title: Imaging fast calcium currents beyond the limitations of electrode techniques.
abstract: The current understanding of Ca(2+) channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca(2+) currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca(2+) channels during physiological changes of membrane potential in complex cells, since the voltage-clamp configuration constrains the membrane potential to a given value. Here, we report for the first time to our knowledge that Ca(2+) currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca(2+) imaging with low-affinity indicators. The optical measurement of the Ca(2+) current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca(2+) channels along the apical dendrite of the CA1 hippocampal pyramidal neuron during the back-propagation of an action potential. To validate the method, we analyzed the voltage dependence of high- and low-voltage-gated Ca(2+) channels. In particular, we measured the Ca(2+) current component mediated by T-type channels, and we investigated the mechanisms of recovery from inactivation of these channels. This method is expected to become a reference approach to investigate Ca(2+) channels in their native physiological environment.
authors.affiliation: Institut national de la santé et de la recherche médicale, Grenoble Institute of Neuroscience, Grenoble, France; Université Joseph Fourier, Laboratoire Interdisciplinare de Physique (Centre National de la Recherche Scientifique UMR 5588), France; Laboratories of Excellence, Ion Channel Science and Therapeutics, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Jaafari
authors.affiliation: Institut national de la santé et de la recherche médicale, Grenoble Institute of Neuroscience, Grenoble, France; Laboratories of Excellence, Ion Channel Science and Therapeutics, France.
authors.forename: Michel
authors.initials: M
authors.lastname: De Waard
authors.affiliation: Institut national de la santé et de la recherche médicale, Grenoble Institute of Neuroscience, Grenoble, France; Université Joseph Fourier, Laboratoire Interdisciplinare de Physique (Centre National de la Recherche Scientifique UMR 5588), France; Laboratories of Excellence, Ion Channel Science and Therapeutics, France. Electronic address: marco.canepari@ujf-grenoble.fr.
authors.forename: Marco
authors.initials: M
authors.lastname: Canepari
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020747:Calcium Channels, T-Type / Q000378:metabolism; D004566:Electrodes; D055724:Electrophysiological Phenomena; D006624:Hippocampus / Q000166:cytology / Q000502:physiology; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D061848:Optical Imaging / Q000295:instrumentation / Q000379:methods*
chemical_list: D020747:Calcium Channels, T-Type; D002118:Calcium