Four novel interaction partners demonstrate diverse modulatory effects on voltage-gated Ca_V2.2 Ca²⁺ channels

Voltage-gated Ca²⁺ channels are embedded in a network of protein interactions that are fundamental for channel function and modulation. Different strategies such as high-resolution quantitative MS analyses and yeast-two hybrid screens have been used to uncover these Ca²⁺ channel nanodomains. We applied the yeast split-ubiquitin system with its specific advantages to search for interaction partners of the Ca_V2.2 Ca²⁺ channel and identified four proteins: reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS) and transmembrane protein 223 (TMEM223). Interactions were verified using the yeast split-ubiquitin system and narrowed down to Ca_V2.2 domain IV. Colocalization studies using fluorescent constructs demonstrated defined regions of subcellular localization. Detailed electrophysiological studies revealed that coexpression of RTN1 modulated Ca_V2.2 channels only to a minor extent. SLC38 accelerated the cumulative current inactivation during a high-frequency train of brief depolarizing pulses. As neurons expressing Ca_V2.2 channels were exposed to high-frequency bursts under physiological conditions, observed regulation may have a negative modulatory effect on transmitter release. Coexpression of PTGDS significantly lowered the average current density and slowed the kinetics of cumulative current inactivation. Since the latter effect was not significant, it may only partly compensate the first one under physiological conditions. Expression of TMEM223 lowered the average current density, accelerated the kinetics of cumulative current inactivation and slowed the kinetics of recovery from inactivation. Therefore, TMEM223 and, to a lesser extent, PTGDS, may negatively modulate Ca²⁺ entry required for transmitter release and/or for dendritic plasticity under physiological conditions.