Engineering selectivity into RGK GTPase inhibition of voltage-dependent calcium channels

Abstract

SignificanceInflux of calcium ions through surface membrane calcium channels that open in response to electrical signals is important for vital biological processes including generation of the heartbeat and nerve cell communication. Blocking such calcium channels in a tissue- and isoform-specific manner is a sought-after treatment strategy for diseases including chronic pain and Parkinson’s disease. Proteins that can be expressed in cells to selectively block different calcium channel types have particular advantages over conventional small-molecule blockers. A four-member family of proteins known as RGK proteins strongly inhibit calcium channels, but do so in a non-selective manner, limiting their potential usefulness. Here we identified mutated RGK proteins that perform as isoform-selective calcium channel blockers, advancing the therapeutic potential of these proteins.

Designer genetically encoded voltage‐dependent calcium channel inhibitors inspired by RGK GTPases

“…2010; Puckerin et al . 2018). A recent elegant study revealed that endogenous Rad in cardiomyocytes constitutively exerts a gating brake on a fraction of Ca V 1.2 channels.…”

“…2012; Puckerin et al . 2016, 2018). In the particular case of Rem inhibition of Ca V 1.2, the β‐binding‐independent mechanism of inhibition is mediated by an interaction of the Rem distal C‐terminus with the α 1C N‐terminus region just upstream of the first transmembrane spanning segment of the channel (Yang et al .…”

“…Consistent with the hypothesis, Rem[R200A/L227A] selectively inhibited Ca V 1.2, but not other Ca V 1/Ca V 2 channels, reconstituted in HEK293 cells (Puckerin et al . 2018). The ability of Rem[R200A/L227A] to discriminate between Ca V 1.2 and Ca V 1.3 was especially notable given the difficulty of identifying small molecules that can effectively distinguish between these two L‐type channel subtypes (Zamponi et al .…”

“…Using a similar logic, we found that Rad[R208A/L235A] selectively blocked Ca V 1.2 and Ca V 2.2, consistent with the finding that Rad inhibits these two channels using both β‐binding‐dependent and β‐binding‐independent mechanisms (Puckerin et al . 2018). Importantly, both Rem[R200A/L227A] and Rad[R208A/L235A] strongly inhibited Ca V 1.2 channels in cardiomyocytes, indicating that the β‐binding‐independent mechanism of inhibition is operational in this native environment.…”

“…Similarly, the two proteins inhibited HVA Ca V channel currents in dorsal root ganglion (DRG) neurons to different extents, reflecting their varying selectivity for Ca V 1.2 and Ca V 1.2/Ca V 2.2 channels, respectively (Puckerin et al . 2018).…”