Alternative splicing of RyR1 alters the efficacy of skeletal EC coupling

Abstract

Alternative splicing of ASI residues (Ala(3481)-Gln(3485)) in the skeletal muscle ryanodine receptor (RyR1) is developmentally regulated: the residues are present in adult ASI(+)RyR1, but absent in the juvenile ASI(-)RyR1 which is over-expressed in adult myotonic dystrophy type 1 (DM1). Although this splicing switch may influence RyR1 function in developing muscle and DM1, little is known about the properties of the splice variants. We examined excitation-contraction (EC) coupling and the structure and interactions of the ASI domain (Thr(3471)-Gly(3500)) in the splice variants. Depolarisation-dependent Ca(2+) release was enhanced by >50% in myotubes expressing ASI(-)RyR1 compared with ASI(+)RyR1, although DHPR L-type currents and SR Ca(2+) content were unaltered, while ASI(-)RyR1 channel function was actually depressed. The effect on EC coupling did not depend on changes in ASI domain secondary structure. Probing RyR1 function with peptides possessing the ASI domain sequence indicated that the domain contributes to an inhibitory module in RyR1. The action of the peptide depended on a sequence of basic residues and their alignment in an alpha-helix adjacent to the ASI splice site. This is the first evidence that the ASI residues contribute to an inhibitory module in RyR1 that influences EC coupling. Implications for development and DM1 are discussed.

Figure 1. Maximal voltage-gated Ca²⁺ calcium release is enhanced in ASI(-)-expressing myotubes

A) Representative L-type Ca²⁺ currents (upper traces) and Ca²⁺ transients (lower traces) elicited by 200ms depolarisations to the indicated potential in ASI(+)RyR1- and ASI(-)RyR1-expressing myotubes. Average voltage-dependence of peak L-type Ca²⁺ current density (B) and intracellular Ca²⁺ transients (C) in dyspedic myotubes expressing ASI(+) (○, n=10) or ASI(-) (●, n=9). D) Representative time-dependence of the first derivative of the Ca²⁺ transient (δF/δt) elicited at +60mV in dyspedic myotubes expressing either ASI(+)RyR1 (black trace) or ASI(-)RyR1 (grey trace). E) Average peak of the first derivative of the Ca²⁺ transient (δF/δt) at +60mV in dyspedic myotubes expressing ASI(+)RyR1 (open bar) or ASI(-)RyR1 (filled bar). ^†p<0.01 compared to ASI(+)RyR1.

Figure 2. Releasable SR Ca²⁺ content is not different in ASI(+)RyR1- and ASI(-)RyR1-expressing myotubes

A) Representative fura-FF ratio (340/380) traces in ASI(+)RyR1- and ASI(-)RyR1-expressing myotubes following sequential addition of 500μM 4-cmc, wash, ICE (10μM ionomycin, 30μM CPA, and 2mM EGTA), and finally a 10mM Ca²⁺ + 10μM ionomycin Ringer's solution used to determine the maximal fura-FF ratio (dotted lines) during Ca²⁺ saturation of the dye. B) Average peak intracellular Ca²⁺ responses to addition of 4-cmc (left), 0 Ca²⁺ total store Ca²⁺ release cocktail (ICE, middle), and 10mM Ca²⁺ + 10μM ionomycin (right) in non-expressing (hatched bars, n=9), ASI(+)RyR1-expressing (open bars, n=29) and ASI(-)RyR1-expressing (filled bars n=22) dyspedic myotubes. ^*p<0.05 compared to ASI(+)RyR1; ^†p<0.01 compared to ASI(+)RyR1. Minor deviations between the traces shown in A not observed in the average data reflect the cell-to-cell variability typically observed in these experiments.

Figure 3. NMR-derived properties of the ASI peptides

Summary of the sequential and medium range connectivities, and ³J_NH-Hα coupling constants identified for each of the ASI peptides. The NOE connectivities are indicated by black lines. The line thickness reflects the strengths of the NOE correlation as strong, medium and weak. Values of ³J_NH-Hα less than 6 Hz are indicated by ↓.

Figure 4. Structure of ASI peptides

A) CD spectra at 5°C for peptides ASI(+) (thin solid line), ASI(-) (thin dashed line), ASI(-) mutant (thick solid line), and ASI-short (thick dashed line) in H₂O, pH5.0. B) Structural representation of ASI(+), ASI(-), and ASI(-)-mutant (overlayed on left) and peptide A (right). Side chains of ASI residues K³⁴⁹⁵ and R³⁴⁹⁹ and peptide A residues K⁶⁷⁷, R⁶⁸¹ and R⁶⁸⁴ are indicated.

Figure 5. The sequence and structure of basic residues K³⁴⁹⁵KKRR³⁴⁹⁹ determines ASI peptide activation of RyR1

A) Effects of peptides ASI(-), ASI(mutant), and ASI(short) on [³H]ryanodine binding with 10μM Ca²⁺ (n=3 for each peptide at each concentration). B-D) OD changes at 710nm, reflecting changes in extravesicular [Ca²⁺] measured using antipyrylazoIII (500μM) with an addition of 1μM thapsigargin (first arrow), then 100μM peptide ASI(-)(second arrow in B), ASI(mutant) (C) or ASI(short) (D), 5μM ruthenium red (third arrow in B), and 1.5μg/ml Ca²⁺ ionophore A23187 (last arrow in B). E) Average rates (nmoles/mg/min, n=3) with peptides ASI(-), ASI(mutant), and ASI(short). The average initial rate of release (peptide minus thapsigargin) is shown in E. It is apparent in D that this initial rate is not substantially increased by the ASI(short) peptide, while the later release rate (1-3min after peptide addition) is greater than that with ASI(mutant). Asterisks (*) in (A) and (D) indicate average values significantly different from control, while the (^#) symbol indicates a significant difference from the wild-type ASI(-) at each peptide concentration.

Figure 6. Peptide ASI(-) activates RyR1 channels at -40mV, but ASI(short) does not. Both peptides at 100μM inhibit channels at +40mV

A) & B) Channel activity (3s) with all-points histograms for the entire 30s record. The probability of the maximum open current (indicated by the broken lines in the channels records or arrows in the all-points histograms) is high only in the presence of ASI(-) at -40mV (in A) or under control conditions at +40mV in (A & B). Upper panels - control activity; lower panels - activity with 100μM peptide. A) ASI(-). B) ASI(short). Channel openings are downward at -40 mV (left) and upward at +40 mV (right). C) Average (n=3) effects of peptides ASI(-) and ASI-short on open probability of native RyR1 at -40mV (left) and +40mV (right). Asterisks (*) in (C) indicate average values significantly different from control (C), while the (^#) symbol indicates values ASI(short) that are significantly different lower those with ASI(-) at the same peptide concentration.

Figure 7. Competition between ASI peptides and peptideA

Relative [³H]ryanodine with 10μM Ca²⁺. A) ASI(+) alone (□) or ASI(+) plus 100μM peptide A (◆) with 10μM Ca²⁺. Average values with ASI(+) alone are significantly greater than control at each peptide concentration. Peptide A produces a significant increase in activity, but there is not further increase as ASI(+)is added with peptide A. B) ASI(-) alone (▪) or ASI(-) plus 100μM peptide A (◆) with 10μM Ca²⁺. Average values with ASI(-) alone are significantly greater that control at each peptide concentration. Peptide A produces a significant increase in activity, but there is not further increase as ASI(-) is added with peptide A. C) Caffeine alone (●) significantly increases [³H]ryanodine at all concentrations tested. There is a further significant increase in activity each caffeine concentration when 100μM peptide A is present with caffeine (◆) and an additional significant increase when 100μM ASI(-) is present with caffeine (▪). Symbols show mean ±SEM (n=3). Some SEM bars do not appear because they fall within the dimensions of the symbols.

Figure 8. ASI peptides and peptide A do not alter the Ca²⁺-dependence of [³H]ryanodine binding

A) & B) Average (n=3) [³H]ryanodine binding (A, pmol/mg; B, % maximum) in the absence of added peptide (●) or with 100μM ASI(+) (□),100μM ASI(-) (▪),100μM peptide A (◆), or 100μM DP4 (▼). In A), the average [³H]ryanodine binding with each of ASI(-), ASI(+) and peptide A are significantly greater than that in the absence of peptide at Ca²⁺ concentrations ≥10μM. Amongst the 3 peptides, average [³H]ryanodine binding with ASI(-) is significantly greater than with either ASI(+) or peptide A when Ca²⁺ was ≥10μM. Average [³H]ryanodine binding with DP4 on the other hand is significantly greater than control with 0.1μM, 1.0μM and 1mM Ca²⁺, no different with 10μM and significantly lower with 100μM Ca²⁺. In B), there are no significant differences between the average normalised [³H]ryanodine binding for control, ASI(-), ASI(+) and peptide A at any Ca²⁺ concentration. [³H]ryanodine binding in the presence of DP4 is significantly higher than that with each of the other peptides in the presence of 0.1 μM, 1.0μM and 1mM Ca²⁺. Some SEM bars do not appear because they fall within the dimensions of the symbols. Asterisks (*) indicate average [³H]ryanodine binding with DP4 that is significantly different from values in the absence of peptide at the indicated Ca²⁺.