  Fluctuation analysis is a method which allows measurement of the single
channel current of ion channels even when it is too small to be resolved
directly with the patch clamp technique. This is the case for voltage-gated
Ca2+ channels (VGCCs). They are present in all mammalian central neurons,
controlling presynaptic release of transmitter, postsynaptic signaling and
synaptic integration. The amplitudes of their single channel currents in a
physiological concentration of extracellular Ca2+, however, are small and not
well determined. But measurement of this quantity is essential for estimating
numbers of functional VGCCs in the membrane and the size of channel-associated
Ca2+ signaling domains, and for understanding the stochastic nature of Ca2+
signaling. Here, we recorded the VGCC current in nucleated patches from layer 5
pyramidal neurons in rat neocortex, in physiological external Ca2+ (1-2 mM).
The ensemble-averaging of current responses required for conventional
fluctuation analysis proved impractical because of the rapid rundown of VGCC
currents. We therefore developed a more robust method, using mean current
fitting of individual current responses and band-pass filtering. Furthermore,
voltage ramp stimulation proved useful. We validated the accuracy of the method
by analyzing simulated data. At an external Ca2+ concentration of 1 mM, and a
membrane potential of -20 mV, we found that the average single channel current
amplitude was about 0.04 pA, increasing to 0.065 pA at 2 mM external Ca2+, 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 are relevant to understanding the stochastic
properties of dendritic Ca2+ spikes in neocortical layer 5 pyramidal neurons.
With the reported method, single channel current amplitude of native VGCCs can
be resolved accurately despite conditions of unstable rundown.
