Biophysical Methods to Analyze Direct G-Protein Regulation of Neuronal Voltage-Gated Calcium Channels
Neuronal voltage-gated calcium channels play an essential role for calcium entry into presynaptic endings responsible for the release of neurotransmitters. In turn, and in order to fine tune synaptic activity, numerous neurotransmitters exert a potent negative feedback over the calcium signal provided by G-protein-coupled receptors that can be recognized by characteristic biophysical modifications of the calcium current. There are two main biophysical approaches to analyze direct G-protein regulation of voltage-gated calcium channels: the so-called double-pulse method, which is indirectly assessed by the gain of current produced by a depolarizing prepulse potential, and the “subtraction” method that allows the analysis of G-protein regulation from the ionic currents induced by regular depolarizing pulses. The later method separates the ionic currents due to nonregulated channels from the ion currents that result from a progressive departure of G-proteins from regulated channels, thereby providing valuable information on the OFF kinetics of G-protein regulation. In this chapter, we introduce these “double pulses” and “subtraction” procedures for use primarily with single cells and also discuss the limitations inherent to these two approaches.
KeywordsCalciumchannel Cav2 channel G-protein-coupled receptor G-proteins Gβγ-dimer Prepulse facilitation Biophysical method
Research in NW’s laboratory is supported by the Czech Science Foundation (grant 15-13556S), the Ministry of Education Youth and Sports (grant 7AMB15FR015), and the Institute of Organic Chemistry and Biochemistry.
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