Abstract
Key points The role of trimeric intracellular cation (TRIC) channels is not known, although evidence suggests they may regulate ryanodine receptors (RyR) via multiple mechanisms. We therefore investigated whether Tric‐a gene knockout (KO) alters the single‐channel function of skeletal RyR (RyR1).We find that RyR1 from Tric‐a KO mice are more sensitive to inhibition by divalent cations, although they respond normally to cytosolic Ca2+, ATP, caffeine and luminal Ca2+.In the presence of Mg2+, ATP cannot effectively activate RyR1 from Tric‐a KO mice.Additionally, RyR1 from Tric‐a KO mice are not activated by protein kinase A phosphorylation, demonstrating a defect in the ability of β‐adrenergic stimulation to regulate sarcoplasmic reticulum (SR) Ca2+‐release.The defective RyR1 gating that we describe probably contributes significantly to the impaired SR Ca2+‐release observed in skeletal muscle from Tric‐a KO mice, further highlighting the importance of TRIC‐A for normal physiological regulation of SR Ca2+‐release in skeletal muscle. The type A trimeric intracellular cation channel (TRIC‐A) is a major component of the nuclear and sarcoplasmic reticulum (SR) membranes of cardiac and skeletal muscle, and is localized closely with ryanodine receptor (RyR) channels in the SR terminal cisternae. The skeletal muscle of Tric‐a knockout (KO) mice is characterized by Ca2+ overloaded and swollen SR and by changes in the properties of SR Ca2+ release. We therefore investigated whether RyR1 gating behaviour is modified in the SR from Tric‐a KO mice by incorporating native RyR1 into planar phospholipid bilayers under voltage‐clamp conditions. We find that RyR1 channels from Tric‐a KO mice respond normally to cytosolic Ca2+, ATP, adenine, caffeine and to luminal Ca2+. However, the channels are more sensitive to the inactivating effects of divalent cations, thus, in the presence of Mg2+, ATP is inadequate as an activator. Additionally, channels are not characteristically activated by protein kinase A even though the phosphorylation levels of Ser2844 are similar to controls. The results of the present study suggest that TRIC‐A functions as an excitatory modulator of RyR1 channels within the SR terminal cisternae. Importantly, this regulatory action of TRIC‐A appears to be independent of (although additive to) any indirect consequences to RyR1 activity that arise as a result of K+ fluxes across the SR via TRIC‐A.
Highlights
There are two subtypes of trimeric intracellular cation channel (TRIC), termed TRIC-A and TRIC-B, and both are found on the endoplasmic/sarcoplasmic reticulum (ER/SR) and the nuclear membranes of most cell types (Yazawa et al 2007)
Evidence suggests that luminal Ca2+ is higher than normal in skeletal muscle from Tric-a KO tissue (Zhao et al 2010) yet Ca2+-release is impaired, so we initially performed experiments using K+ as the permeant ion so that we could investigate whether the luminal Ca2+ sensitivity of the single ryanodine receptor (RyR) channels was modified
Adding 1 mM ATP to the cytosolic channel side led to similar increases in Po in channels from WT and Tric-a KO tissue, indicating that the response of RyR1 to ATP was not altered in Tric-a KO tissue
Summary
There are two subtypes of trimeric intracellular cation channel (TRIC), termed TRIC-A and TRIC-B, and both are found on the endoplasmic/sarcoplasmic reticulum (ER/SR) and the nuclear membranes of most cell types (Yazawa et al 2007). It was subsequently suggested that the RyR channels may be able to pass most or all of their own counter-current (Gillespie & Fill 2008; Gillespie et al 2009) If this is so, the necessity for the SR K+ channel to pass counter-ion flux is not as critical as first assumed, equilibration of K+ across the SR will still be important. Because the Tric-a KO mouse survives until adulthood, we isolated SR membranes from the mature skeletal muscle and incorporated them into bilayers to investigate whether the gating or conductance of the RyR channels are modified by the absence of TRIC-A. The results obtained show that the RyR from Tric-a KO mice exhibit modified gating properties that prevent the channels from responding normally to activators such as ATP or to phosphorylation by protein kinase A (PKA)
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