title: Comparison of read-through effects of aminoglycosides and PTC124 on rescuing nonsense mutations of HERG gene associated with long QT syndrome.
abstract: Aminoglycosides promote the readthrough of premature stop codons introduced by nonsense mutations to produce full-length proteins in genetic disease models. The read-through effects of different aminoglycosides and PTC124 on HERG gene have yet to be adequately elucidated. The wild-type (WT) or mutant genes were transiently transfected in HEK293 cells. The read-through effect was examined by adding drugs into culture medium for 24 h. Western blot analysis and patch clamping were performed to evaluate the expression and function of the genes. The mRNA levels were determined using qPCR. The results showed that G418 and PTC124 significantly increased the protein expression of R1014X mutant in a dose-dependent manner and produced a full-length protein. The maximal protein levels after G418, gentamicin or PTC124 treatment were 39.1±2.4, 18.6±0.3 or 10.3±1.0%, respectively, of the WT level. Tobramycin did not exhibit a read-through effect. The mRNA levels, however, did not differ between WT and mutant gene. The tail current densities of R1014X channels at 40 mV were 22.57±2.26 pA/pF for G418, 16.21±1.49 pA/pF for gentamicin and 9.62±0.73 pA/pF for PTC124. The leftward shift of the activation curve was corrected only by G418 and gentamicin. The read-through effects of W927X, R863X and E698X revealed that as the mutation site approached the N-terminal, the rescue efficiency was decreased. The above results suggest that aminoglycosides and PTC124 induced different effects on rescue nonsense mutations of the HERG gene. The mutation site was a significant factor in determining the pharmacological rescue efficiency.
authors.affiliation: State Key Laboratory of Cardiovascular Disease, Physiology and Pathophysiology Laboratory, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China.
authors.forename: Haiyun
authors.initials: H
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Cardiovascular Disease, Physiology and Pathophysiology Laboratory, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Cardiovascular Disease, Physiology and Pathophysiology Laboratory, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China.
authors.forename: Jian
authors.initials: J
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Cardiovascular Disease, Physiology and Pathophysiology Laboratory, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China.
authors.forename: Yinhui
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Cardiovascular Disease, Physiology and Pathophysiology Laboratory, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China.
authors.forename: Roumu
authors.initials: R
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Cardiovascular Disease, Physiology and Pathophysiology Laboratory, Fuwai Hospital, National Center for Cardiovascular Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100037, P.R. China.
authors.forename: Jielin
authors.initials: J
authors.lastname: Pu
mesh_terms: D000617:Aminoglycosides / Q000008:administration & dosage; D018389:Codon, Nonsense / Q000235:genetics*; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D008133:Long QT Syndrome / Q000235:genetics* / Q000473:pathology; D010069:Oxadiazoles / Q000008:administration & dosage; D012333:RNA, Messenger / Q000096:biosynthesis*
chemical_list: D000617:Aminoglycosides; D018389:Codon, Nonsense; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D010069:Oxadiazoles; D012333:RNA, Messenger; C515878:ataluren