Identification of Kv11.1 isoform switch as a novel pathogenic mechanism of long-QT syndrome

Abstract

Background: The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. The relative expression of the full-length Kv11.1a isoform and the C-terminally truncated Kv11.1a-USO isoform plays an important role in regulation of channel function. The formation of C-terminal isoforms is determined by competition between the splicing and alternative polyadenylation of KCNH2 intron 9. It is not known whether changes in the relative expression of Kv11.1a and Kv11.1a-USO can cause long-QT syndrome.

Methods and results: We identified a novel KCNH2 splice site mutation in a large family. The mutation, IVS9-2delA, is a deletion of the A in the AG dinucleotide of the 3' acceptor site of intron 9. We designed an intron-containing full-length KCNH2 gene construct to study the effects of the mutation on the relative expression of Kv11.1a and Kv11.1a-USO at the mRNA, protein, and functional levels. We found that this mutation disrupted normal splicing and resulted in exclusive polyadenylation of intron 9, leading to a switch from the functional Kv11.1a to the nonfunctional Kv11.1a-USO isoform in HEK293 cells and HL-1 cardiomyocytes. We also showed that IVS9-2delA caused isoform switch in the mutant allele of mRNA isolated from patient lymphocytes.

Conclusions: Our findings indicate that the IVS9-2delA mutation causes a switch in the expression of the functional Kv11.1a isoform to the nonfunctional Kv11.1a-USO isoform. Kv11.1 isoform switch represents a novel mechanism in the pathogenesis of long-QT syndrome.

Keywords: long QT syndrome; potassium channels.

Figure 1

A, The pedigree of a family with the IVS9-2delA mutation. The arrow indicates the proband. The QTc (ms), age at diagnosis and KCNH2 1956T/C SNP associated with each genotyped individual are shown. B, Sequence analysis revealing a deletion of an A (indicated by arrow) in the conserved AG dinucleotide of the 3′ acceptor site of intron 9. The sequences of WT and the IVS9-2delA mutation are shown below the sequence electropherogram.

Figure 2

Analysis of the IVS9-2delA mutation using the short KCNH2 gene construct. Flp-In HEK293 cells were stably transfected with WT and IVS9-2delA short KCNH2 gene constructs and analyzed by RPA and immunoblot. A, The structure of the short KCNH2 gene construct showing the position of the IVS9-2delA mutation in the 3′ acceptor site of intron 9. B, Analysis of mRNA by RPA. C, RPA signals were quantified, normalized to hygromycin resistance gene (Hygro), and plotted as the relative expression of the total WT (1a+1a-USO) Kv11.1 mRNA. Data are presented as individual (WT, open symbols; mutant, filled symbols) and as median values (black bars). Circle, triangle and square symbols represent total, 1-a, and 1-a-USO mRNA respectively (n=4, *P<0.05, Mann-Whitney U test). D, Immunoblot analysis of WT the IVS9-2delA mutation. Cell lysates were subjected to SDS-PAGE and immunoblotted with an antibody against the N-terminus of Kv11.1. The expression level of hygromycin B phosphotransferase (HPH) encoded by hygromycin B resistant gene served as a loading control. E, The level of protein bands was quantified, normalized to HPH, and plotted as the relative expression of the total WT (1a+1a-OSU) Kv11.1 protein (n=4, *P<0.05, Mann-Whitney U test).

Figure 3

Functional properties of WT and IVS9-2delA mutant Kv11.1 channels. A, Representative currents from Flp-In HEK293 cells stably transfected with WT and IVS9-2delA mutant short KCNH2 gene constructs. Kv11.1 current was activated from a holding potential of −80 mV with 4-s depolarizing test potentials between −70 and +50 mV. Tail current (indicated by arrows) was recorded at −50 mV following each depolarizing pulse. B, Current–voltage plot of tail current densities of WT (circles, n=10) and IVS9-2delA (squares, n=9). Data are plotted as mean±SEM.

Figure 4

RT-PCR analysis of the IVS9-2delA mutation. Flp-In HEK293 cells were stably transfected with WT and IVS9-2delA mutant in short KCNH2 gene (lanes 1 and 2) or short KCNH2 gene with intron 9 poly(A) signal deletion (del poly(A), lanes 3 and 4). RT- PCR was performed using primers in exon 9 and exon 11 as indicated by arrows. In the absence of intron 9 poly(A) signal, the IVS9-2delA mutation results in cryptic splicing (indicated by arrowhead) and complete intron 9 retention.

Figure 5

Analysis of the IVS9-2delA mutation in HL-1 cardiomyocytes. HL-1 cardiomyocytes were transiently transfected with HA-tagged WT and IVS9-2delA short KCNH2 gene constructs and analyzed by RPA and immunoblot. A, Analysis of mRNA by RPA. B, RPA signals were quantified, normalized to hygromycin resistance gene (Hygro), and plotted as the relative expression of the total WT (1a+1a-USO) Kv11.1 mRNA. Data are presented as individual (WT, open symbols; mutant, filled symbols) and as median values (black bars). Circle, triangle and square symbols represent total, 1-a, and 1-a-USO mRNA respectively (n=4, *P<0.05, Mann-Whitney U test). C, Immunoblot analysis of WT and IVS9-2delA mutation. Cell lysates were subjected to SDS-PAGE and immunoblotted with an antibody against the HA epitope. The expression level of hygromycin B phosphotransferase (HPH) encoded by hygromycin B resistant gene served as a loading control. D, The level of protein bands was quantified, normalized to HPH, and plotted as the relative expression of the total WT (1a+1a-USO) Kv11.1 protein (n=4, *P<0.05, Mann-Whitney U test).

Figure 6

Functional properties of WT and IVS9-2delA mutant Kv11.1 channels expressed in HL-1 cardiomyocytes. A, Representative currents from untransfected HL-1 cardiomyocytes and HL-1 cardiomyocytes transiently transfected with either WT or IVS9-2delA mutant short KCNH2 gene constructs. Kv11.1 current was activated from a holding potential of −50 mV with 2-s depolarizing test potentials between −40 and +40 mV. Tail current was recorded at −50 mV following each depolarizing pulse. Kv11.1 current was blocked by 5 µmol/L E-4031 and expressed as E-4031 sensitive current. B, Current–voltage plot of E-4031 sensitive tail current of untransfected (triangles, n=7), WT (circles, n=8) and IVS9-2delA (squares, n=7) current. Data are plotted as mean±SEM.

Figure 7

Allele-specific expression of WT and mutant mRNA isolated from lymphocytes. A, Schematic diagrams are shown for RT-PCR primers for Kv11.1a (1a), Kv11.1a-USO (1a-USO) and total (1a+1a-USO). The position of the synonymous SNP, 1956T/C, is indicated. B, Sequence analysis of PCR products from the proband and a normal subject of the family. Arrow indicates the expression of only the 1956C peak in the 1a PCR product from the proband. C, Analysis of allele ratio of 1956C and 1956T using the PeakPicker program. The dot plot shows the allele ratios of 1956T to 1956C in 1a (triangle), 1a-USO (square) and total (circle) PCR products from three normal subjects (open symbols) and four mutation carriers (filled symbols) of the family (*P<0.05, Mann-Whitney U test).