Imaging Fast Calcium Currents beyond the Limitations of Electrode Techniques
2014
The current understanding of Ca2+ channel function is derived from the use of the patch-clamp technique. In particular, the measurement of fast cellular Ca2+ currents is routinely achieved using whole-cell voltage-clamp recordings. However, this experimental approach is not applicable to the study of local native Ca2+ 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 Ca2+ currents from individual cells can be quantitatively measured beyond the limitations of the voltage-clamp approach using fast Ca2+ imaging with low-affinity indicators. The optical measurement of the Ca2+ current was correlated with the membrane potential, simultaneously measured with a voltage-sensitive dye to investigate the activation of Ca2+ 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 Ca2+ channels. In particular, we measured the Ca2+ 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 Ca2+ channels in their native physiological environment.
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