Prepulse facilitation of the honeybee Ca_V4 channel is produced by a shift in channel activation and requires an intact inactivation sequence

The recently characterized honeybee Ca_V4 channel is a high-voltage–activated Ca²⁺ channel ortholog to the DSC1 channel identified in Drosophila. While sequence similarities to Na_V channels are obvious, permeation properties and current kinetics are more closely aligned with those of Ca_V channels. Ca_V4 exhibits a distinctive cation-dependent inactivation pattern, a hallmark of Ca²⁺ channel behavior, and nonetheless displays sensitivity to a Na⁺ channel–specific regulator, veratrine. Calcium channel facilitation is a phenomenon whereby the probability of calcium channel opening increases with successive depolarization pulses, resulting in an enhanced Ca²⁺ influx during repetitive or sustained electrical activity. In this study, we have identified an additional specific property of Ca_V4 in the form of an atypical voltage-dependent facilitation of the Ca²⁺ or Ba²⁺ currents by strong pre-depolarizations or prepulses (pPs). This physiologically relevant phenomenon, known as pP-induced facilitation (PiF), is subject to positive regulation by the amplitude of the pP but to negative regulation by its duration. It produces a hyperpolarizing shift of the I–V curve without any change in the reversal potential and macroscopic or single channel conductance. PiF is thus more pronounced for small depolarizations and almost absent when channels reach their maximal open probability. A mutation that affects the inactivation of the Ca_V4 channel prevents the occurrence of PiF. This previously undocumented form of facilitation appears exclusive to Ca_V4 channels. A strong pP may lock Ca_V4 channels in a pre-open state, rendering them more susceptible to activation and thereby shifting the activation curve toward more negative potentials. This, in turn, would accelerate channel opening and increase current amplitude. Lastly, we show that the inactivation particle of Ca_V4 (MFLT sequence, equivalent to the IFMT motif in human Na_V, or MFMT in Apis Na_V channel), in addition to its role in the initiation of the voltage-dependent inactivation, also modulates PiF.