Allosterically Coupled Calcium and Magnesium Binding Sites are Unmasked by Ryanodine Receptor Chimeras

Abstract

We studied cation regulation of wild type ryanodine receptor type 1 (_WTRyR1), type 3 (_WTRyR3) and RyR3/RyR1 chimeras (Ch) expressed in 1B5 dyspedic myotubes. Using [³H]ryanodine binding to sarcoplasmic reticulum (SR) membranes, Ca²⁺ titrations with _WTRyR3 and three chimeras show biphasic activation that is allosterically coupled to attenuated inhibition relative to _WTRyR1. Chimeras show biphasic Mg²⁺ inhibition profiles at 3 and 10μM Ca²⁺, no observable inhibition at 20μM Ca²⁺ and monophasic inhibition at 100μM Ca²⁺. Ca²⁺ imaging of intact myotubes expressing Ch-4 exhibit caffeine-induced Ca²⁺ transients with inhibition kinetics that are significantly slower than those expressing _WTRyR1 or _WTRyR3. Four new aspects of RyR regulation are evident: 1) high affinity (H) activation and low affinity (L) inhibition sites are allosterically coupled, 2) Ca²⁺ facilitates removal of the inherent Mg²⁺ block, 3) _WTRyR3 exhibits reduced cooperativity between H activation sites when compared to _WTRyR1, and 4) uncoupling of these sites in Ch-4 results in decreased rates of inactivation of caffeine-induced Ca transients.

Three isoforms of wild type ryanodine receptors (_WTRyR1, 2 and 3) are expressed in specialized regions of endoplasmic/sarcoplasmic reticulum (ER/SR) in most mammalian cells where they function as Ca²⁺ release channels that produce local and global Ca²⁺ signals [1] and 2]. Fluctuating physiological cation concentrations, especially Ca²⁺ and Mg²⁺, tightly regulate all three _WTRyR isoforms [3], [4], [5], [6], [7], [8] and [9]. Cytoplasmic Ca²⁺ ranging from nM to μM enhances the open probability of _WTRyRs, whereas >100 μM Ca²⁺ or Mg²⁺ depresses channel activity [5], [10] and [11]. The “bell shaped” regulation of _WTRyR by Ca²⁺ is thought to be responsible for physiological and pathophysiological Ca²⁺-induced Ca²⁺ release (CICR) phenomena observed in many cell types, including muscle and neurons [1], [2], [12], [13] and [14].

_WTRyR isoforms preferentially bind the plant alkaloid ryanodine with nM affinity when in the open state [3], [6], and [7]. Ca²⁺ and Mg²⁺ titrations in [³H]ryanodine binding and Ca²⁺ release experiments have revealed Hill coefficients >1, indicating multiple, coordinated regulation of CICR channels by multiple cation binding sites [15]. Mg²⁺ inhibition studied with channels reconstituted in lipid bilayer membranes [16] and Ca²⁺ release from SR membrane vesicles [4] and [11] suggest dual mechanisms of Mg²⁺ inhibition through competition with Ca²⁺ for high affinity (H) activation sites and binding at low affinity (L) cation inhibition sites. At physiological concentrations, free Mg²⁺ (1–2mM) is likely to occupy both H and L sites, providing a basal level of _WTRyR inhibition that must be overcome for EC-coupling to occur [17] and [18]. Studies have suggested that the physical coupling between _WTRyR and the dihydropyridine receptor (DHPR) may remove the Mg²⁺ block during EC-coupling [18]; whereas, other authors have suggested oxidation of _WTRyR sulfhydryl groups may override Mg²⁺ inhibition [19]. Functional overlap in Ca²⁺ and Mg²⁺ interactions at H and L regulation sites have confounded cation regulation studies of _WTRyR1 and resulted in conclusions partially derived from extrapolation. A system that permits more direct analysis of cytoplasmic Ca²⁺ and Mg²⁺ regulation would greatly facilitate mechanistic interpretations about RyR cation regulation in health and disease.

RyR3/RyR1 chimeras were designed to identify regions of _WTRyR1 directly associated with the DHPR during EC-coupling [20] and [21]. In this report, [³H]ryanodine binding experiments on _WTRyR1, _WTRyR3, and a subset of these chimeras (Ch-4: _WTRyR₁ 1681-3770; Ch-17: 1681–2217; Ch-21: 1924–2446) reveal a biphasic cytoplasmic Ca²⁺ activation profile, suggesting variable cooperativity between H activation sites, revealing coupled but separated interactions at H activation and L inhibition sites. This study provides direct insight into Ca²⁺ and Mg²⁺ regulation, suggests new aspects of _WTRyR function, and establishes the chimeras as models for future studies of _WTRyRs and cation regulation.

Materials and Methods

Chimeric RyR1/RyR3 constructs

Specific primers were designed for PCR amplification of the selected fragments using _WTRyR1 as a template. Amplified fragments from _WTRyR1 encoding aa 1681–2217 (Ch-17), 1924–2446 (Ch-21) and 1681–3770 (Ch-4) were inserted, in frame, into the endogenous restriction site(s) of HSV-RyR3 plasmid as described previously [20]. All chimeric constructs were cloned into the HSV-1 amplicon vector and packaged using a helper virus-free packaging system [22].

Cell culture, infection and membrane preparation

1B5 myoblasts were cultured and differentiated into myotubes as described previously [20] and [23]. Plates with differentiated myotubes were infected with virion containing wild type and RyR1/RyR3 chimeric cDNA for 2 h and membrane extracts were prepared 36 h after infection. Myotubes were homogenized and membrane fractions obtained by differential centrifugation as described previously [24].

[³H]Ryanodine binding assay

High affinity binding of [³H]ryanodine ([³H]Ry; 56 Ci/mmol; New England Nuclear, Boston, MA) to membranes (10–50μg/ml protein) was performed in the presence of 250mM KCl, 20mM Hepes pH 7.4 and 5nM [³H]Ry [6]. Free Ca²⁺ and Mg²⁺ concentrations buffered with EGTA were determined using the Bound And Determined software [25]. The binding reaction was equilibrated at 37 °C for 3h. Non-specific binding was assessed in the presence of 5μM unlabeled ryanodine. Bound ligand was separated from free by filtration through Whatman GF/B glass fiber filters using a Brandel cell harvester (Gaithersburg, MD), washed with ice-cold buffer, placed into 5 ml scintillation cocktail (ScintiVerse; Fisher Scientific) and radioactivity counted.

Equations for binding analysis

Curve fitting was generated by Microcal^™ Origin^® Version 6.0 using the equations:

Activation:

$$\text{a})y=\frac{(Bmax)\left(x^n\right)}{k^n+x^n}\text{b})y=\frac{(B{max}_1)\left(x^{n_1}\right)}{{k_1}^{n_1}+x^{n_1}}+\frac{(B{max}_2)\left(x^{n_2}\right)}{{k_2}^{n_2}+x^{n_2}}$$

where, Bmax = maximum bound, k = EC₅₀ and n = Hill coefficient

Inhibition:

$$\text{c})y=\frac{A_1-A_2}{1+{\left(\frac{x}{xo}\right)}^p}+A_2\text{d})y=A_2+\frac{(A_1-A_2)f}{1+{10}^{(x-logx{o}_1)}}+\frac{(A_1-A_2)(1-f)}{1+{10}^{(x-logx{o}_2)}}$$

where A1 = top asymptote, A2 = bottom asymptote, xo = IC₅₀, f = fraction and p = power

Ca²⁺ imaging

Calcium imaging was performed during the stable phase of transduced protein expression in the myotubes 36–48h post-infection as previously described [20]. Ca²⁺ release was induced with 20s perfusion of 20mM caffeine. Monophasic exponential decay curves were fitted with GraphPad Prism^® Version 4.0 according to following function: y=Span · e^(−k·X) + Plateau were k= rate constant.

Results and Discussion

To examine cation regulation, both _WTRyRs and RyR3/RyR1 chimeras were expressed in dyspedic 1B5 myotubes, a cell line void of all _WTRyR isoforms, but containing the accessory proteins necessary for normal _WTRyR function [26]. Regulation of _WTRyRs and chimeras in membrane fractions was assessed using [³H]Ry binding to probe cation regulation of channel conformation permitting direct analysis of cytoplasmic Ca²⁺ and Mg²⁺ regulation of RyR conformation [6] without confounding influences of varying luminal Ca²⁺ [27].

Ca²⁺ regulation

Ca²⁺ activation of _WTRyR1 and _WTRyR3 determined by [3H]Ry binding (Fig. 1A) indicated monophasic activation of _WTRyR1 (EC₅₀=0.54±0.05μM) and biphasic activation of _WTRyR3 (EC₅₀₍₁₎ and EC₅₀₍₂₎=0.36±0.06μM and 20.0±5.78μM) with a plateau from 4 – 8 μM (inset). Similar analyses of Ch-21, -17 and -4 revealed accentuated biphasic Ca²⁺ responses, with plateaus of 20–100μM, 10–100μM and 3–100μM, respectively (Fig. 1B). Ch-21, −17 and −4 exhibited EC₅₀₍₁₎ values of 0.90±0.19μM, 0.39±0.09μM and 0.18±0.03μM, and EC₅₀₍₂₎ values of 397±190 μM, 510±174μM, and 761±192μM, respectively (Table 1).

Fig. 1.

Ca²⁺ regulation of wild type and chimeric RyR3/RyR1 expressed in dyspedic 1B5 myotubes, determined by [³H]Ry binding (see METHODS). (A) Activation of RyR1 () and RyR3 () curve fit with Hill and biphasic Hill equations, respectively. (B) Activation of Ch-4 (), Ch-17 (---◇---) and Ch-21 (—▽—) curve fit with a biphasic Hill equation. (C) Inhibition of RyR1 () and RyR3 () curve fit with a monophasic equation. (D) Inhibition of Ch-4 (), Ch-17 (---◇---) and Ch-21 (—▽—) with a biphasic equation. Data points are the mean values, error bars are ± standard deviations. Activation profiles for _WTRyR1, _WTRyR3 and Ch-4 were the combined result of 2–4 experiments performed in duplicate or triplicate from multiple 1B5 membrane preparations.

Table 1.

Curve fit statistics for [³H]Ry binding analyses of Ca²⁺ activation

Construct	EC50(1) (μM )	EC50(2) (μM )	Hill Coeff. 1	Hill Coeff. 2	r2
WTRyR1	0.54 ± 0.05	N/A	2.42 ± 0.48	N/A	0.982
WTRyR3	0.36 ± 0.06	20.0 ± 5.78	3.26 ± 1.75	1.29 ± 0.40	0.985
Ch-4	0.18 ± 0.03	761 ± 192	1.04 ± 0.17	1.78 ± 0.65	0.986
Ch-17	0.39 ± 0.09	510 ± 174	1.20 ± 0.33	3.08 ± 2.90	0.961
Ch-21	0.90 ± 0.19	397 ± 190	1.03 ± 0.19	3.06 ± 4.32	0.985

The biphasic profiles suggest _WTRyR3 and chimeras possess reduced cooperativity between H activation sites compared to _WTRyR1. The Ca²⁺ dependence of _WTRyR3 has been previously published based on immunoprecipitated protein and the resulting data fitted using a single-site model indicating monophasic activation of _WTRyR3 by Ca²⁺ [28] and [29]. Two significant methodological differences distinguish the present study. First, the use of SR membranes from _WTRyR3-expressing 1B5 myotubes preserves known interactions with lumenal proteins such as calsequestrin [26] that are known to contribute to cation regulation of _WTRyR1 and _WTRyR2 [27] and [30]. Second, the extremely broad titrations in previous studies with immunopurified _WTRyR3 consisted of 2–4 data points covering several log range of Ca²⁺ concentrations and would have missed the biphasic activation of _WTRyR3 by Ca²⁺ if present after immunopurification. The present study is the first report of a distinctly biphasic activation for _WTRyR3 and may result from an altered conformation relative to _WTRyR1 that is exaggerated in the chimeras. A slight variation in protein conformation would explain the deviation in chimeric Ca²⁺ activation from that of _WTRyR1 and 3, an unpredictable response based on primary sequence alone. It is also noteworthy that previously published whole cell Ca²⁺ imaging experiments indicate these chimeras expressed in 1B5 cells are functional, with all three constructs exhibiting calcium-induced Ca²⁺ release (CICR) and Ch-4 and −21 additionally engaging skeletal-type EC-coupling [20]. Interestingly, a recent [³H]Ry-binding study with membranes isolated from rat ventricular muscle showed a similar biphasic Ca²⁺ activation curve for RyR2, which was reverted to a monophasic binding profile in the presence of Mg²⁺ [31]. Collectively these results support a mechanism by which coordinated Ca²⁺ activation sites regulate RyR conformations that bind ryanodine with high affinity.

[³H]Ry binding analysis of Ca²⁺ inhibition through L sites revealed an attenuated response that correlates to H site cooperativity (Fig. 1C & D). Compared to _WTRyR1, multiple aspects of Ca²⁺inhibition were shifted in _WTRyR3 and chimeric constructs. First, the onset of inhibition is attenuated in _WTRyR3 and Ch-21 to ~ 1mM and in Ch-17 and −4 to ~3–4mM vs. _WTRyR1 at 100μM. Additionally, the extent of inhibition observed in the chimeras is shifted, where Ch-21, −17, and −4 were respectively 84%, 60% and 39% of the near complete inhibition observed in _WTRyR1 and _WTRyR3. Finally, as summarized in Table 2, the IC₅₀ values suggested attenuated inhibition compared to _WTRyR1 (0.84±0.08mM) in _WTRyR3 (3.08±0.88mM), Ch-21 (4.11±0.27 mM), Ch-17 (5.09±4.13mM) and Ch-4 (5.86±1.18mM) (Table 2). The extent of attenuated Ca²⁺ inhibition through L sites appeared related to H site cooperativity, in which channels with the greatest reduction in H site cooperativity exhibit the largest attenuation in L site mediated inhibition. These data strongly suggests H and L sites are allosterically coupled.

Table 2.

Curve fit statistics for [³H]Ry binding analyses of Ca²⁺ inhibition

Construct	IC50 (mM)	r2
WTRyR1	0.84 ± 0.08	0.997
WTRyR3	3.08 ± 0.88	0.999
Ch-4	5.86 ± 1.18	0.985
Ch-17	5.09 ± 4.13	0.999
Ch-21	4.11 ± 0.27	0.994

The observations presented heretofore illustrate a model of RyR regulation by Ca²⁺ in which the binding of at least one H site by Ca²⁺ induced a plateau in [³H]Ry binding. This plateau was undetected in _WTRyR1 due to cooperative binding of Ca²⁺ to at least one additional H site, but was observed in _WTRyR3 and further exacerbated in chimeras where there was a reduction in cooperativity between H sites. Binding of Ca²⁺ to the additional H site(s) induced a conformational transition to a state of maximal ryanodine binding. Further increases of Ca²⁺ occupied L sites and decreased [³H]Ry binding. This data indicates H and L sites are allosterically coupled, as the onset of L site mediated inhibition did not occur until complete H site occupation, and complete inhibition through L sites required near-complete cooperativity between H sites.

Mg²⁺ inhibition

In light of Ca²⁺ regulation through coupled but separated H and L sites, we examined with [³H]Ry the role of Mg²⁺ in Ch-4 cation regulation (Fig. 2; Table 3), with curve statistics summarized in Table 3. In the presence of 3 and 10 μM Ca²⁺, Mg²⁺ produced biphasic responses with IC₅₀₍₁₎ values of 0.40±0.13mM and 0.64±0.09mM, respectively. The IC₅₀₍₂₎ values for Mg²⁺ at 3 and 10μM Ca²⁺ were 5.71±0.31mM and 4.18±0.16mM, respectively. At 100μM Ca²⁺, the first phase of Mg²⁺ inhibition was not detected and only an incomplete (~40% of maximum), monophasic inhibition (IC₅₀=4.06±0.40mM) was observed. The biphasic Mg²⁺ profiles at 3 and 10μM Ca²⁺ likely represent competition with Ca²⁺ for H sites in the first phase and binding to L sites in the second. Competition by Mg²⁺ for H sites is supported by a shifting first phase for Mg²⁺ inhibition at 3 and 10μM Ca²⁺ that is not observed at 100μM Ca²⁺. Binding of Mg²⁺ to L sites is described by a monophasic inhibition at 100μM Ca²⁺ (IC₅₀=4–6mM), a range that corresponds to the observed Ca²⁺ inhibition through L sites (5.86+ 1.18mM) in Fig. 1D. Theses results provide more direct biochemical evidence in support of the dual mechanism of Mg²⁺ inhibition originally proposed by Meissner and Laver based on experiments with _WTRyR [4], [16] and [32].

Fig. 2.

Mg²⁺ inhibition at variable Ca²⁺ of Ch-4 expressed in dyspedic 1B5 myotubes, determined by ³H-ryanodine binding (see METHODS). In the presence of 3 () and 10 μM (···□···) Ca²⁺ curve fit with a biphasic equation. In the presence of 20μM Ca²⁺ (—▲—) fit linearly (m = −0.0032±0.0029 and b = 0.611±0.015) and at 100μM Ca²⁺ (---◆---) curve fit with a monophasic equation. Data points are the mean value for at least two experiments, error bars are ± standard deviations.

Table 3.

Curve fit statistics for [³H]Ry binding analyses of Ch-4 Mg²⁺ inhibition at variable Ca²⁺

[Ca2+]	IC50(1) (mM)	IC50(2) (mM)	r2
3 μM	0.40 ± 0.13	5.71 ± 0.31	0.964
10 μM	0.64 ± 0.09	4.18 ± 0.16	0.998
20 μM	No Inhibition
100 μM	4.06 ± 0.40	N/A	0.881

An intriguing finding was that Mg²⁺ titrations in the presence of 20μM Ca²⁺ produced no observable inhibition in [³H]Ry binding experiments. Loss of Mg²⁺ inhibition would be possible through competition at H sites and a conformational state at the Ca²⁺ biphasic plateau, which reduces the affinity of Mg²⁺ at L sites relative to conformations at low (3 and 10μM) and high (100μM) Ca²⁺. Alternatively, Mg²⁺ binding could impart an activating effect on H sites, as suggested for rat RyR2 [31]. This further supports allosteric coupling between H and L sites and suggests Ca²⁺ may contribute along with DHPR [18] and oxidation [19] to relieving the inherent physiological block provided by Mg²⁺.

Physiological relevance of the observations presented in this work depend on the idea that chimeric characteristics unmasked by the reduced H site cooperativity are inherent to _WTRyR, but are difficult to observe in wild type (especially _WTRyR1) due to overlapping interactions at H and L sites. For _WTRyR3 however, the present results would predict unique functional effects at Ca²⁺ concentrations corresponding to the activation plateau (4–8μM) shown in Fig. 1A. Indeed, at 5–10μM Ca²⁺, _WTRyR3 was shown to exhibit increased subconductance behavior relative to _WTRyR1 [28] and [33]. This correlation is additionally supported by increased subconductance behavior of brain _WTRyRs at specific Ca²⁺ and ATP concentrations [34].

Ca²⁺ release from SR stores

To extend the [³H]Ry binding results, we examined the effect of altered Ch-4 cation regulation on Ca²⁺ release in transduced 1B5 myotubes. In these cells, transduced Ch-4 was shown to engage skeletal-type EC-coupling and respond to caffeine stimulation [20] and [24]. We therefore compared the kinetics associated with caffeine-induced Ca²⁺ release from myotubes expressing _WTRyR1, _WTRyR3, and Ch-4. Since Ch-4 cation inhibition is attenuated at basal levels of intracellular Ca²⁺ or Mg²⁺ due to incomplete occupation of H sites, we predict a prolonged Ca²⁺ response following a caffeine-induced Ca²⁺ transient for myotubes expressing Ch-4 compared to those expressing _WTRyR1 or _WTRyR3. Figure 3 shows the average Ca²⁺ transients induced by 20 mM caffeine in 1B5 myotubes expressing each construct and loaded with Fluo-4. Upon caffeine application a fast phase of Ca²⁺ release and subsequent slower phase of cytoplasmic Ca²⁺ removal was followed by a fast Ca²⁺ removal phase after caffeine withdrawal. Whereas all constructs showed seemingly identical activation kinetics for caffeine-induced Ca²⁺ release, Ch-4-expressing cells presented a noticeable reduction in the slow Ca²⁺ removal phase and hence prolonged Ca²⁺ response when compared to cells expressing either _WTRyR1 or _WTRyR3. Analysis of the slow Ca²⁺ removal phase using a monophasic exponential decay reveals that the rate constant (k) for Ch-4 expressing myotubes is nearly 2.5 fold slower than those measured for _WTRyR1 or _WTRyR3 myotubes (Fig. 3). Since saturating concentrations of caffeine were used to induce Ca²⁺ release, the differences in k values observed in this study were most likely associated to the differences in Ca²⁺ and Mg²⁺ regulation of Ch-4 and not to potential differences in sensitivity to the agonist between the three constructs.

Fig 3.

Caffeine-induced Ca²⁺ release kinetics of 1B5 myotubes expressing Ch-4, _WTRyR1 or _WTRyR3. Average Ca²⁺ transients, normalized by the peak amplitude, are presented for each construct (Mean±SE). For clarity trend line for Ch-4 is displayed without SE. Rate constant were calculated using only the decay portion of each Ca²⁺ transient.

Conclusion

The results with RyR1/RyR3 chimeras presented here provide novel insights into the allosteric mechanisms by which Ca²⁺ and Mg²⁺ regulate RyRs, especially interactions between H and L cation binding sites. This allosteric coupling underscores the importance of considering the contribution of Mg²⁺ in addition to Ca²⁺ when assessing RyR function, as physiological levels of Mg²⁺ likely occupy _WTRyR L sites and may influence Ca²⁺ activation through H sites. Furthermore, these results suggest that observed differences between _WTRyRs and/or chimeric constructs should be interpreted with awareness of specific channel responsiveness to and the concentrations of Ca²⁺ and Mg²⁺. This is made apparent by variable responses to the same cation concentrations among wild type and chimeric RyRs. The contribution of RyR effectors such as FKBP, ATP, DHPR and oxidation in the context of the cation allosteric regulation presented here is yet to be determined. Additionally, the association of subconductance behavior to Ca²⁺ concentrations in the activation plateau needs to be further examined. This work provides new insights into _WTRyR regulation and function, and establishes RyR chimeras, particularly Ch-4, as a model for future studies of RyR cation regulation and subconductance behavior.