Guo Guang Du

Topology of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum (RyR1)

To define the topology of the skeletal muscle ryanodine receptor (RyR1), enhanced GFP (EGFP) was fused in-frame to the C terminus of RyR1, replacing a series of C-terminal deletions that started near the beginning or the end of predicted transmembrane helices M1–M10. The constructs were expressed in HEK-293 (human embryonic kidney cell line 293) or mouse embryonic fibroblast (MEF) cells, and confocal microscopy of intact and saponin-permeabilized cells was used to determine the subcellular location of the truncated fusion proteins. The fusion protein truncated after M3 exhibited uniform cytoplasmic fluorescence, which was lost after permeabilization, indicating that proposed M′, M′′, M1, M2, and M3 sequences are not membrane-associated. The fusion protein truncated at the end of the M4–M5 loop and containing M4 was membrane-associated. All longer truncated fusion proteins were also associated with intracellular membranes. Mapping by protease digestion and extraction of isolated microsomes demonstrated that EGFP positioned after either M5, the N-terminal half of M7 (M7a), or M8 was located in the lumen, and that EGFP positioned after either M4, M6, the C-terminal half of M7 (M7b), or M10 was located in the cytoplasm. These results indicate that RyR1 contains eight transmembrane helices, organized as four hairpin loops. The first hairpin is likely to be made up of M4a–M4b. However, it could be made up from M3–M4, which might form a hairpin loop even though M3 alone is not membrane-associated. The other three hairpin loops are formed from M5–M6, M7a–M7b, and M8–M10. M9 is not a transmembrane helix, but it might form a selectivity filter between M8 and M10.

Functional consequences of mutations of conserved, polar amino acids in transmembrane sequences of the Ca2+ release channel (ryanodine receptor) of rabbit skeletal muscle sarcoplasmic reticulum.

The potential role in Ca2+ release channel function of highly conserved, polar, and small amino acids in predicted transmembrane sequences in the rabbit skeletal muscle ryanodine receptor (RyR1) was investigated through mutagenesis. Acidic amino acids Asp3987, Glu4032, Asp4815, Asp4917, Asp4938, and Asp4969 and amidated residues Asn4034, Asn4037, Asn4574, Asn4805, Asn4806, and Gln4933, and Gly4033 were mutated to Ala, and Ala3988 was mutated to Val. When expressed in HEK-293 cells and challenged with either caffeine or 4-chloro-m-cresol, mutants E4032A, N4806A, D4815A, and D4917A did not respond, indicating that Ca2+ release channel function was impaired. None of these mutants exhibited specific binding of [3H]ryanodine. Mutants N4805A and Q4933A showed a diminished response to both caffeine and 4-chloro-m-cresol, but [3H]ryanodine binding was not altered. Other mutant responses and the responses of mutants E4032D, N4806Q or D, D4815N or E, and D4938N or E were unaltered when compared with RyR1. However, mutants E4032Q, D4917N or E, and Q4933N or E displayed neither caffeine nor 4-chloro-m-cresol response nor [3H]ryanodine binding. Sedimentation assays indicated that the nonfunctional mutants did contain tetrameric complexes, implying that defects in the assembly of a functional channel did not occur with specific mutations in transmembrane sequences. These results support the view that amino acids Glu4032 (M2), Asn4806 (M7), Asp4815 (M7), Asp4917 (M10), and Gln4933 (M10) are involved in channel function and regulation.

Ca2+ Inactivation Sites Are Located in the COOH-terminal Quarter of Recombinant Rabbit Skeletal Muscle Ca2+ Release Channels (Ryanodine Receptors)

Ca2+ activation of skeletal (RyR1) and cardiac (RyR2) muscle Ca2+ release channels (ryanodine receptors) occurs with EC50 values of about 1 μm. Ca2+ inactivation occurs with an IC50 value of about 3.7 mm for RyR1, but RyR2 shows little inactivation, even at >100 mm Ca2+. In an attempt to localize the low affinity Ca2+ binding sites responsible for Ca2+ inactivation in RyR1, chimeric RyR1/RyR2 molecules were constructed. Because [3H]ryanodine binds only to open channels, and because channel opening and closing are Ca2+-dependent, the Ca2+ dependence of [3H]ryanodine binding was used as an indirect measurement of Ca2+ release channel opening and closing. IC50 values for [3H]ryanodine binding suggested that Ca2+ affinity for the low affinity Ca2+ inactivation sites was unchanged in a chimera in which a glutamate-rich sequence (amino acids 1743–1964) in RyR1 was replaced with the corresponding, less acidic sequence from RyR2. Ca2+ affinity (IC50) for low affinity Ca2+ inactivation sites was intermediate in RyR1/RyR2 chimeras containing RyR2 amino acids 3726–4186 (RF9), 4187–4628 (RF10), or 4629–5037 (RF11), was closer to RyR2 values in RyR1 chimeras with longer RyR2 replacements (RF9/10 or RF10/11), and was indistinguishable from RyR2 in RyR1 containing all three RyR2 replacements (RF9/10/11). These data suggest that multiple low affinity Ca2+ binding sites or multiple components of a low affinity Ca2+ binding site are located between amino acids 3726 and 5037 and that their effects on Ca2+ inactivation of the release channel are cooperative. Measurement of Ca2+activation of [3H]ryanodine binding showed that chimeras RF10, RF9/10, and RF9/10/11 were more sensitive to Ca2+than was either RyR1 or RyR2. Measurement of caffeine activation of Ca2+ release in vivo showed that chimeras RF9, RF10, RF9/10, RF10/11, and RF9/10/11 were more sensitive to caffeine than wild-type RyR1. These results suggest that Ca2+ and caffeine activation sites also involve COOH-terminal sequences in RyR1 and RyR2.

Ca2+ Signaling in HEK-293 and Skeletal Muscle Cells Expressing Recombinant Ryanodine Receptors Harboring Malignant Hyperthermia and Central Core Disease Mutations

Malignant hyperthermia (MH) and central core disease (CCD) are caused by mutations in the RYR1 gene encoding the skeletal muscle isoform of the ryanodine receptor (RyR1), a homotetrameric Ca2+ release channel. Rabbit RyR1 mutant cDNAs carrying mutations corresponding to those in human RyR1 that cause MH and CCD were expressed in HEK-293 cells, which do not have endogenous RyR, and in primary cultures of rat skeletal muscle, which express rat RyR1. Analysis of intracellular Ca2+ pools was performed using aequorin probes targeted to the lumen of the endo/sarcoplasmic reticulum (ER/SR), to the mitochondrial matrix, or to the cytosol. Mutations associated with MH caused alterations in intracellular Ca2+ homeostasis different from those associated with CCD. Measurements of luminal ER/SR Ca2+ revealed that the mutations generated leaky channels in all cases, but the leak was particularly pronounced in CCD mutants. Cytosolic and mitochondrial Ca2+ transients induced by caffeine stimulation were drastically augmented in the MH mutant, slightly reduced in one CCD mutant (Y523S) and completely abolished in another (I4898T). The results suggest that local Ca2+ derangements of different degrees account for the specific cellular phenotypes of the two disorders.

HEK-293 cells possess a carbachol- and thapsigargin-sensitive intracellular Ca2+ store that is responsive to stop-flow medium changes and insensitive to caffeine and ryanodine.

Because HEK-293 cells are widely used for the functional expression of channels, exchangers and transporters involved in Ca(2+) homoeostasis, the properties of intracellular Ca(2+) stores and the methods used for measuring intracellular Ca(2+) release in HEK-293 cells were evaluated. Ca(2+) imaging was used to show caffeine-, carbachol- and thapsigargin-induced Ca(2+) release in HEK-293 cells transfected with ryanodine receptor (RyR) cDNA, but only carbachol- and thapsigargin-induced Ca(2+) release in untransfected HEK-293 cells. Intracellular Ca(2+) release in untransfected HEK-293 cells was also observed if medium changes were performed by aspirating and replacing fresh medium (stop-flow), but not if medium changes were performed by a continuous over-flow procedure. Stop-flow medium-change-induced Ca(2+) release in HEK-293 cells was independent of caffeine and ryanodine, demonstrating that it did not occur through RyR channels. Consistent with these observations was the observation that the level of expression of endogenous RyR proteins was below the limits of detection by Western blotting or [(3)H]ryanodine binding. Thus the level of endogenous expression of RyR is so low in HEK-293 cells as to provide a negligible background in relation to functional analysis of recombinant RyR molecules. These results are inconsistent with those of Querfurth et al. [Querfurth, Haughey, Greenway, Yacono, Golan and Geiger (1998) Biochem. J. 334, 79-86], who reported higher levels of endogenous RyR expression in untransfected HEK-293 cells.

Central core disease mutations R4892W, I4897T and G4898E in the ryanodine receptor isoform 1 reduce the Ca2+ sensitivity and amplitude of Ca2+-dependent Ca2+ release.

Three CCD (central core disease) mutants, R4892W (Arg4892-->), I4897T and G4898E, in the pore region of the skeletal-muscle Ca2+-release channel RyR1 (ryanodine receptor 1) were characterized using a newly developed assay that monitored Ca2+ release in the presence of Ca2+ uptake in microsomes isolated from HEK-293 cells (human embryonic kidney 293 cells), co-expressing each of the three mutants together with SERCA1a (sarcoplasmic/endoplasmic-reticulum Ca2+-ATPase 1a). Both Ca2+ sensitivity and peak amplitude of Ca2+ release were either absent from or sharply decreased in homotetrameric mutants. Co-expression of wild-type RyR1 with mutant RyR1 (heterotetrameric mutants) restored Ca2+ sensitivity partially, in the ratio 1:2, or fully, in the ratio 1:1. Peak amplitude was restored only partially in the ratio 1:2 or 1:1. Reduced amplitude was not correlated with maximum Ca2+ loading or the amount of expressed RyR1 protein. High-affinity [3H]ryanodine binding and caffeine-induced Ca2+ release were also absent from the three homotetrameric mutants. These results indicate that decreased Ca2+ sensitivity is one of the serious defects in these three excitation-contraction uncoupling CCD mutations. In CCD skeletal muscles, where a mixture of wild-type and mutant RyR1 is expressed, these defects are expected to decrease Ca2+-induced Ca2+ release, as well as orthograde Ca2+ release, in response to transverse tubular membrane depolarization.

Ryanodine sensitizes the cardiac Ca2+ release channel (ryanodine receptor isoform 2) to Ca2+ activation and dissociates as the channel is closed by Ca2+ depletion

In single-channel recordings, the rabbit cardiac Ca2+ release channel (RyR2) is converted to a fully open subconductance state with about 50% of full conductance by micromolar concentrations of ryanodine. At +30 mV, corresponding to a luminal to cytoplasmic cation current, the probability of opening (Po) of ryanodine-modified channels was only marginally altered at pCa 10 (pCa = −log10 Ca concentration). However, at −30 mV, the Po was highly sensitive to Ca2+ added to the cis (cytoplasmic) side and, at pCa 10, was reduced to less than 0.27. The EC50 value for channel opening was about pCa 8. No significant Ca2+ inactivation was observed for ryanodine-modified channels at either −30 mV or +30 mV. The opening of unmodified Ca2+ channels is Ca2+ sensitive, with an EC50 value of about pCa 6 (two orders of magnitude less sensitive than ryanodine-modified channels) and IC50 values of pCa 2.2 at −30 mV and 2.5 at +30 mV. Mg2+ decreased the Po of ryanodine-modified channels at low Ca2+ concentrations at both −30 and +30 mV. Caffeine, ATP, and ruthenium red were modulators of the Po of ryanodine-modified channels. In a [3H]ryanodine binding assay, [3H]ryanodine dissociation from the high-affinity binding site was found to be Ca2+ sensitive, with an IC50 of pCa 7.1. High concentrations of unlabeled ryanodine prevented [3H]ryanodine dissociation, but ruthenium red accelerated dissociation. These results suggest that ryanodine sensitizes Ca2+ activation of the Ca2+ release channel and desensitizes Ca2+ inactivation through an allosteric interaction. [3H]Ryanodine dissociates from the high-affinity site when the channel is closed by removal of Ca2+, implying that high-affinity ryanodine and Ca2+ binding sites are linked through either short- or long-range interactions, probably involving conformational changes.

Role of the Sequence Surrounding Predicted Transmembrane Helix M4 in Membrane Association and Function of the Ca2+ Release Channel of Skeletal Muscle Sarcoplasmic Reticulum (Ryanodine Receptor Isoform 1)

The role of the sequence surrounding M4 in ryanodine receptors (RyR) in membrane association and function was investigated. This sequence contains a basic, 19-amino acid M3/M4 loop, a hydrophobic 44–49 amino acid sequence designated M4 (or M4a/M4b), and a hydrophilic M4/M5 loop. Enhanced green fluorescent protein (EGFP) was inserted into RyR1 and truncated just after the basic sequence, just after M4, within the M4/M5 loop, just before M5 and just after M5. The A52 epitope was inserted into RyR2 and truncated just after M4a. Analysis of these constructs ruled out a M3/M4 transmembrane hairpin and narrowed the region of membrane association to M4a/M4b. EGFP inserted between M4a and M4b in full-length RyR2 was altered conformationally, losing fluorescence and gaining trypsin sensitivity. Although it was accessible to an antibody from the cytosolic side, tryptic fragments were membrane-bound. The expressed protein containing EGFP retained caffeine-induced Ca2+ release channel function. These results suggest that M4a/M4b either forms a transmembrane hairpin or associates in an unorthodox fashion with the cytosolic leaflet of the membrane, possibly involving the basic M3/M4 loop. The expression of a mutant RyR1, Δ4274–4535, deleted in the sequence surrounding both M3 and M4, restored robust, voltage-gated L-type Ca2+ currents and Ca2+ transients in dyspedic myotubes, demonstrating that this sequence is not required for either orthograde (DHPR activation of sarcoplasmic reticulum Ca2+ release) or retrograde (RyR1 increase in DHPR Ca2+ channel activity) signals of excitation-contraction coupling. Maximal amplitudes of L-currents and Ca2+ transients with Δ4274–4535 were larger than with wild-type RyR1, and voltage-gated sarcoplasmic reticulum Ca2+ release was more sensitive to activation by sarcolemmal voltage sensors. Thus, this region may act as a negative regulatory module that increases the energy barrier for Ca2+ release channel opening.