Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, C101

Oral Communications

Single-channel characterisation of the trimeric intracellular (TRIC) membrane proteins reveals the identity of the sarcoplasmic reticulum K+-channel

S. J. Pitt1, K. Park2, M. Nishi3, T. Urashima3, S. Aoki3, D. Yamazaki3, J. Ma2, H. Takeshima3, R. Sitsapesan1

1. Department of Physiology and Pharmacology, NSQI and Bristol Heart Institute, University of Bristol, Bristol, United Kingdom. 2. Department of Physiology and Biophysics, Robert Wood Medical School, Piscataway, New Jersey, United States. 3. Department of Biological Chemistry, Kyoto University, Kyoto, Japan.


Two related trimeric intracellular membrane proteins (named TRIC-A and TRIC-B) are located in the sarco/endoplasmic (SR) reticulum and nuclear membranes of many cells types including muscle cells (1). Knockout mice lacking both TRIC-A and TRIC-B die of embryonic heart failure due to impairment of SR Ca2+-release. TRIC-A was shown to be a K+-permeable ion-channel. Here we investigated if TRIC-B could also function as an ion-channel, and if the TRIC channels share properties with the previously identified SR K+-channel (2). We performed a detailed comparison of the single-channel properties of purified skeletal TRIC-A and TRIC-B, with those K+ currents observed with native SR vesicles from sheep cardiac or rabbit skeletal muscle. Purified TRIC-A and TRIC-B, prepared using previously described techniques (1) or SR vesicles were incorporated into phosphatidylethanolamine lipid bilayers under voltage clamp conditions as previously described (3). Student’s t-test was used to assess the difference between mean values. We found that TRIC-B behaves as a monovalent cation-selective channel. In symmetrical solutions of 210 mM KCl (pH 7.2), TRIC-B displays a single-channel conductance of 138±8pS (n=6) with sub-conductance levels of 59±6 pS and 35±4 pS (≥4). TRIC-A exhibits a fully open state conductance of 192±21 pS and a sub-conductance level of 129±12 pS (n=6). Under identical recording conditions, we found that the K+-current fluctuations observed after incorporating cardiac or skeletal SR into bilayers, result from the gating of both TRIC-A and TRIC-B channels, thus solving the identity of ‘the SR K+-channel’ first observed more than 30 years ago (2). We demonstrate that TRIC-A is strongly regulated by trans-membrane voltage having a significantly higher Po at positive potentials (0.71±0.16 at +30 mV) compared to negative potentials (0.05±0.04 at -30 mV) (n=3; P<0.05). TRIC-B is inhibited by micromolar trans luminal Ca2+ (Po decreases from 0.005±0.002 to 0.001±0.0005, n=6; P<0.001). This inhibition is overcome by the addition of micromolar cis cytosolic Ca2+ causing a significant increase in Po (0.033±0.021, n=6; P<0.01). Neither cytosolic or luminal Ca2+ at micromolar concentrations had any significant effect on TRIC-A. In summary, our characterisation of purified TRIC-A and TRIC-B channels has exposed the molecular identity of the SR K+-channel and identified new mechanisms of regulating SR K+-fluxes. Further work is underway to expand our understanding of how TRIC channels are regulated, their essential physiological role and how this integrates into the overall picture of SR/ER Ca2+-release and re-uptake processes.

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