RGK Proteins Preferentially Inhibit Fast-Inactivating Voltage-Gated Calcium Channels: Implications for Human Disease

Abstract

Voltage-gated calcium channels (VGCC) are critical for nerve, heart, and skeletal muscle function, and their mutations can cause neurological and cardiovascular disease. Some of these mutations alter channel inactivation, leading to aberrant Ca2+ influx into cells. For example, Timothy syndrome (TS) is characterized by severe cardiomyopathy, and is often accompanied by autism spectrum disorder. The abnormalities arise from a point mutation in the L-type VGCC that slows channel inactivation. On the other hand, some types of migraine and epilepsy are associated with point mutations that speed the inactivation of P/Q-type VGCC. RGK proteins (Rad, Rem1, Rem2 and Gem/Kir) are small GTPases that strongly inhibit L-, N-, P/Q- and R-type VGCC. They are difficult to study because their overexpression nearly completely abolishes VGCC currents. Thus, we studied RGK-mediated inhibition using two-electrode voltage clamp (TEVC) in Xenopus oocytes, where we titrated the amount of injected RNA to achieve a more physiological level of VGCC inhibition of ∼50%. Under these conditions, we discovered that mutations that speed inactivation, such as those that cause familial hemiplegic migraine, turn channels hypersensitive to Gem inhibition. Remarkably, when we challenged the non-inactivating TS channel with Gem, we found that it was insensitive to inhibition. Furthermore, expressing VGCC with the β2a subunit, a Ca2+ channel auxiliary subunit known to slow channel inactivation, also weakened Gem inhibition.This could potentially occur if Gem were to trap channels in the inactivated state, thereby inhibiting inactivating channels, while sparing non-inactivating channels. Our additional findings corroborate this since channel recovery from inactivation is much slower in the presence of Gem.These results shed light on a new type of VGCC regulation. In addition, our results point to RGKs as potential players in calcium channelopathies.