Abstract 15452: Regulation of Kv11.1 C-terminal Isoform Expression by Branch Point of Intron 9
The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart and mutations in KCNH2 cause long QT syndrome type 2 (LQT2). Two Kv11.1 C-terminal isoforms are expressed in the heart. Splicing of KCNH2 intron 9 generates the full-length, functional Kv11.1a isoform, while activation of an alternative polyadenylation site within intron 9 leads to the expression of the non-functional, C-terminally truncated Kv11.1a-USO isoform. Only one-third of KCNH2 pre-mRNA is processed to Kv11.1a in the heart due to inefficient intron 9 splicing. The mechanisms that regulate the alternative processing of KCNH2 intron 9 are not fully understood. In this study, we investigate the role of the branch point at the 3’-splice site of intron 9 in the regulation of Kv11.1 isoform expression. Compared to the consensus sequence, the branch point of KCNH2 intron 9 has one mismatch. To test whether the non-consensus branch point sequence contributes to the inefficient splicing of intron 9, we changed the branch point from CTGAG to the consensus sequence CTGAC in an intron-containing full-length KCNH2 gene construct. RNase protection assay, immunoblot and patch clamp analyses showed that the change to consensus sequence in intron 9 branch point increased the expression of the functional Kv11.1a isoform and concomitantly decreased the expression of the non-functional Kv11.1a-USO isoform, resulting in a significant increase in the Kv11.1 channel current. We also studied the LQT2 branch point mutation IVS9-28A>G. We found that the IVS9-28A>G mutation disrupted normal splicing and resulted in exclusive polyadenylation of intron 9, leading to a complete switch from the functional Kv11.1a isoform to the non-functional Kv11.1a-USO isoform. Our results demonstrate isoform switch as a pathogenic mechanism for the IVS9-28A>G mutation and support the conclusion that the KCNH2 intron 9 branch point plays an important role in regulation of the Kv11.1 C-terminal isoform expression and Kv11.1 channel function.
Author Disclosures: Q. Gong: None. M.R. Stump: None. Z. Zhou: None.
- © 2014 by American Heart Association, Inc.