in Fig.  8.  Similarly, the smaller amounts of sub-conductance activity were comparable in RyR1 and RyR2 isolated from muscle tissue and compared in Fig. 1. In each case, sub-state activity was similar in constructs being compared.
Fig. 5
Fig. 5

ASI residues enhance the effect of β1a on recombinant RyR1 channel activity in lipid bilayers. a, b Three second (3 s) traces of ASI(+)RyR1 (a) or ASI(−)RyR (b) activity at +40 mV, opening upwards from the closed (c) to maximum open (o) level, before (top panel; control, cis 10 μM [Ca2+], no ATP) and after addition of 10 nM β1a subunit (middle panel) or 50 nM β1a subunit (bottom panel) to the cis chamber. c Average relative P o (log10 rel P o ) were calculated in the same ways as described for averaged relative P o in Fig. 2a, left. d Average P o . c and d Single channel parameters were calculated from ~180 s of channel activity (at +40 and −40 mV). Data in d is shown for 0 nM β1a (black bar), 10 nM β1a subunit (dark grey bar), and 50 nM β1a subunit (light grey bar). Error bars indicate + SEM, n = 9–12 experiments/bar. *p < 0.05 vs control or 0 nM β1a subunit determined using paired (c) or un-paired (d) Student’s t-test, # p < 0.05 vs 10 nM β1a subunit on RyR1 ASI(+) determined by ANOVA

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 [18]. 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 [18].

Depolarization-dependent Ca2+ release was measured simultaneously with DHPR L-type Ca2+ currents (Fig. 6a, b). Peak L-current density (Fig. 6c) and maximal DHPR Ca2+ conductance (G max) were significantly reduced in RyR1 K-Q mutant-expressing myotubes compared to WT RyR1-expressing myotubes (Table 1). Consistent with an earlier report [18], maximal voltage-induced SR Ca2+ release was also significantly reduced in RyR1 K-Q mutant-expressing myotubes (Fig. 6d and Table 1). In addition, the maximum rate of depolarization-induced Ca2+ release (approximated from the peak of the first derivative of the fluo-4 fluorescence trace elicited during a test depolarization at 30 mV) was significantly reduced in RyR1 K-Q-expressing myotubes compared to WT RyR1-expressing myotubes (Fig. 6e). These findings indicate that the RyR1 K-Q mutation substantially reduces voltage-induced SR Ca2+ release, with a small effect on maximal L-channel conductance. It should be noted that the reduced Ca2+ release is unlikely to result from reduced expression of RyR1 K-Q as it was previously shown that peak 4-chloro-m-cresol stimulated SR Ca2+ release was similar in WT RyR1- and RyR1 K-Q-expressing myotubes [18]. In addition, we found that WT RyR1 and RyR1 K-Q exhibited a similar punctate pattern and DHPR co-localization in expressing myotubes, consistent with similar levels of WT and K-Q expression and junctional localization (Additional file 1: Figure S1 and Additional file 2).
Fig. 6
Fig. 6

PBM mutation diminishes depolarization-induced SR Ca2+ release and DHPR Ca2+ currents in dyspedic myotubes. Dyspedic myotubes were transfected with either WT RyR1 or RyR1 K-Q mutant. a, b Representative L-type currents (lower trace) and Ca2+ transient (upper trace) obtained following depolarization to the indicated potentials of dyspedic myotubes expressing either a WT RyR1 or b RyR1 K-Q mutant. c Voltage dependence of average (±SEM) peak L-type Ca2+ current density (pA/pF) as a function of voltage. The data were fit (continuous line) with a modified Boltzmann function. d Voltage dependence of average (±SEM) peak Ca2+ transient amplitude as a function of voltage. The data were fit (continuous line) with a Boltzmann function. c, d Average (±SEM) values of the parameters from individual fits to each myotube are shown in Table 1. e The average (±SEM) peak of the first derivative of the fluo-4-fluorescence trace elicited during the test depolarization at 30 mV. ce n = 10–12 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 [44].

The effects of cytoplasmic Ca2+ and ATP were similar (p = 0.356–0.894) between +40 and -40 mV, and the data were combined. A decrease in cis free [Ca2+] from 1 mM to 10 μM caused a 1.7-fold increase in WT RyR1 P o and a similar 1.6-fold increase in RyR1 K-Q P o , (log10 rel P o of 0.22 ± 0.06 [p = 0.013] and 0.20 ± 0.09 [p = 0.048], respectively, n = 7 for each). Similar increases in P o with a decrease in cis free [Ca2+] from 1 mM to 10 μM have been reported previously for recombinant WT RyR1 channels in lipid bilayers [44] and for [3H]ryanodine binding to RyR1 [33]. Addition of 2 mM Na2 ATP to the cis solution increased WT RyR1 activity by 2.2-fold and RyR1 K-Q activity by 2.5-fold (log10 rel P o of 0.34 ± 0.12 [p = 0.032] and 0.40 ± 0.14 [p = 0.037], respectively, n = 7 for each). As observed for recombinant WT RyR1, prominent sub-state activity was also observed for recombinant RyR1 K-Q channels (Fig. 7a, b). The similar conductance, sub-state activity, and regulation by Ca2+ and ATP between WT RyR1 and RyR1 K-Q channels indicate that the K-Q mutation does not markedly alter RyR1 function in the absence of the β1a subunit.
Fig. 7
Fig. 7

The K-Q mutation abolishes β1a activation of RyR1 activity. a, b Three second (3 s) traces of WT RyR1 (a) or RyR K-Q mutant (b) activity at +40 mV, opening upward from the closed (c) state to the maximum open (o) level, before (top panel; control, cis 10 μM [Ca2+] and 2 mM ATP) and after addition of 100 nM β1a subunit (bottom panel) to the cis chamber. Open probability (P o ) is shown at the right hand corner of each trace

As before (Figs. 1, 2, 3, 4 and 5), cis addition of 100 nM β1a significantly increased WT RyR1 channel activity (Fig. 7a). In marked contrast, the activity of RyR1 K-Q channels was unaffected by addition of 100 nM β1a (Fig. 7a). As effects of the β1a subunit on WT or RyR1 K-Q channels did not differ (p = 0.677–0.991) between +40 and −40 mV, values at these two potentials were again combined in the average data. On average, addition of 10 or 100 nM β1a significantly increased WT RyR1 relative P o by 1.8- and 1.9-fold, respectively (Fig. 8a), due to a significant increase in mean open time (Fig. 8b) and decrease in mean closed time (Fig. 8c). On the other hand, neither the relative P o nor the mean open or closed times of RyR1 K-Q channels were significantly altered by addition of either 10 or 100 nM β1a subunit (Fig. 8a–c). Thus, the ability of the β1a subunit to activate RyR1 was abolished by neutralizing the polybasic residues within the K4395-R3502 region, indicating that the PBM is required for the functional effect of β1a subunit on RyR1 activity.
Fig. 8
Fig. 8

Effect of β1a subunit on RyR1 in lipid bilayers is abolished for the K-Q mutation. a–c (left) Average relative P o (log10 rel P o ; a), mean open time (log10 To; b) or mean closed time (log10 rel T c ; c) were calculated in the same ways as described for averaged relative P o in Fig. 2a, left. a–c (right) The average of the single channel parameter values shown to the right of the corresponding relative values. a–c Single-channel parameters were calculated from ~180 s of channel activity (at +40 and −40 mV). Data are shown without β1a (0 nM β1a) (black bar), 10 nM β1a subunit (dark grey bar), and 100 nM β1a subunit (light grey bar), where applicable. Error bars indicate ± −SEM. n = 5–14 experiments/bar. *p < 0.05 vs control determined by paired (left) or un-paired (right) Student’s t-test, # p < 0.05 vs WT RyR2 determined by ANOVA


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 [45] 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 [46], 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 [18] 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 [18], 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 [21]. 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 [27]. 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 [20]. Finally, alanine substitution of β1a subunit hydrophobic triplet residues (L496, L500, and W503) only partially reduces depolarization-induced Ca2+ release in β1a null myotubes [14], despite this mutation fully abolishing β1a modulation of RyR1 activity in vitro [21].

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 [27]. 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 [43]. 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


dihydropyridine receptor




polybasic motif


skeletal ryanodine receptor


cardiac ryanodine receptor


sarcoplasmic reticulum



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.

Authors’ Affiliations

Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA
John Curtin School of Medical Research, Australian National University, Canberra, Australian Capital, PO Box 334, Canberra, ACT 2601, Australia
Department of Physiology and Pharmacology, University of Rochester Medical Center, Rochester, NY, USA
Discipline of Biomedical Sciences, Centre for Research in Therapeutic Solutions, University of Canberra, Canberra, ACT 2601, Australia


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