Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line.
The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA-MB-453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a-e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNA's, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNA's encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer.
Institute for Biophysics, Center of Physiological Medicine, Medical University of Graz, Graz, Austria.
Valerie
V
Wagner
Elke
E
Stadelmeyer
Monika
M
Riederer
Peter
P
Regitnig
Astrid
A
Gorischek
Trevor
T
Devaney
Kurt
K
Schmidt
Helmut A
HA
Tritthart
Koret
K
Hirschberg
Thomas
T
Bauernhofer
Wolfgang
W
Schreibmayer
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002999:Clone Cells; D005260:Female; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D017393:RNA Editing / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014982:Xenopus laevis
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; C497066:KCNJ3 protein, human; D020033:Protein Isoforms; D012333:RNA, Messenger