Regulation of skeletal ryanodine receptors by dihydropyridine receptor II–III loop C-region peptides: relief of Mg²⁺ inhibition

Abstract

The aim of the present study was to explore interactions between surface-membrane DHPR (dihydropyridine receptor) Ca²⁺ channels and RyR (ryanodine receptor) Ca²⁺ channels in skeletal-muscle sarcoplasmic reticulum. The C region (⁷²⁵Phe-Pro⁷⁴²) of the linker between the 2nd and 3rd repeats (II–III loop) of the α₁ subunit of skeletal DHPRs is essential for skeletal excitation–contraction coupling, which requires a physical interaction between the DHPR and RyR and is independent of external Ca²⁺. Little is known about the regulatory processes that might take place when the two Ca²⁺ channels interact. Indeed, interactions between C fragments of the DHPR (C peptides) and RyR have different reported effects on Ca²⁺ release from the sarcoplasmic reticulum and on RyR channels in lipid bilayers. To gain insight into functional interactions between the proteins and to explore different reported effects, we examined the actions of C peptides on RyR1 channels in lipid bilayers with three key RyR regulators, Ca²⁺, Mg²⁺ and ATP. We identified four discrete actions: two novel, low-affinity (>10 μM), rapidly reversible effects (fast inhibition and decreased sensitivity to Mg²⁺ inhibition) and two slowly reversible effects (high-affinity activation and a slow-onset, low-affinity inhibition). Fast inhibition and high-affinity activation were decreased by ATP. Therefore peptide activation in the presence of ATP and Mg²⁺, used with Ca²⁺ release assays, depends on a mechanism different from that seen when Ca²⁺ is the sole agonist. The relief of Mg²⁺ inhibition was particularly important since RyR activation during excitation–contraction coupling depends on a similar decrease in Mg²⁺ inhibition.

INTRODUCTION

During EC (excitation–contraction) coupling, the action potential in the transverse tubule membrane of striated muscle fibres leads to a release of Ca²⁺ from the SR (sarcoplasmic reticulum). Two key components of this process are the DHPR (dihydropyridine receptor; an L-type Ca²⁺ channel), which is the voltage sensor in the transverse tubule membrane [1–4], and the RyR (ryanodine receptor), which is the Ca²⁺ release channel in the SR. Molecular interactions between these two Ca²⁺ channels play a key role in EC coupling and are fundamentally different in skeletal and cardiac muscles [5]. In the heart, transverse tubule depolarization triggers an influx of Ca²⁺ through the DHPR, which activates RyRs and calcium release [6–8]. In skeletal muscles, the influx of Ca²⁺ is not required [5] and RyR activation depends on a physical link between the DHPR and RyR [9] that is dependent on cytoplasmic levels of Ca²⁺, ATP and Mg²⁺ [13–16].

A picture of how RyRs are regulated by the DHPR in skeletal muscles, in conjunction with intracellular Ca²⁺, Mg²⁺ and ATP is emerging. Isolated RyRs are inhibited by cytoplasmic Mg²⁺ (the half-inhibitory concentration of Mg²⁺, K_i, is ∼0.2 mM at physiological ionic strength [10]) and activated by Ca²⁺ (∼1 μM [11]) as well as ATP (∼0.3 mM, even in the absence of Ca²⁺; [12]). During transverse tubule depolarization and activation of the RyR by the DHPR, (i) the sensitivity of RyRs to inhibition by Mg²⁺ is markedly reduced [13,14], (ii) ATP is a key activator of calcium release from the SR [15] and (iii) although Ca²⁺ is not the major RyR activator in EC coupling, it augments RyR activation [16,17]. These findings support Lamb and Stephenson's proposition [13] that, during EC coupling, DHPRs somehow relieve Mg²⁺ inhibition and thus permit RyR activation by ATP and Ca²⁺. The regions of the DHPR that interact physically with the RyR have been examined in myocytes/myotubes as well as in cell-free systems. In the first case, cDNAs encoding chimaeric constructs of skeletal and cardiac DHPR α₁ subunits injected into dysgenic myotubes (i.e. myotubes lacking DHPRs [1,3,18]) revealed that the cytoplasmic loop between repeats II and III of the skeletal DHPR α₁ subunit (⁶⁶⁶Glu-Leu⁷⁹¹) [2], and specifically residues ⁷²⁵Phe-Pro⁷⁴², are the crucial determinants of skeletal EC coupling [19]. The recombinant II–III loop activates isolated skeletal but not cardiac RyRs [20]. Shorter synthetic sequences [21], A_S (⁶⁷¹Thr-Leu⁶⁹⁰) and C_S (⁷²⁴Glu-Pro⁷⁶⁰), also interact with isolated RyRs [34–39]. A_S activates skeletal RyRs [21–25] or inhibits them by blocking the ion-conduction pathway [23]. In spite of the importance of residues ⁷²⁵Phe-Pro⁷⁴² for skeletal EC coupling [19], the regulation of RyRs by the corresponding synthetic peptide is not well understood. Ryanodine binding and Ca²⁺ release assays show that C_S is a much weaker activator of RyRs than A_S [21,22,26], whereas single-channel studies show that C_S strongly activates RyRs [25,27].

In the present study, we have further defined the interactions between C_S and skeletal RyRs (RyR1) in the presence of Ca²⁺, Mg²⁺ and ATP, which are key physiological regulators of the RyR. Importantly, Mg²⁺ and ATP were present in previous experiments examining Ca²⁺ release from SR [21,22,26], but were not present in single-channel studies [25,27]. To characterize further the interaction with the RyR, we have compared the effects of C_S with those of related peptides, C_C (the cardiac C sequence), since replacement of the crucial region in the α_1C subunit with its skeletal counterpart restores skeletal EC coupling [19]. We also examined the two halves of C_S, the C_S1 (⁷²⁵Phe-Gly⁷⁴³) and C_S2 (⁷⁴⁰Asp-Pro⁷⁶⁰). C_S2 is more potent (K_i∼290 μM) compared with C_S1 (K_i∼3 mM) in reversing the effects of A_S (30 μM) on Ca²⁺ release from SR vesicles, [³H]ryanodine binding to RyRs and changes in MCA (4-methylcoumaryl-7-amide) fluorescence [22]. However, unlike C_S2, only the C_S1 region is crucial for skeletal-type EC coupling in myocytes [19].

EXPERIMENTAL

Peptides

Peptides with the sequences shown in Figure 1 were synthesized as in [23].

Figure 1. Sequences of the skeletal and cardiac DHPR C regions and the synthetic DHPR C-region peptides used in the present study.

Note that several residues are given (double spaced) on either side of the skeletal C region used here to illustrate points made in the Discussion section.

Isolation of SR vesicles, lipid bilayers, solutions, single-channel recording and analysis

These techniques have been described previously in [28,29]. The cis (cytoplasmic) bath contained either 230 mM CsCH₃O₃S+20 mM CsCl+10 mM TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid; pH 7.4 adjusted with CsOH) (250 mM Cs⁺ solution) or 80 mM CsCH₃O₃S and 20 mM CsCl, 10 mM TES (100 mM Cs⁺ solution). The trans (luminal) solution contained either 30 mM CsCH₃O₃S+20 mM CsCl+10 mM TES (50 mM Cs⁺ solution) or 250 mM Cs⁺ solution+0.1–1.0 mM CaCl₂. In experiments where vesicle fusion was performed with cis and trans baths containing 250 mM Cs⁺ solution, 500 mM mannitol was added to the cis bath to produce the osmotic gradient necessary for vesicle fusion. [Ca²⁺] was determined using a Ca²⁺ electrode (Fluka, Rolconkoma, NY, U.S.A.). [Mg²⁺] was estimated from the total amount of MgCl₂ using published association constants [30] and the program ‘Bound and Determined’ [31]. Lipids were obtained from Avanti Polar Lipids (Alabaster, AL, U.S.A.), ATP, CsCH₃O₃S, CsCl and MgCl₂ from Sigma, CaCl₂ from BDH Chemicals (Poole, Dorset, U.K.) and TES, CsOH and DIDS (di-isothiocyanostilbene-2,2′-disulphonic acid) from MP Biomedicals (Seven Hills, NSW, Australia).

Positive current is defined as flow of positive charge from cytoplasm to lumen (cis to trans). Electrical potential is expressed as V_cytoplasm−V_lumen (i.e. V_cis−V_trans). Channel activity was recorded at +40 mV. Channels were exposed to different conditions by flowing solutions over the bilayer using the local perfusion system with an exchange time of 1–2 s, depending on the flow rate [32].

DIDS modification

In one experiment, RyRs were modified by DIDS to slow the gating kinetics of the channels. The RyRs were almost fully activated by 30–90 s exposure to 500 μM DIDS at 1 mM cytoplasmic Ca²⁺ (P₀±S.E.M.=0.94±0.01, n=11). Removal of DIDS decreased P₀ to approx. 0.8 because the reversible component of DIDS activation was removed [32].

Statistics

The means were weighted with the number of channels in the bilayer. The S.E.M. was determined as:

where N is the number of experiments, n_i the number of RyRs in the bilayer in the ith experiment, x_i the test value in the ith experiment and X the weighted mean of the test values x_i, where

RESULTS

Activation of RyRs by 100 nM–1 μM DHPR C-region peptides

In the present study, RyRs were activated under control and test conditions either by cytoplasmic Ca²⁺ alone (100 μM) or by cytoplasmic ATP (2 mM) in the presence of free [Ca²⁺] between 20 and 200 μM. It has previously been shown that C_S (≤10 μM) activates RyRs at cytoplasmic [Ca²⁺] over the range 1 nM–100 μM [27]. We show here that activation after adding peptide C_S to the cytoplasmic side of the RyR depended on the presence of ATP (Figure 2). In the absence of ATP, the addition of 1 μM C_S to the cytoplasmic side of the channels substantially increased RyR activity, whereas removal of peptide did not return P₀ to control levels within the time frame of the experiments (Figure 2A). In contrast, in the presence of ATP, RyRs had a P₀∼0.8 and were not stimulated further by the addition of 1 μM C_S (Figure 2B).

Figure 2. RyRs are activated by the DHPR C-region peptide C_S in the cytoplasmic bath.

(A) Current traces in a single experiment on two RyRs. The current baselines are denoted by the broken lines and channel openings are seen as upward current transitions. Channels were recorded (a) in cis/trans 250 mM/50 mM Cs⁺, 0.1 mM/1 mM Ca²⁺, (b) during exposure to 1 μM C_S, (c) 15 s after removal of C_S from the solution, (d) during a second exposure to 1 μM C_S and (e) after removal again of the peptide. Reapplication of C_S produced no additional increase in channel activity. (B) Two RyRs are activated by 2 mM ATP+0.5 mM Ca²⁺ (free [Ca²⁺]=200 μM) in the cytoplasmic bath (a) under control conditions and (b) during exposure to 1 μM C_S.

RyR activation by the four peptides (C_S, C_S1, C_S2 and C_C) was measured during rapid application of 100 nM peptide to bilayers by local perfusion for 30–90 s. These exposures were brief enough and at sufficiently low concentrations that the inhibitory effects of the peptides (see below) were minor. Peptides were removed by flowing control solution over the bilayer for 30–120 s. Under control conditions, in the presence of 100 μM cytoplasmic Ca²⁺, the RyRs had P₀ values of approx. 0.1. Application of each of the peptides increased channel activity with efficacies in the order C_S>C_S1∼C_S2>C_C (Figure 3A, black bars). Curiously, current levels marginally increased after the removal of peptides (Figure 3A, open bars).

Figure 3. RyR activation by the DHPR C-region peptides C_S, C_S1, C_S2 and C_C.

(A) Channel activity is expressed as P₀. The degree of activation in response to peptide application (black bars) and peptide washout (open bars), normalized to control values before the addition of peptide. The four data groups on the left show the effect of 100 nM peptide in the presence of 100 μM Ca²⁺. The data on the right show the effect of 1 μM C_S in the presence of 2 mM ATP+0.5 mM Ca²⁺ (free [Ca²⁺]=200 μM). Control values of P₀ and the number of experiments are given in Table 1. (B) Time course of RyR activation. Ensemble traces were compiled from the RyR current summed from all experiments. Initially, RyRs were partially activated in the presence of 100 μM cytoplasmic Ca²⁺ with P₀ values of approx. 0.1. The peptide was applied at zero time for 20–30 s. The exponential time constants, activation rates and number of experiments are given in Table 1.

The kinetics of RyR activation by 100 nM of each of the peptides was measured by compiling ensemble current traces from all experiments (Figure 3B). The exponential time constants for activation (τ_a) and their associated rate constants are given in Table 1. The activation time constant for C_S2 was similar to the time of solution exchange and, thus, was a lower estimate of the actual activation rates. Activation by C_S, C_S1 and C_C was significantly slower.

Table 1. Activation time constants (τ_a) for the DHPR C-region peptides.

Values of τ_a were obtained from exponential fits to the current-activation time courses shown in Figure 3. P_min is the mean P₀ before peptide application. N is the total number of bilayer experiments and n the total number of channels summed for each current time course.

	Pmin	N	n	τa (s)	kon (M−1·s−1)
CS	0.12	4	88	7.63±0.25	(13.1±0.4)×105
CC	0.09	9	375	4.7±0.07	(21±0.3)×105
CS1	0.08	10	216	3.80±0.15	(26±1)×105
CS2	0.07	6	80	0.92±0.05	(10.9±0.6)×106

In the presence of ATP, C_S did not increase RyR activation (Figures 3A and 4). The results presented in Figure 4 show the concentration dependence of RyR activation by ATP in the presence and absence of C_S. In the absence of C_S (Figure 4, open circles), ATP (with 100 μM Ca²⁺) increased P₀ from 0.19±0.07 to 0.63±0.14, whereas in the presence of C_S (Figure 4, filled circles), RyRs had P₀ of 0.4±0.1 and ATP could increase this to 0.75±0.11. Hill fits to the data showed that K_a for ATP activation was 400 μM both in the presence and absence of C_S.

Figure 4. Activation of RyRs in the presence and absence of C_S.

ATP (2 mM) alone (○, N=6), ATP+1 μM C_S (●, N=4), ATP+75 μM C_S (■, N=4). The curves show Hill fits (Hill coefficient=2) to each data set. The fitted Hill parameters are: (······), K_a=0.4 mM, P_max=0.71, P_min=0.13; (——), K_a=0.4 mM, P_max=0.75 and P_min=0.41; and (----), K_a=0.23 mM, P_max=0.72 and P_min=0.1. The baths contained (cis/trans) 250 mM/50 mM Cs⁺ and 0.1 mM/1 mM Ca²⁺ (free).

Fast inhibition of RyRs is most apparent with ≥10 μM DHPR C-region peptides

Fast inhibition of RyRs described in this section has not been reported previously. The existence of fast inhibition was indicated by several previous observations. First, we noticed that application of ≥10 μM C_S to RyRs caused less initial activation when compared with that induced by 100 nM peptide (e.g. Figure 3). Secondly, RyR activity increased when 100 nM or 1 μM of the peptides was removed (e.g. Figures 2A and 3A), indicating that activity was decreased in the presence of the peptide. In seven other experiments, application of 10 μM C_S increased P₀ from 0.03±0.01 to 0.07±0.02 and then P₀ increased further to 0.12±0.03 on washout of C_S. These observations showed that RyRs were reversibly inhibited by C_S and that the inhibition masked peptide activation. To measure the concentration dependence of inhibition, RyRs were permanently (within the time frame of the experiment) stimulated at the beginning of each experiment by a brief (30 s) application of 10 μM C_S in the presence of 100 μM Ca²⁺. When C_S was re-applied, P₀ decreased in a concentration-dependent manner (Figure 5B, triangles) with K_i=28 mM and n_i=1.5. When the same method was used in the presence of ATP, RyRs were inhibited by C_S with a lower affinity, K_i=190 μM and n_i=2.2 (Figure 5B, circles), in 100 or 250 mM Cs⁺ solutions. Figure 4 shows that, in the absence of ATP, RyRs are inhibited by 75 μM C_S compared with 1 μM C_S and that this inhibition is markedly alleviated by [ATP] as low as 0.2 mM.

Figure 5. Fast inhibition of RyRs by the DHPR C-region peptide C_S.

(A) The gating of four RyRs are recorded at +40 mV. Channel activity during application and withdrawal of 100 μM C_S reveals reversible peptide-induced inhibition. Before the control recording, the channels were exposed to 10 μM C_S for 30 s so that the channels were activated by the peptide in the control and test situations. The broken line denotes the current baselines in each record. The baths contained (cis/trans) 250 mM/50 mM Cs⁺ and 0.1 mM/0.1 mM Ca²⁺. (B) Concentration dependences of RyR inhibition by C_S, normalized to control values under three experimental conditions. Cytoplasmic solutions contained 250 mM Cs⁺ and 0.1 mM Ca²⁺ (△; P₀ for control=0.24±0.05, N=5); 100 mM Cs⁺ and 2 mM ATP, 0.1 mM Ca²⁺ (free [Ca²⁺]=40 μM) (○; P₀ for control=0.74±0.13, N=5); or 250 mM Cs⁺, 2 mM ATP, 0.1 mM Ca²⁺ (free [Ca²⁺]=20 μM) (●; P₀ for control=0.42±0.11, N=3). Curves show Hill fits to the data with K_i=28 mM and n_i=1.5 (······) and K_i=190 μM and n_i=2.2 (——).

In an effort to define the kinetics of inhibition more clearly, we used channels whose gating kinetics were slowed by DIDS modification (see the Experimental section). DIDS-modified RyRs had a lower frequency of channel closures that made it easier to distinguish the closing events due to peptide inhibition (Figure 6A). The DIDS-modified RyRs were exposed to cycles of peptide application (10–60 s) and washout (20–90 s). Peptide inhibition occurred within 15–30 s of peptide application (Figures 6A and 6B). Peptide inhibition was fully reversed within 20–60 s of washout. Rates of RyR inhibition and recovery were determined from exponential fits to the current response. The apparent affinities for the DHPR C-region peptides, calculated from the on- and off-rates of inhibition, are given in Table 2. C_S and C_C (50 μM) each produced similar inhibition of DIDS-modified RyRs (Figure 6C). Peptides C_S1 and C_S2 at 50 μM were much weaker inhibitors than C_S or C_C. C_S2 was a more potent inhibitor than C_S1 at 150 μM.

Figure 6. Fast inhibition of DIDS-modified RyRs by DHPR C-region peptides.

(A) The gating of a single, DIDS-modified RyR during application and withdrawal of 50 μM C_S under conditions described in Figure 5(A). Baselines are shown by the broken line. (B) Inhibition of seven DIDS-modified RyRs in one bilayer subjected to application and removal of 150 μM peptides C_S1 and C_S2. The solid line labelled ‘i_max’ denotes the current when all channels are open. (C) DIDS-modified RyRs inhibited by DHPR C-region peptides. Data are normalized to P₀ values in the absence of a peptide. The control values and number of experiments are given in Table 2.

Table 2. Time constants for inhibition (τ_i) and recovery (τ_r) and the associated reaction rates k_on and k_off for the DHPR C-region peptides on DIDS-modified RyRs.

P_max is the value of P₀ in the absence of peptide and N the total number of experiments and n the total number of channels over which the averages for τ were taken.

	Pmax	N	n	τi (s)	kon (M−1·s−1)	N	n	τr (s)	koff (s−1)	Ki (μM) (koff/kon)
50 μM CS	0.85±0.01	14	923	3.3±0.2	(6.1±0.4)×103	13	809	12.9±0.7	(7.8±0.1)×10−2	13
50 μM CC	0.87±0.02	8	488	2.4±0.2	(8.3±0.7)×103	8	488	23.4±2.2	(4.3±0.4)×10−2	5.2
150 μM CS1	0.81±0.02	7	76	1.0±0.1	(6.7±0.7)×103	4	48	3.6±0.3	(2.8±0.2)×10−1	42
150 μM CS2	0.79±0.02	5	67	3.5±0.2	(1.9±0.1)×103	4	60	10.2±0.7	(9.8±0.7)×10−2	52

Slow inhibition by 50–150 μM DHPR C-region peptides

The C peptides also induced a very slow low-affinity inhibition. It was shown previously that when C_S was applied to RyRs in bilayers at sufficiently high concentrations (50–150 μM) for several minutes, it produced channel closure that can take several minutes to reverse, if at all [27]. In the present study, we report that C_S1, C_S2 and C_C at 50–150 μM induced a similar inhibition. C_S1 inhibited 9/13 channels within 30–420 s, C_S2 inhibited 3/9 channels within 60–260 s and C_C inhibited 2/7 channels within approx. 40 s.

Ca²⁺/Mg²⁺ inhibition of RyRs is modified by 25–50 μM DHPR C-region peptides

Inhibition of RyRs by cytoplasmic Mg²⁺ is an important regulator of Ca²⁺ release in muscles. We have shown that Mg²⁺ inhibits RyRs by binding to either the Ca²⁺-activation sites (A-sites) or to low-affinity sites (I-sites) where Ca²⁺, Mg²⁺ and other bivalent cations can bind and inhibit the channel [33,34]. The effect of DHPR C-region peptides on Ca²⁺/Mg²⁺ inhibition of RyRs was measured to determine whether the peptides could mimic the change in the bivalent ion affinity of I-sites, which is so integral to EC coupling (see the Introduction section). To avoid effects of high-affinity peptide activation, RyRs were subjected at the start of each experiment to two cycles of exposure to peptides and washout to ensure that the channels were permanently activated by the peptide. To measure inhibition at the I-sites, RyRs were locally perfused with solutions containing various [Ca²⁺] or [Mg²⁺] in the presence of 2 mM ATP. Ca²⁺ and Mg²⁺ concentration dependences were obtained from the peptide-modified RyRs, both in the presence and absence of a peptide.

Figure 7 shows that application of C_S rapidly and reversibly alleviates Ca²⁺ inhibition of RyRs in the presence of ATP. C_S caused a small decrease in activity of channels that were not Ca²⁺-inhibited [i.e. when the cytoplasmic [Ca²⁺] was 40 μM (Figure 7A)]. Increasing cytoplasmic [Ca²⁺] to 1 mM resulted in a decrease in P₀ due to Ca²⁺ inhibition at the I-sites (Figure 7B). Addition of C_S to the inhibited channels caused a rapid increase in the current, which decreased as soon as the peptide was removed. The presence of C_S (50 μM) decreased the sensitivity of RyRs to inhibition by Ca²⁺ under high (250 mM) and low (100 mM) ionic strength conditions (Figure 7C). The half-inhibitory concentration K_i and Hill coefficient n_i for Ca²⁺ and Mg²⁺ are given in Table 3. The K_i for I-site inhibition is shifted to higher bivalent cation concentrations when the ionic strength is higher (Figure 7C and [35]). This ionic strength dependence was retained in the presence of C_S.

Figure 7. Alleviation of Ca²⁺ inhibition by C_S.

(A, B) The gating of three RyRs recorded at +40 mV. The cytoplasmic solution contained 250 mM Cs⁺ and 2 mM ATP and the indicated free [Ca²⁺]. In these experiments, luminal [Ca²⁺]=1 mM. (A) Channel activity in 40 μM Ca²⁺ is slightly inhibited by application of 50 μM C_S (note the increased frequency of short closures). (B) The presence of 1 mM Ca²⁺ has two effects on channel activity. It slightly decreased the channel conductance by partially blocking the Cs⁺ flow and decreased the open probability of channel opening (the latter being Ca²⁺ inhibition). During application of 50 μM C_S in the presence of 1 mM Ca²⁺, channel activity reversibly increased as a result of decreased Ca²⁺ inhibition. The broken lines denote the current baselines in each record and the solid lines labelled ‘i_max’ denote the current when all channels are open. (C) Ca²⁺ inhibition normalized to P₀ at 10–40 μM Ca²⁺ in the presence of 250 mM Cs⁺ (○, ●) and 100 mM Cs⁺ (□, ■) and in the absence (○, □) or presence of 50 μM C_S (●, ■). The data are the mean P₀. The Hill fit parameters for all the experiments, including normalizing values at low [Ca²⁺] and [Mg²⁺], are given in Table 3.

Table 3. Summary of the parameters of Hill fits to the [Ca²⁺] and [Mg²⁺] dependences of P₀ in the absence and presence of DHPR C-region peptides.

P_max is the P₀ at low bivalent-ion concentration, K_i the half-inhibitory concentration of X²⁺ and n_i the Hill coefficient. N is the number of bilayers and n the number of channels measured.

DHPR C-region peptide	Experimental conditions	X2+	Pmax	Ki (μM)	ni	N	n
–	250 mM Cs+, 2 mM ATP	Ca2+	0.86±0.03	980±055	3.0±0.6	5	51
50 μM CS	250 mM Cs+, 2 mM ATP	Ca2+	0.84±0.06	2300±400	2.6±1.1	4	31
–	100 mM Cs+, 2 mM ATP	Ca2+	0.73±0.06	210±037	2.0±0.5	3	40
50 μM CS	100 mM Cs+, 2 mM ATP	Ca2+	0.49±0.05	460±071	4.5±2.8	3	40
–	100 mM Cs+, 2 mM ATP	Mg2+	0.69±0.07	250±40	2.3±0.7	5	70
50 μM CS	100 mM Cs+, 2 mM ATP	Mg2+	0.46±0.07	420±110	2.7±2.0	6	65
25 μM CS1	100 mM Cs+, 2 mM ATP	Mg2+	0.58±0.06	570±120	1.7±1.4	4	75
25 μM CS2	100 mM Cs+, 2 mM ATP	Mg2+	0.62±0.07	350±41	3.2±2.0	4	12
*Native	250 mM Cs+	Ca2+		710±80		18
*MHS	250 mM Cs+	Ca2+		1160±220		13
*Native	100 mM Cs+	Ca2+		204±32		18
*MHS	100 mM Cs+	Ca2+		957±250		9

* Native and MHS refer to normal and malignant hyperthermia-susceptible RyRs respectively from pig skeletal muscles [39].

Similarly, DHPR C-region peptides decreased RyR sensitivity to Mg²⁺ inhibition. Mg²⁺ inhibition was measured with 10–40 μM free cytoplasmic Ca²⁺ to saturate the A-sites and ensure that inhibition was mainly due to Mg²⁺ binding to the low-affinity I-sites [33]. Figure 8 shows the effect of DHPR C-region peptides on Mg²⁺ inhibition at low ionic strength. C_S (50 μM) caused a decrease in RyR activity in the absence of Mg²⁺ (Figure 8A). In the presence of 0.3 mM Mg²⁺, the peptide addition had a stimulating effect (Figure 8B). Comparison of these records indicates that C_S decreased the amount of Mg²⁺ inhibition in these channels (Figure 8C). Experiments using 25 μM C_S1 and C_S2 also showed that these peptides shifted Mg²⁺ inhibition to higher concentrations (Figure 8C and Table 3). After washout of these peptides, the K_i values for Ca²⁺ and Mg²⁺ inhibition at high and low ionic strengths were similar to those seen previously in native RyRs (Table 3), indicating that Ca²⁺/Mg²⁺ inhibition was not altered during high-affinity activation by C_S.

Figure 8. Alleviation of Mg²⁺ inhibition by DHPR C-region peptides.

(A, B) The gating of five RyRs recorded at +40 mV. The cytoplasmic solution contained 100 mM Cs⁺ and 2 mM ATP and the indicated [Mg²⁺] and luminal [Ca²⁺]=1 mM. (A) Channel activity in 10 μM Ca²⁺ is partially inhibited in the presence of 50 μM C_S. (B) In the presence of 0.3 mM Mg²⁺, 50 μM C_S slightly increased channel activity. Comparison of the traces in (A, B) shows that Mg²⁺ produced less inhibition in the presence of C_S. The broken lines denote the current baselines in each record and the solid lines labelled ‘i_max’ denote the current when all channels are open. (C) Mg²⁺ inhibition in 100 mM Cs⁺ in the presence and absence of peptides. Data are normalized to P₀ at 10 μM Ca²⁺ and zero Mg²⁺. In the presence of peptides (filled symbols), RyRs are less sensitive to bivalent ion inhibition. The broken and solid curves are Hill fits to ○ and ● respectively. The Hill fit parameters for all the experiments, including normalizing values at low [Ca²⁺] and [Mg²⁺], are given in Table 3.

DISCUSSION

In the present study, we find that regulation of RyRs by DHPR C-region peptides is strongly dependent on the cytoplasmic Ca²⁺, Mg²⁺ and ATP. This dependence can explain previous differences between the effects of the peptides on Ca²⁺ release from SR and on single-channel activity. A significant finding was that the DHPR C-region peptides decreased the apparent affinity of the RyR for Mg²⁺ inhibition. This is a particularly important result since the C region of the DHPR II–III loop is the determinate of skeletal-type EC coupling [2], a process that is proposed to activate RyRs by relieving their Mg²⁺ inhibition [13]. The experiments revealed other novel effects of these peptides on RyR activity. In the present study, the inhibition of RyRs by C_S seen previously [27] is shown to be due to two mechanisms that are clearly distinguished by their kinetics. One mechanism produces inhibition with onset and recovery times of minutes after peptide application and removal. On the time scale of the bilayer experiments, this inhibition process is non-reversible. The other mechanism causes reversible inhibition with onset and recovery times of seconds. Fast inhibition occurs at the same time as, and partially masks, a slowly reversible activation process and both effects are substantially decreased in the presence of ATP.

A DHPR II–III loop peptide relieves Ca²⁺/Mg²⁺ inhibition

The ability of the C-region peptide to decrease Mg²⁺ inhibition reveals a tantalizing parallel between the actions of the peptide and changes in RyR activity during EC coupling in skeletal muscles. It is surprising that the mere addition of the C_S section of the DHPR II–III loop to the RyR in bilayers can mimic an effect of the DHPR on RyRs during EC coupling. The 2-fold decrease in RyR affinity for Mg²⁺ caused by C_S is much smaller than the 10–100-fold decrease that occurs during EC coupling in skinned muscle fibres [36] and in triad preparations [37]. However, the effect of C_S indicates that, in this very different isolated system, there is a loose interaction between the DHPR C-region peptides and the RyR, which can affect Ca²⁺/Mg²⁺ inhibition. Such an interaction is consistent with a recent finding by Gallant [38] that the Arg⁶¹⁵→Cys mutation in pig skeletal RyRs not only decreases the apparent affinity of the RyR for Ca²⁺ and Mg²⁺ inhibition [39] but also decreases the activating effect of C_S. Although the Arg⁶¹⁵→Cys mutation and C_S both relieve Ca²⁺/Mg²⁺ inhibition, the peptide and the mutation differ in their ionic strength dependences. The Arg⁶¹⁵→Cys mutation has a larger effect on Ca²⁺/Mg²⁺ inhibition at low ionic strength (Table 3), whereas C_S has the same effects both at high and low ionic strength.

Different peptide mechanisms are revealed by different experimental conditions

Effects of C-region peptides depend on the type of RyR agonists present. A slowly reversible activation by C_S is seen when RyRs are activated by Ca²⁺ alone (without ATP; e.g. Figure 3). In the presence of ATP, this type of activation is absent. However, if Mg²⁺ is also present, C_S can reversibly activate RyRs by decreasing their sensitivity to Mg²⁺ inhibition (e.g. Figure 8). These findings can reconcile disparate reports in the literature about the effect of these peptides on RyR1. This and other single-channel studies [25,27] show that RyR activation by peptide C_S is much stronger and occurs with higher affinity than that revealed by measurements of Ca²⁺ release from SR vesicles where K_a>10 μM (C. S. Haarmann, A. F. Dulhunty and D. R. Laver, unpublished work; [21,22,26]). So far, single-channel studies have measured the effects of C_S under conditions where Ca²⁺ is the sole agonist, whereas Ca²⁺ release experiments were performed in the presence of ATP and Mg²⁺ since these ligands are required for vesicle loading. Therefore C_S-induced activation of SR Ca²⁺ release could be due to the relief of Mg²⁺ inhibition, which is a mechanism different from the high-affinity activation seen with Ca²⁺ alone. In the present study, we show that C_S2 is a stronger and faster inhibitor of RyR than C_S1, which may also explain why C_S2 is more potent than C_S1 in reversing the activation by A_S [22].

C_S has no effect on Ca²⁺ release in skinned fibres but decreases depolarization-induced Ca²⁺ release, suggesting that C_S competes with the II–III loop for its activation site on the RyR [24]. It is unlikely that fast inhibition by C_S could occur in these skinned fibre experiments due to the presence of ATP in the cytoplasmic bathing solution. It is intriguing that reversible inhibition is regulated by the physiological agonist ATP. Physiologically, reversible inhibition in the absence of ATP and its abolition in the presence of ATP could contribute to the decrease in Ca²⁺ release seen during fatigue where ATP levels are decreased.

Activation by C_S (⁷²⁴Glu-Pro⁷⁶⁰) in the present study appears to differ from that seen with a similar peptide (⁷²⁰Leu-Glu⁷⁶⁵) [25]. C_S activates with K_a<100 nM and is effectively non-reversible, whereas activation by residues ⁷²⁰Leu-Glu⁷⁶⁵ had K_a∼10 μM and is readily reversible. The additional amino acids at each end of C_S (LKDV and PLAEL; Figure 1) appear to decrease significantly its binding affinity. This is not surprising given the frequency of charged amino acids near the ends of C_S and in additional amino acids in ⁷²⁰Leu-Glu⁷⁶⁵. In a parallel situation, a significant loss of activity is seen when the predominantly positively charged peptide A_S (⁶⁷¹Thr-Leu⁶⁹⁰) is modified by the addition of residues 691–710, which contain several negative charges [40].

DHPR C region in EC coupling

Two requirements for changing cardiac to skeletal EC coupling in dysgenic myotubes expressing CaV1.2 are the localization of DHPRs opposite every other RyR and the activation of RyRs via an external Ca²⁺-independent interaction with the DHPR. Both these requirements are fulfilled when the C-region sequence is skeletal, in an otherwise cardiac context [2,19,41]. Some effects of C-region peptides are consistent with interactions between DHPRs and RyRs that may contribute to skeletal EC coupling. First, the localizing interaction is expected to be of high affinity and specific for the skeletal C region. Indeed, RyR activation by C_S is greatest with the skeletal DHPR peptides that bind and activate with high affinity and have low off rates. Secondly, the activating interaction in EC coupling might be the desensitization of RyRs to Mg²⁺ inhibition [36]. Although the latter effect is not specific for the DHPR isoform, isoform specificity may depend on co-localization of DHPRs and RyRs (via the DHPR C region or by other regions that may, or may not, be conserved between CaV1.1 and CaV1.2). There is a further parallel between our results and EC coupling studies. We find that activation by C_S is stronger than that by either C_S1 or C_S2. This is consistent with a stronger EC coupling if the whole C region is of skeletal sequence compared with the situation when C_S1 is skeletal and C_S2 is cardiac [19]. There appears to be a lack of C-region isoform specificity in fast inhibition, which indicates that both cardiac and skeletal DHPRs and RyRs could interact physically with each other if they were appropriately targeted. This appropriate targeting appears to be dependent on the C_S1 region [19].

In conclusion, the action of these peptides critically depends on the presence of the key physiological regulators, Ca²⁺, Mg²⁺ and ATP, so that peptide effects on isolated RyRs might not equate to their action in muscle but rather provide pointers to mechanisms underlying EC coupling. The manifold effects of the peptides do suggest that the C region of the DHPR is capable of a complex influence over RyR activity in resting muscles and during EC coupling.