Abstract
The sarcoplasmic reticulum (SR) of skeletal muscle contains K(+), Cl(-), and H(+) channels may facilitate charge neutralization during Ca(2+) release. Our recent studies have identified trimeric intracellular cation (TRIC) channels on SR as an essential counter-ion permeability pathway associated with rapid Ca(2+) release from intracellular stores. Skeletal muscle contains TRIC-A and TRIC-B isoforms as predominant and minor components, respectively. Here we test the physiological function of TRIC-A in skeletal muscle. Biochemical assay revealed abundant expression of TRIC-A relative to the skeletal muscle ryanodine receptor with a molar ratio of TRIC-A/ryanodine receptor ∼5:1. Electron microscopy with the tric-a(-/-) skeletal muscle showed Ca(2+) overload inside the SR with frequent formation of Ca(2+) deposits compared with the wild type muscle. This elevated SR Ca(2+) pool in the tric-a(-/-) muscle could be released by caffeine, whereas the elemental Ca(2+) release events, e.g. osmotic stress-induced Ca(2+) spark activities, were significantly reduced likely reflecting compromised counter-ion movement across the SR. Ex vivo physiological test identified the appearance of "alternan" behavior with isolated tric-a(-/-) skeletal muscle, i.e. transient and drastic increase in contractile force appeared within the decreasing force profile during repetitive fatigue stimulation. Inhibition of SR/endoplasmic reticulum Ca(2+ ATPase) function could lead to aggravation of the stress-induced alternans in the tric-a(-/-) muscle. Our data suggests that absence of TRIC-A may lead to Ca(2+) overload in SR, which in combination with the reduced counter-ion movement may lead to instability of Ca(2+) movement across the SR membrane. The observed alternan behavior with the tric-a(-/-) muscle may reflect a skeletal muscle version of store overload-induced Ca(2+) release that has been reported in the cardiac muscle under stress conditions.
Highlights
In the course of searching for membrane proteins participating in E-C coupling of striated muscle, we identified two isoforms of trimeric intracellular cation (TRIC) channel that were expressed in the sarcoplasmic reticulum (SR) membrane [6]
We show that TRIC-A is expressed in the SR membrane of skeletal muscle at a high molecular ratio to ryanodine receptor (RyR)
In the absence of TRIC-A-mediated Kϩ permeability, chronic SR Ca2ϩ overload creates instability for Ca2ϩ movement across the SR membrane, which leads to the appearance of “mechanical alternans” in the mutant muscle under stress conditions, a phenotype that can be aggravated by the sarcoplasmic/endoplasmic reticulum Ca2ϩ-ATPase (SERCA) inhibitor
Summary
Animals—Wild type rabbits (Japanese white, 3 kg) were used for microsome isolation from the hindlimb. 3– 4-Month-old wild type or tric-aϪ/Ϫ male mice of the same genetic background (F1:J1 embryonic stem cells ϫ C57BL/6J) were used for all other experiments. Western Blotting—Total cellular protein was extracted from hindlimb, extensor digitorum longus (EDL), and soleus of wild type and tric-aϪ/Ϫ mice in a buffer containing 0.3 M sucrose, 20 mM Tris-HCl (pH 7.4) and protease inhibitors. Measurement of caffeine-induced Ca2ϩ transient was performed on microscopic system of a PTI spectrofluorometer (Photon Technology International, Princeton, NJ), and changes in [Ca2ϩ]i were determined as changes in the ratio of Fura-2 fluorescence at excitation wavelengths of 350 and 380 nm, following exposure to 20 mM caffeine in Tyrode solution without Ca2ϩ. Contractility of Isolated Muscles—As previously described [12], intact EDL and soleus muscle bundles were dissected from wild type and tric-aϪ/Ϫ mice and mounted in Radnotti glass chambers with built-in platinum stimulating electrodes (Monrovia, CA), bathing in 2.5 mM Ca2ϩ Tyrode solution with 10 mM glucose, saturated with 100% O2. A value of p Ͻ 0.05 was used as criteria for statistical significance and other p values were as specified in the figure legends
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