Biophysical Methods to Analyze Direct G-Protein Regulation of Neuronal Voltage-Gated Calcium Channels
Abstract
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.