PHYSIOLOGICAL RESEARCH ISSN 0862-8408  2003 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax +420 241 062 164 E-mail: physres@biomed.cas.cz http://www.biomed.cas.cz/physiolres Physiol. Res. 52: 571-578, 2003 Kinetics of Cardiac RyR Channel Gating Studied at High Temporal Resolution M. DURA, I. ZAHRADN˝K, A. ZAHRADN˝KOV` Institute of Molecular Physiology and Genetics, Slovak Academy of Sciences, Bratislava, Slovak Republic Received August 30, 2002 Accepted November 1, 2002 Summary Measurements of ryanodine receptor (RyR) activity during dynamic changes of calcium concentration have suggested that RyR has at least four calcium binding sites, and that activation transpires as an increase in the activity within the high open probability H-mode. Binding of several Ca 2+ ions within the H-mode should manifest itself in the steady-state RyR activity by the presence of multiple closed times. However, previously only two closed times were detected in the H-mode of RyR activity. Here we recorded steady-state activity of single cardiac RyRs with high temporal resolution and compared it to data simulated under the same conditions using our previously published model of RyR gating. At a 10 kHz resolution, the closed time histograms of both experimental and simulated data had three exponential components. The closed times of simulated data were not significantly different from those obtained experimentally. After filtering at 2 kHz, only two exponential closed time components with time constants not significantly different from those previously published could be detected in both experimental and simulated records. The conformity of the steady-state experimental data to the model derived from the dynamic data provides further support for the idea that RyRs need binding of multiple Ca 2+ ions to open. Key words Ryanodine receptor • Gating • Kinetics • Calcium release • Cardiac muscle Introduction Calcium ions play a central role in cardiac muscle contraction. Increasing intracellular concentration of Ca 2+ causes a release of further Ca 2+ ions from sarcoplasmic reticulum (SR) through the ryanodine receptor calcium release channels (RyRs). This process, called calcium-induced calcium release, is believed to be controlled mainly by the entry of Ca 2+ through the voltage sensitive Ca 2+ channels (DHPR receptors) on the sarcolemma (Bers 2002, Noble 2002). The homotetrameric RyR protein is localized in discrete junctional domains of the SR near specialized domains of the surface membrane and T tubules that contain DHPR receptors (Franzini-Armstrong et al. 1999). Considering the intracellular RyR localization, the most direct and compelling data about SR Ca 2+ release channel regulation come from measurements of single RyR channels reconstituted in planar lipid bilayers (BLMs). Ryanodine receptor channels exhibit several patterns of activity under steady-state conditions (Ashley and Williams 1990, Percival et al. 1994, ZahradnkovÆ