Single voltage-gated calcium channel current in rat neocortical layer 5 pyramidal neurons in physiological calcium: voltage ramp fluctuation analysis

Voltage-gated calcium channels (VGCCs) are present in all mammalian central neurons, controlling presynaptic release of transmitter, postsynaptic signalling and synaptic integration. The amplitudes of their single open-channel currents in a physiological concentration of extracellular calcium, however, are not well determined, being too small to resolve directly in patch-clamp recordings. However, measurement of this quantity is essential for estimating numbers of functional VGCCs in the membrane and the size of channel-associated calcium signalling domains, and for understanding the stochastic nature of calcium signalling. Here, we recorded the voltage-gated calcium channel current in nucleated patches from layer 5 pyramidal neurons in rat neocortex, in physiological external calcium (1-2 mM). The pharmacological profile of the current was consistent with R-type or Q-type channels, and a small L-type component. In order to reliably separate the calcium current fluctuations from other background currents for fluctuation analysis, despite their small amplitude and rapid rundown, we developed a method using band-pass filtered current responses to ramp membrane potential stimulation. We validated the accuracy of the method by analysing simulated data. At external calcium concentration of 1 mM, and a membrane potential of -20 to -15 mV, we found that single channel current amplitude was about 0.04 pA, increasing to 0.065 pA at 2 mM external calcium, and 0.12 pA at 5 mM. The relaxation time constant of the fluctuations was in the range 0.2-0.8 ms. The results demonstrate that single channel current amplitude of native voltage-gated calcium channels can be resolved accurately and straightforwardly.