Large Ca²⁺-dependent facilitation of Ca_V2.1 channels revealed by Ca²⁺ photo-uncaging

Key points: Ca_V2.1 channels constitute a dominant Ca²⁺ entry pathway into brain neurons, triggering downstream Ca²⁺-dependent processes such as neurotransmitter release. Ca_V2.1 is itself modulated by Ca²⁺, resulting in activity-dependent enhancement of channel opening termed Ca²⁺-dependent facilitation (CDF). Real-time Ca²⁺ imaging and Ca²⁺ uncaging here reveal that CDF turns out to be strikingly faster, more Ca²⁺ sensitive, and larger than anticipated on previous grounds. Robust resolution of the quantitative profile of CDF enables deduction of a realistic biophysical model for this process. These results suggest that Ca_V2.1 CDF would figure most prominently in short-term synaptic plasticity and cerebellar Purkinje cell rhythmicity. Ca_V2.1 (P-type) voltage-gated Ca²⁺ channels constitute a major source of neuronal Ca²⁺ current, strongly influencing rhythmicity and triggering neurotransmitter release throughout the central nervous system. Fitting with such stature among Ca²⁺ entry pathways, Ca_V2.1 is itself feedback regulated by intracellular Ca²⁺, acting through calmodulin to facilitate channel opening. The precise neurophysiological role of this calcium-dependent facilitation (CDF) remains uncertain, however, in large measure because the very magnitude, Ca²⁺ dependence and kinetics of CDF have resisted quantification by conventional means. Here, we utilize the photo-uncaging of Ca²⁺ with Ca_V2.1 channels fluxing Li⁺ currents, so that voltage-dependent activation of channel gating is no longer conflated with Ca²⁺ entry, and CDF is then driven solely by light-induced increases in Ca²⁺. By using this strategy, we now find that CDF can be unexpectedly large, enhancing currents by as much as twofold at physiological voltages. CDF is steeply Ca²⁺ dependent, with a Hill coefficient of approximately two, a half-maximal effect reached by nearly 500 nm Ca²⁺, and Ca²⁺ on/off kinetics in the order of milliseconds to tens of milliseconds. These properties were established for both native P-type currents in cerebellar Purkinje neurons, as well as their recombinant channel counterparts under heterologous expression. Such features suggest that CDF of Ca_V2.1 channels may substantially enhance the regularity of rhythmic firing in cerebellar Purkinje neurons, where regularity is believed crucial for motor coordination. In addition, this degree of extensive CDF would be poised to exert large order-of-magnitude effects on short-term synaptic plasticity via rapid modulation of presynaptic Ca²⁺ entry.