Functional Characterization of Two Variants at the Intron 6—Exon 7 Boundary of the KCNQ2 Potassium Channel Gene Causing Distinct Epileptic Phenotypes

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  FIGURE 1. Localization of KCNQ2 variants herein investigated and effects on KCNQ2 mRNAs expression. (A) Localization of the two variants herein investigated in the KCNQ2 gene, falling at the last nucleotide of intron 6 (KCNQ2 (C)928-1G > C; blue circle; already reported in Soldovieri et al., 2014) or at the first nucleotide of exon 7 (KCNQ2 (C)928G > (A); already reported in Soldovieri et al., 2020). (B) Representative agarose gel run and (C) electrophoresis profiles of major bands obtained from RT-PCR experiments performed on Kv7.2 mRNAs expressed in father- or probands-deriving lymphoblasts, as indicated; the mRNA fragment of 295 bp (B) or 218 bp (C) are expected when the ag acceptor site at the end of intron 6 is used, while those of 265 bp (B) or 188 bp (C) bands are obtained using the alternative splice site in the exon 7. Panels in C have been obtained cropping the regions of interest from the complete electrophoresis profiles, reported in Supplementary Figure S2 Topology of a Kv7.2 subunit showing the 6 transmembrane segments (called from S1 to S6) and four intracellular domains at the C-terminus (called from A to D), binding many regulatory molecules. Middle and left panels show…

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  FIGURE 2. Functional and biochemical characterization of Kv7.2 G310S and Kv7.2 G310Δ10 subunits. (A) Maximal current densities measured in non transfected cells (NT) or in cells expressing wild-type or mutant Kv7.2 subunits, as indicated. * = p < 0.05 versus NT cells. (B) Western blot analysis of proteins from total lysates (upper panels) or biotinylated plasma membrane fractions (lower panels) from CHO cells transfected with the indicated constructs. Higher and lower blots were probed with anti-Kv7.2 or anti-α-tubulin antibodies, as indicated. Numbers on the left correspond to the molecular masses of the protein markers. ODKv7.2 TOT/ODTUB ratios were 1.00 ± 0.20, 1.54 ± 0.19, or 0.66 ± 0.25; n = 4; p > 0.05 among each other; ODKv7.2 BIOT/ODTUB ratios were 1.00 ± 0.50, 5.2 ± 1.50, or 3.33 ± 0.45 in Kv7.2-, Kv7.2 p.G310S-, or Kv7.2 p.G310Δ10-transfected cells, respectively; n = 4; p < 0.05 versus Kv7.2. (C,D) Quantification of maximal currents measured in CHO cells expressing either Kv7.2 variant with Kv7.2 (C) and/or Kv7.3 (D) subunits, as indicated. * = p < 0.05 versus the black bar in each panel; ** = p < 0.05 versus Kv7.3; *** = p < 0.05 Kv7.2 + Kv7.3 (0.5:1). (E) Representative current…

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  FIGURE 3. Functional characterization of heteromeric Kv7.2 + Kv7.3 channels incorporating Kv7.2 G310S or Kv7.2 G310Δ10 subunits expressed in neurons. (A) Macroscopic currents from neurons expressing wild-type or mutant Kv7.2 + Kv7.3 subunits, as indicated. (B,C) Current density/voltage curves measured in non transfected neurons (NT) or expressing the indicated channels incorporating Kv7.2 G310Δ10 (B) or Kv7.2 G310S (C) subunits, as indicated.

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  FIGURE 4. Functional study of the modulation of Kv7.2 G310S or Kv7.2 G310Δ10 subunits by PIP2 and calmodulin. (A,B) Representative traces (A) and quantification (B) of currents measured in CHO cells co-expressing homomeric wild-type or mutant Kv7.2 subunits with PIP5K, CaM, and/or CaM1234, as indicated. * = p < 0.05 versus Kv7.2. (C) Time-dependence of current decrease (left panel) and recovery (right panel) of currents measured in CHO cells co-expressing the indicated channels with DrVSP.