Modulation of voltage-gated CaV2.2 Ca2+ channels by newly identified interaction partners

Abstract

Voltage-gated Ca²⁺ channels are typically integrated in a complex network of protein-protein-interactions, also referred to as Ca²⁺ channel nanodomains. Amongst the neuronal Ca_V2 channel family, Ca_V2.2 is of particular importance due to its general role for signal transmission from the periphery to the central nervous system, but also due to its significance for pain perception. Thus, Ca_V2.2 is an ideal target candidate to search for pharmacological inhibitors but also for novel modulatory interactors. In this review we summarize the last years findings of our intense screenings and characterization of the six Ca_V2.2 interaction partners, tetraspanin-13 (TSPAN-13), reticulon 1 (RTN1), member 1 of solute carrier family 38 (SLC38), prostaglandin D2 synthase (PTGDS), transmembrane protein 223 (TMEM223), and transmembrane BAX inhibitor motif 3 (Grina/TMBIM3) containing protein. Each protein shows a unique way of channel modulation as shown by extensive electrophysiological studies. Amongst the newly identified interactors, Grina/TMBIM3 is most striking due to its modulatory effect which is rather comparable to G-protein regulation.

Keywords: Cav2.2; G protein; Grina/TMBIM3; calcium channel; channel modulation.

Figure 1.

Schematic depiction of the role of presynaptic Ca_V2.2 in signal transmission in a nociceptive synapse. Modified from [22] with permission.

Figure 2.

Schematic representation of the Ca_V2.2 α1 subunit (a) and parts of amino acid sequence of rat Ca_V2.2 α1 subunit (UniProtKB – Q02294) (b). Three loci (pink blocks in A) were identified as putative interaction sites with Gβγ subunits. Site on N-terminus (amino acids 1–95) includes amino acids 45–55 (red line in B) [36] with specific role of amino acids 52 and 54 [37] and amino acids 56–90 (blue line in B) [35]. Two interaction sites were identified in the loop connecting domains I and II. First part overlaps with the α1-interaction site (red block in A) consisting of amino acids 379–396 (red letters in B) [33] and involves amino acids 353–389 (red line in B) [18,34]. Depicted is amino acid sequence of the I–II loop. The entire interaction site includes also amino acids ³⁵³GVLS³⁵⁶ from the IS6 segment. Second is located further downstream and includes amino acids 410–428 (blue line in B) [18]. Role of C-terminus is less clear and putative binding region was not unambiguously identified. Domain IV interacts with Grina/TMBIM3 (gray block in A) but is not narrowed down in more detail.

Figure 3.

Prepulse facilitation protocol used in CHO cells stably transfected with human α1-Ca_V2.2, α₂δ-1, and β1 subunits coexpressed with Gβγ subunits of G proteins (left) or with Grina/TMBIM3 (right) relieved current inhibition and accelerated activation kinetics in a similar manner [46]. In the bottom row, current traces recorded, before (black) and after (red) depolarizing prepulse were normalized to the same amplitude to facilitate comparison of current kinetics.

Figure 4.

Prepulse facilitation was applied in CHO cells stably transfected with human α1-Ca_V2.2, α₂δ-1, and β1 subunits coexpressed with Gβγ subunits of G proteins (olive) and with Grina/TMBIM3 protein (red). In panel (a) length of depolarizing pulse was gradually increased and prepulse facilitation evolved with increasing pulse length due to unbinding of Gβγ subunits from the channel. In panel (b) time intervals after depolarizing pulses were increased and prepulse facilitation faded away due to rebinding of Gβγ subunits. In both paradigms kinetics of effects was similar.

Figure 5.

CHO cells were transfected with C-terminal EGFP-tagged Grina/TMBIM3 and as a control with EGFP-tagged Tspan13. Tspan13 is a four transmembrane protein with cytosolic oriented N- and C-termini. Following transfection, both proteins show the same staining pattern, demonstrating that Grina/TMBIM3 is localized in the plasma membrane. However, only permeabilized cells allow an antibody staining of EGFP-tag indicating a cytosolic orientation of the Grina/TMBIM3 C-terminus.