Bilayer measurement of endoplasmic reticulum Ca2+ channels
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390Cold Spring Harbor Protocols (Impact Factor: 4.63). 11/2013; 2013(11). DOI: 10.1101/pdb.top066225
Reconstitution of ion channels into planar lipid bilayers (also called black lipid membranes or BLM) is the most widely used method to conduct physiological studies of intracellular ion channels, including endoplasmic reticulum (ER) calcium (Ca(2+)) channels. The two main types of Ca(2+) release channels in the ER membrane are ryanodine receptors (RyanRs) and inositol(1,4,5)-trisphosphate receptors (InsP3Rs). Use of the BLM reconstitution technique enabled the initial description of the functional properties of InsP3R and RyanR at the single-channel level more than 20 years ago. Since then, BLM reconstitution methods have been used to study physiological modulation and to perform structure-function analysis of these channels, and to study pathological changes in the function of InsP3R and RyanR in various disease states. The BLM technique has also been useful for studies of other intracellular Ca(2+) channels, such as ER Ca(2+) leak presenilin channels and NAADP-gated lysosomal Ca(2+) channels encoded by TPC2. In this article, basic protocols used for BLM studies of ER Ca(2+) channels are introduced.
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ABSTRACT: In this study, we provide the first description of the biophysical and pharmacological properties of ryanodine receptor type 1 (RyR1) expressed in a native membrane using the on-nucleus configuration of the patch clamp technique. A stable cell line expressing rabbit RyR1 was established (HEK-RyR1) using the FLP-in 293 cell system. In contrast to untransfected cells, RyR1 expression was readily demonstrated by immunoblotting and immunocytochemistry in HEK-RyR1 cells. In addition, the RyR1 agonists 4-CMC and caffeine activated Ca(2+) release that was inhibited by high concentrations of ryanodine. On nucleus patch clamp was performed in nuclei prepared from HEK-RyR1 cells. Raising the [Ca(2+)] in the patch pipette resulted in the appearance of a large conductance cation channel with well resolved kinetics and the absence of prominent subconductance states. Current versus voltage relationships were ohmic and revealed a chord conductance of ∼750pS or 450pS in symmetrical 250mM KCl or CsCl, respectively. The channel activity was markedly enhanced by caffeine and exposure to ryanodine resulted in the appearance of a subconductance state with a conductance ∼40% of the full channel opening with a Po near unity. In total, these properties are entirely consistent with RyR1 channel activity. Exposure of RyR1 channels to cyclic ADP ribose (cADPr), nicotinic acid adenine dinucleotide phosphate (NAADP) or dantrolene did not alter the single channel activity stimulated by Ca(2+), and thus, it is unlikely these molecules directly modulate RyR1 channel activity. In summary, we describe an experimental platform to monitor the single channel properties of RyR channels. We envision that this system will be influential in characterizing disease-associated RyR mutations and the molecular determinants of RyR channel modulation.Cell Calcium 08/2014; 56(2). DOI:10.1016/j.ceca.2014.05.004 · 3.51 Impact Factor
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ABSTRACT: Atrial myocytes in a number of species lack transverse tubules. As a consequence the intracellular calcium signals occurring during each heart beat exhibit complex spatio-temporal dynamics. These calcium patterns arise from saltatory calcium waves that propagate via successive rounds of diffusion and calcium-induced calcium release. The many parameters that impinge on calcium-induced calcium release and calcium signal propagation make it difficult to know a priori whether calcium waves will successfully travel, or be extinguished. In this study, we describe in detail a mathematical model of calcium signalling that allows the effect of such parameters to be independently assessed. A key aspect of the model is to follow the triggering and evolution of calcium signals within a realistic three-dimensional cellular volume of an atrial myocyte, but with a low computational overhead. This is achieved by solving the linear transport equation for calcium analytically between calcium release events and by expressing the onset of calcium liberation as a threshold process. By being able to follow the evolution of a calcium wave in three dimensions, the model makes non-intuitive predictions about calcium signal propagation. For example, our modelling illustrates that the boundary of a cell produces a wave-guiding effect that enables calcium ions to propagate further and for longer, and can subtly alter the pattern of calcium wave movement. The high spatial resolution of the modelling framework allows the study of any arrangement of calcium release sites. We demonstrate that even small variations in randomly positioned release sites cause highly heterogeneous cellular responses. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. Copyright © 2015. Published by Elsevier B.V.Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 03/2015; 1853(9). DOI:10.1016/j.bbamcr.2015.02.019 · 5.02 Impact Factor
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