K V 1.2 is tetrameric, voltage-dependent, potassium-selective channels that regulate neuronal excitability. Variants of K V 1.2-encoding KCNA2 cause severe neurological disorders. Proline-substitution variants p.H310P and p.P407R affect residues highly conserved among K V -channels, at the C-terminus of helix S4 or the pore PVP-motif, respectively. In BPS2024 (Gkotsi et al.), we showed that both variants were loss-of-function, diminishing macroscopic currents of “homozygous-mutant” cells; and dominant-negative, suppressing conductance in “heterozygous” cells. Here, we investigate their trafficking and biosynthesis using immunocytochemistry and proximity-labeling proteomics. Surface trafficking was quantified by flow cytometry of transiently-transfected HEK293T cells using a fluorescent antibody against an extracellular epitope tag. K V 1.2(H310P) subunits showed no significant trafficking ( p = 0.58 compared to negative control, n = 8), whereas K V 1.2(P407R) did traffic, albeit partially (44 ± 4.1%, n = 7) relative to wild-type (100 ± 2.3%, n = 8). In “heterozygous” cells transfected with both wild-type and variant subunits, K V 1.2 (wild-type) trafficking was halved by K V 1.2(H310P) (49 ± 5.8%, n = 9) and K V 1.2(P407R) (48 ± 3.9%, n = 7), suggesting that some wild-type subunits were retained in non-trafficking hetero-tetramers. Wild-type subunits did not change the levels of either variant-subunit trafficking ( p >0.05, n = 7–8). The differential trafficking deficiency by the two variants anticipates diverse interactomes during biogenesis. We are testing this hypothesis by BioID-based proximity labeling and mass spectrometry. In preliminary results, K V 1.2(H310P) interacted less with Rab-1B, compared to wild-type. Rab-1B regulates ER-Golgi vesicle trafficking, suggesting the ER retention of K V 1.2(H310P)-subunits. K V 1.2(P407R) interacted less with peptidylprolyl isomerase B (PPIB), implicating the latter in PVP-motif folding. Both variants cause loss of KCNA2 function and act in a dominant-negative manner. Regarding p.H310P, this is likely due to strong trafficking impairment of variant subunits and wild-type/variant heteromers. The milder trafficking deficiency of p.P407R does not account for the complete, or 20-fold, suppression of macroscopic conductance in homozygous-mutant and heterozygous cells, respectively; we suggest that p.P407R produces both trafficking and gating defects.
Miguel et al. (Sun,) studied this question.