Suzanne M. Curtis

The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling.

The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC50 between 0.5 and 1.0 μm and activated the skeletal RyR1 with an AC50 between 1 and 10 μm when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca2+ fluxes observed during sperm capacitation.

Activation and Inhibition of Skeletal RyR Channels by a Part of the Skeletal DHPR II-III Loop: Effects of DHPR Ser687 and FKBP12

Peptides, corresponding to sequences in the N-terminal region of the skeletal muscle dihydropyridine receptor (DHPR) II-III loop, have been tested on sarcoplasmic reticulum (SR) Ca2+ release and ryanodine receptor (RyR) activity. The peptides were: A1, Thr671-Leu690; A2, Thr671-Leu690 with Ser687 Ala substitution; NB, Gly689-Lys708 and A1S, scrambled A1 sequence. The relative rates of peptide-induced Ca2+ release from normal (FKBP12+) SR were A2 > A1 > A1S > NB. Removal of FKBP12 reduced the rate of A1-induced Ca2+ release by approximately 30%. A1 and A2 (but not NB or A1S), in the cytoplasmic (cis) solution, either activated or inhibited single FKBP12+ RyRs. Maximum activation was seen at -40 mV, with 10 microM A1 or 50 nM A2. The greatest A1-induced increase in mean current (sixfold) was seen with 100 nM cis Ca2+. Inhibition by A1 was greatest at +40 mV (or when permeant ions flowed from cytoplasm to SR lumen) with 100 microM cis Ca2+, where channel activity was almost fully inhibited. A1 did not activate FKBP12-stripped RyRs, although peptide-induced inhibition remained. The results show that peptide A activation of RyRs does not require DHPR Ser687, but required FKBP12 binding to RyRs. Peptide A must interact with different sites to activate or inhibit RyRs, because current direction-, voltage-, cis [Ca2+]-, and FKBP12-dependence of activation and inhibition differ.

Characteristics of irreversible ATP activation suggest that native skeletal ryanodine receptors can be phosphorylated via an endogenous CaMKII.

Phosphorylation of skeletal muscle ryanodine receptor (RyR) calcium release channels by endogenous kinases incorporated into lipid bilayers with native sarcoplasmic reticulum vesicles was investigated during exposure to 2 mM cytoplasmic ATP. Activation of RyRs after 1-min exposure to ATP was reversible upon ATP washout. In contrast, activation after 5 to 8 min was largely irreversible: the small fall in activity with washout was significantly less than that after brief ATP exposure. The irreversible activation was reduced by acid phosphatase and was not seen after exposure to nonhydrolyzable ATP analogs. The data suggested that the channel complex was phosphorylated after addition of ATP and that phosphorylation reduced the RyRs sensitivity to ATP, adenosine, and Ca(2+). The endogenous kinase was likely to be a calcium calmodulin kinase II (CaMKII) because the CaMKII inhibitor KN-93 and an inhibitory peptide for CaMKII prevented the phosphorylation-induced irreversible activation. In contrast, phosphorylation effects remained unchanged with inhibitory peptides for protein kinase C and A. The presence of CaMKIIbeta in the SR vesicles was confirmed by immunoblotting. The results suggest that CaMKII is anchored to skeletal muscle RyRs and that phosphorylation by this kinase alters the enhancement of channel activity by ATP and Ca(2+).

Porin-type1 proteins in sarcoplasmic reticulum and plasmalemma of striated muscle fibres

SummaryThe location of porin-type1 proteins in mammalian striated muscle has been assessed using immunogold electron microscopy with an anti-porin 31HL monoclonal antibody as the primary antibody. Gold particles were found on the mitochondrial outer membrane, the sarcoplasmic reticulum and plasmalemma in longitudinal sections of rat and rabbit skeletal muscle and rabbit and sheep cardiac muscle. The relative densities of gold particles in the mitochondrial outer membrane, sarcoplasmic reticulum and plasmalemma were 7:3:1 in white sternomastoid muscle, for example. Skeletal and cardiac sarcoplasmic reticulum vesicles, which had been fractionated by discontinuous sucrose density centrifugation, were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. The anti-porin 31HL monoclonal antibody detected a band of relative molecular mass (Mr) 31 000 in all muscle sarcoplasmic reticulum vesicle fractions and also in liver mitochondria. The intensity of immunostaining of the sarcoplasmic reticulum fractions was 2.5–10% that of mitochondrial outer membranes per μg of membrane protein blotted. Contamination of the sacroplasmic reticulum fractions by mitochondrial outer membrane was <0.75% as determined from the specific activity of monoamine oxidase. Thus, only a small part of the porin detected in sarcoplasmic reticulum vesicles can be attributed to mitochondrial contamination. These results show that porin-type1 immunoreactivity is not restricted to mitochondria but found in the sarcoplasmic reticulum and plasmalemma of both mammalian skeletal and cardiac muscle.

Structural determinants for activation or inhibition of ryanodine receptors by basic residues in the dihydropyridine receptor II-III loop.

The structures of peptide A, and six other 7-20 amino acid peptides corresponding to sequences in the A region (Thr671- Leu690) of the skeletal muscle dihydropyridine receptor II-III loop have been examined, and are correlated with the ability of the peptides to activate or inhibit skeletal ryanodine receptor calcium release channels. The peptides adopted either random coil or nascent helix-like structures, which depended upon the polarity of the terminal residues as well as the presence and ionisation state of two glutamate residues. Enhanced activation of Ca2+ release from sarcoplasmic reticulum, and activation of current flow through single ryanodine receptor channels (at -40 mV), was seen with peptides containing the basic residues 681Arg Lys Arg Arg Lys685, and was strongest when the residues were a part of an alpha-helix. Inhibition of channels (at +40 mV) was also seen with peptides containing the five positively charged residues, but was not enhanced in helical peptides. These results confirm the hypothesis that activation of ryanodine receptor channels by the II-III loop peptides requires both the basic residues and their participation in helical structure, and show for the first time that inhibition requires the basic residues, but is not structure-dependent. These findings imply that activation and inhibition result from peptide binding to separate sites on the ryanodine receptor.

The three-dimensional structural surface of two beta-sheet scorpion toxins mimics that of an alpha-helical dihydropyridine receptor segment.

An alpha-helical II-III loop segment of the dihydropyridine receptor activates the ryanodine receptor calcium-release channel. We describe a novel manipulation in which this agonists activity is increased by modifying its surface structure to resemble that of a toxin molecule. In a unique system, native beta-sheet scorpion toxins have been reported to activate skeletal muscle ryanodine receptor calcium channels with high affinity by binding to the same site as the lower-affinity alpha-helical dihydropyridine receptor segment. We increased the alignment of basic residues in the alpha-helical peptide to mimic the spatial orientation of active residues in the scorpion toxin, with a consequent 2-20-fold increase in the activity of the alpha-helical peptide. We hypothesized that, like the native peptide, the modified peptide and the scorpion toxin may bind to a common site. This was supported by (i) similar changes in ryanodine receptor channel gating induced by the native or modified alpha-helical peptide and the beta-sheet toxin, a 10-100-fold reduction in channel closed time, with a < or = 2-fold increase in open dwell time and (ii) a failure of the toxin to further activate channels activated by the peptides. These results suggest that diverse structural scaffolds can present similar conformational surface properties to target common receptor sites.

Effects of ivermectin and midecamycin on ryanodine receptors and the Ca2+‐ATPase in sarcoplasmic reticulum of rabbit and rat skeletal muscle

1 Ryanodine receptor (RyR) Ca2+ channels in the sarcoplasmic reticulum (SR) of skeletal muscle are regulated by the 12 kDa FK506‐ (or rapamycin‐) binding protein (FKBP12). Rapamycin can also activate RyR channels with FKBP12 removed, suggesting that compounds with macrocyclic lactone ring structures can directly activate RyRs. Here we tested this hypothesis using two other macrocyclic lactone compounds, ivermectin and midecamycin. 2 Rabbit skeletal RyRs were examined in lipid bilayers. Ivermectin (cis, 0.66–40 μm) activated six of eight native, four of four control‐incubated and eleven of eleven FKBP12‐‘stripped’ RyR channels. Midecamycin (cis, 10–30 μm) activated three of four single native channels, six of eight control‐incubated channels and six of seven FKBP12‐stripped channels. Activity declined when either drug was washed out. 3 Neither ivermectin nor midecamycin removed FKBP12 from RyRs. Western blots of terminal cisternae (TC), incubated for 15 min at 37 °C with 40 μm ivermectin or midecamycin, showed normal amounts of FKBP12. In contrast, no FKBP12 was detected after incubation with 40 μm rapamycin. 4 Ivermectin reduced Ca2+ uptake by the SR Ca2+‐Mg2+‐ATPase. Ca2+ uptake by TC fell to ∼40% in the presence of ivermectin (10 μm), both with and without 10 μm Ruthenium Red. Ca2+ uptake by longitudinal SR also fell to ∼40% with 10 μm ivermectin. Midecamycin (10 μm) reduced Ca2+ uptake by TC vesicles to ∼76% without Ruthenium Red and to ∼90% with Ruthenium Red. 5 The rate of rise of extravesicular [Ca2+] increased ∼2‐fold when 10 μm ivermectin was added to TC vesicles that had been partially loaded with Ca2+ and then Ca2+ uptake blocked by 200 nm thapsigargin. Ivermectin also potentiated caffeine‐induced Ca2+ release to ∼140% of control. These increases in Ca2+ release were not seen with midecamycin. 6 Ivermectin, but not midecamycin, reversibly reduced Ca2+ loading in four of six skinned rat extensor digitorum longus (EDL) fibres to ∼90%, and reversibly increased submaximal caffeine‐induced contraction in five of eight fibres by ∼110% of control. Neither ivermectin nor midecamycin altered twitch or tetanic tension in intact EDL muscle fibres within 20 min of drug addition. 7 The results confirm the hypothesis that compounds with a macrocyclic lactone ring structure can directly activate RyRs. Unexpectedly, ivermectin also reduced Ca2+ uptake into the SR. These effects of ivermectin on SR Ca2+ handling may explain some effects of the macrolide drugs on mammals.

Arg(615)Cys substitution in pig skeletal ryanodine receptors increases activation of single channels by a segment of the skeletal DHPR II-III loop.

The effect of peptides, corresponding to sequences in the skeletal muscle dihydropyridine receptor II-III loop, on Ca(2+) release from sarcoplasmic reticulum (SR) and on ryanodine receptor (RyR) calcium release channels have been compared in preparations from normal and malignant hyperthermia (MH)-susceptible pigs. Peptide A (Thr(671)-Leu(690); 36 microM) enhanced the rate of Ca(2+) release from normal SR (SR(N)) and from SR of MH-susceptible muscle (SR(MH)) by 10 +/- 3.2 nmole/mg/min and 76 +/- 9.7 nmole/mg/min, respectively. Ca (2+) release from SR(N) or SR(MH) was not increased by control peptide NB (Gly(689)-Lys(708)). AS (scrambled A sequence; 36 microM) did not alter Ca (2+) release from SR(N), but increased release from SR(MH) by 29 +/- 4.9 nmoles/mg/min. RyR channels from MH-susceptible muscle (RyR(MH)) were up to about fourfold more strongly activated by peptide A (> or =1 nM) than normal RyR channels (RyR(N)) at -40 mV. Neither NB or AS activated RyR(N). RyR(MH) showed an approximately 1.8-fold increase in mean current with 30 microM AS. Inhibition at +40 mV was stronger in RyR(MH) and seen with peptide A (> or = 0.6 microM) and AS (> or = 0.6 microM), but not NB. These results show that the Arg(615)Cys substitution in RyR(MH) has multiple effects on RyRs. We speculate that enhanced DHPR activation of RyRs may contribute to increased Ca(2+) release from SR in MH-susceptible muscle.