Kir2.2 p.Thr140Met: a genetic susceptibility to sporadic periodic paralysis

Abstract

Introduction: Periodic paralyses (PP) are recurrent episodes of flaccid limb muscle weakness. Next to autosomal dominant forms, sporadic PP (SPP) cases are known but their genetics are unclear.

Methods: In a patient with hypokalemic SPP, we performed exome sequencing to identify a candidate gene. We sequenced this gene in 263 unrelated PP patients without any known causative mutations. Then we performed functional analysis of all variants found and molecular modelling for interpretation.

Results: Exome sequencing in the proband yielded three heterozygous variants predicted to be linked to disease. These encoded p.Thr140Met in the Kir2.2 potassium channel, p.Asp229Asn in protein kinase C theta, and p.Thr15943Ile in titin. Since all hitherto known causative PP genes code for ion channels, we studied the Kir2.2-encoding gene, KCNJ12, for involvement in PP pathogenesis. KCNJ12 screening in 263 PP patients revealed three further variants, each in a single individual and coding for p.Gly419Ser, p.Cys75Tyr, and p.Ile283Val. All four Kir2.2 variants were functionally expressed. Only p.Thr140Met displayed relevant functional alterations, i.e. homo-tetrameric channels produced almost no current, and hetero-tetrameric channels suppressed co-expressed wildtype Kir2.1 in a dominant-negative manner. Molecular modelling showed Kir2.2 p.Thr140Met to reduce movement of potassium ions towards binding sites in the hetero-tetramer pore compatible with a reduced maximal current. MD simulations revealed loss of hydrogen bonding with the p.Thr140Met substitution.

Discussion: The electrophysiological findings of p.Thr140Met are similar to those found in thyrotoxic PP caused by Kir2.6 mutations. Also, the homologous Thr140 residue is mutated in Kir2.6. This supports the idea that Kir2.2 p.Thr140Met conveys susceptibility to SPP and should be included in genetic screening.

Keywords: Kir2.1 channel; Kir2.2 channel; hetero-tetramer; periodic paralysis; susceptibility gene.

Figure 1.

Sanger sequencing proband. Electropherogram of both genes, KCNJ12 (top) and KCNJ18 (bottom). The presence of a heterozygous c.419C > T base change coding for p.Thr140Met in the coding region of KCNJ12 was confirmed using Sanger sequencing (red box). In KCNJ18, no base change was identified at this position. Additionally, two positions were marked (black boxes) to highlight the differences in wildtype sequences of these two genes in this highly homologous region proving the specificity of our amplification.

Figure 2.

Functional expression. Functional characterization of Kir2.2-WT and four missense variants expressed in tsA 201 cells. A) Representative current traces. Note that p.Thr140Met channels do not produce a current. B) Fluorescence image of the tsa 201 cell expressing WT and p.Thr140Met Kir2.2 fusion proteins with GFP (Left). Mean grey values from the membrane areas after subtraction of background showed comparable signal intensity between WT (n = 18) and p.Thr140Met (n = 19) (Right). C) Normalized current-voltage relationships for untransfected cells (n = 9), WT (n = 19), p.Cys75Tyr (n = 15), p.Thr140Met (n = 16), p.Ile283Val (n = 15), p.Gly419Ser (n = 8) and Kir2.2-WT+Kir2.2- p.Thr140Met (n = 10) recorded in 30mM external K+. D) Normalized current-voltage relationships for WT (n = 10), p.Cys75Tyr (n = 11), p.Thr140Met (n = 15), p.Ile283Val (N = 11) and p.Gly419Ser (n = 11) recorded in 5mM external K+. Data are shown as means ± SEM.

Figure 3.

Co-expression of Kir2.1. Functional characterization of hetero-tetramers between Kir2.1 and Kir2.2-WT and four missense variants expressed in tsA 201 cells. A) Representative current traces. B) Normalized current-voltage relationships for WT Kir2.1 (n = 17), WT/WT Kir2.1 + Kir2.2 (n = 14), WT Kir2.1+Kir2.2-p.Cys75Tyr (n = 17), WT Kir2.1 + Kir2.2-p.Thr140Met (n = 19), WT Kir2.1 + Kir2.2-p.Ile283Val (n = 17) and WT Kir2.1 + Kir2.2- p.Gly419Ser (n = 16) recorded in 5mM external K+. Data are shown as means ± SEM.

Figure 4.

Homology models. Homology models of Kir2.2 and Kir2.1 incorporated into a tetramer of equimolar ratio A). Pore helical regions are colored orange (Kir2.2) or green (Kir2.1), with homologous pore helix residues Thr140 (Kir2.2) and Thr139 (Kir2.1) shown in white with corresponding arrows; potassium ions are included with chain A (Kir2.2, right) from the original 4lp8 pdb coordinates. Top view of same prior to equilibration is shown in B). After equilibration for energy minimization, top views show relative re-distribution of potassium ions for the WT equimolar heterotetramer C), and with variant p.Thr140Met incorporated into 2 Kir2.2 chains D) or 3 Kir2.2 chains E). The Kir2.2 p.Thr140Met variant resulted in less movement of the potassium ions towards binding sites in the pore helix.

Figure 5.

Molecular dynamics simulations. Models of equimolar Kir2.2 and Kir2.1 were incorporated into a POPC membrane, solvated and ionized, and subjected to an electric field for molecular dynamics simulations. The pore helix of one Kir2.2 chain and the TIGYG selectivity filter are shown in panels A) (WT 2.2) and B) (Thr140M 2.2), along with potassium ions (cyan). Distance measurements between the pore helix Glu139 (yellow) and Thr140 (blue, panel A) or Met140 (red, panel B) are depicted by the white arrows, and are calculated from MD trajectories shown in panel C). Hydrogen bonding between Glu139 and the Ile144 of the selectivity filter are calculated from trajectories shown in panel D). Reduced distance between pore helix residues Glu139/Met140 in the variant compared to Glu139/Thr140 in WT is correlated with an absence of hydrogen bonding between Glu139 and the target Ile144 of the selectivity filter.