Cloning and characterisation of GIRK1 variants resulting from alternative RNA editing of the KCNJ3 gene transcript in a human breast cancer cell line

Abstract

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.

KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients

“…Further, it is unknown how the GIRK1 protein might interact with the estrogen receptor. GIRK1 is a non-functional channel when expressed as a homomer [37], located intracellularly as it accumulates in the endoplasmatic reticulum upon overexpression [38]. Thus, it remains to be determined whether and how GIRK1 and ER act together in same signaling pathways and further studies are needed to elucidate the mechanism of action of KCNJ3 upregulation in breast cancer.…”

Critical evaluation ofKCNJ3gene product detection in human breast cancer: mRNA in situ hybridisation is superior to immunohistochemistry

“…However, our data point towards differences in performance and robustness of the detection methods used and not towards biological reasons for highly differential mRNA and protein expression. Remarkably, few studies succeeded in immunohistochemical staining of GIRK1 on FFPE human tissue using Ab#48 and Ab#5 315 16 In retrospect, these previous studies might have overestimated GIRK1 expression, since anti-GIRK1 antibodies might lead to unspecific background staining.…”

“…8); Ab#3: rabbit polyclonal antibody directed against the N-terminus (N-T) of GIRK1 (generated by Kurt Schmidt); Ab#4: polyclonal goat antibody from Santa Cruz (#Sc-16131); Ab#5: rabbit polyclonal antibody from Alomone (#APC-005) and Ab#6: mouse monoclonal antibody from Alomone (#ALM-031). Sheep anti-rabbit/horseradish peroxidase (HRP), sheep anti-mouse/HRP (both kindly provided by Amir-Hassan Zarnani, Avicenna Research Institute, Tehran, Iran; both 1:10 000 dilution) and donkey anti-goat IgG-HRP antibodies (Santa Cruz Biotechnology, #Sc-2020; 1:5000 dilution) served as secondary antibodies.…”

“…8 HL-1 cells were purchased from William C. Claycomb and maintained as described 9. Cells were kept in a humidified atmosphere at 37°C and 5% CO 2 .…”

GIRK Channel Plasticity and Implications for Drug Addiction

“…The existence of splice variants of the GIRK1 and GIRK2 subunits contributes to the molecular diversity of GIRK channels (Wei et al, 1998;Zhu et al, 2001). Several splice variants of GIRK1 have been described; however, there is limited knowledge about the expression and functional roles of these isoforms (Nelson, Marino, & Allen, 1997;Steinecker, Rosker, & Schreibmayer, 2007;Wagner et al, 2010;Zhu et al, 2001). A number of splice variants of GIRK2 have been identified (Isomoto, Kondo, & Kurachi, 1997;Lesage et al, 1994Lesage et al, , 1995Wei et al, 1998), and three of these isoforms (GIRK2a, GIRK2b, and GIRK2c) are widely expressed in the brain (Isomoto et al, 1997;Wei et al, 1998).…”