TRIC-B Channels Exhibit Labile Gating Properties; Evidence from TRIC-A Knockout Mice

Abstract

Trimeric intracellular cation channels (TRIC-A and TRIC-B) are located in the sarcoplasmic/endoplasmic reticulum (SR/ER) of most cells. Identifying the distinct biophysical properties of TRIC-A and TRIC-B is difficult because both channels are present in most tissues, yet this is crucial for delineating their individual physiological roles. Skeletal muscle SR vesicles (LSR) from TRIC-A knockout mice were incorporated into artificial membranes under voltage-clamp conditions as previously described [Pitt et al., 2010, Biophys. J. 99, 417-426] and single-channel recordings of native TRIC-B were obtained in symmetrical solutions of 210 mM K-PIPES, pH 7.2. The maximum single-channel conductance of TRIC-B was 197 ± 2 pS (n=32; SEM). TRIC-B channels always exhibited sub-conductance gating states and while these were of a variable nature, the predominant sub-conductance levels were found at 156 ± 3 pS (n=17; SEM), 125 ± 2 pS (n=19; SEM), 96 ± 2 pS (n=19; SEM) and 62 ± 2 pS (n=27; SEM). TRIC-B channel gating was voltage-dependent and channels were inhibited at negative holding potentials. For example, the probability of dwelling in the full open channel level was 0.0478±0.0194 at +30 mV but only 0.0010±0.0008 at −30 mV (n=6; SEM; ∗p<0.05). Application of 300 mM KCl to the cytosolic channel side produced a parallel shift in the current-voltage relationship and a shift in the reversal potential to approximately −20 mV indicating that TRIC-B is not permeable to anions. Our study demonstrates that the single-channel properties of TRIC-B channels are exceptionally labile. This intrinsic variability may be important for enabling flexible physiological regulation of monovalent cation fluxes across the SR membrane.Supported by the British Heart Foundation and the Japan Society for the Promotion of Science.