Our results clearly corroborate that overexpression of GIRK1 protein exerts profound effects on wound healing, chemoinvasion and cellular motility in the MCF-7 breast cancer cell line suggesting a role to promote invasion and metastasis. Induction of angiogenesis was also affected. Most noteworthy is the fact that all vital parameters affected by GIRK1 overexpression are manipulated in opposite direction, depending on the GIRK1 variant tested. Overexpression of either GIRK1a or GIRK1c reinforces vital properties of MCF-7 cells towards the malignant phenotype, while GIRK1d overexpression seemingly counteracts upon the opposite direction. Hence, differential features of GIRK1 variant proteins could be responsible for this antithetical behavior and comparison of their established functional properties may provide insight. While homo- and heterotetrameric K
+ channels containing the full length GIRK1a subunit have lengthily been studied [
1], little is known on the function(s) and essentially nothing on the possible (patho)physiological role of the smaller GIRK1c and GIRK1d variants. In the few studies undertaken so far by several groups using different expression systems, homotetramers composed of GIRK1c or GIRK1d subunits proved themselves to be inactive as ion channels (despite of expression at the protein level), and, in addition, entirely silenced both homo- and heterotetrameric GIRK complexes by acting as dominant negative constituents (see [
12] for functional testing of splice variants and a subsumption of existing literature). Thereupon we suggest that the effect of GIRK1d that is contrary to the effect of GIRK1a overexpression is due to the dominant negative effect of GIRK1d on the function of GIRK complexes. Reinforcement of the malignant phenotype via GIRK signaling takes, to some degree, already place in the native MCF-7
WT cell line, where both mRNA and protein have been shown to exist [
12,
13], although at a much smaller scale when compared to the overexpressors. Overexpression of GIRK1d might impair endogenous GIRK signaling in MCF-7
WT, weakening cellular behavior related to the activation of invasion, metastatic spread and induction of angiogenesis. At the same time, overexpression of GIRK1a would enhance and reinforce the biological activities of preexisting GIRK complexes, in line with the results of the prevailing study. Also the finding of prolonged G
0/G
1 period in MCF-7 cells upon GIRK1d overexpression supports the view of a dominant negative action on endogenous GIRK complexes. The functional role of GIRK1c overexpression remains, however, enigmatic. Our study reveals for the first time a function for the GIRK1c variant, other than the constitutive negative properties reported in previous publications. Here we report that the functional outcome of GIRK1c overexpression rather resembles the one produced by overexpression of the GIRK1a subunit. Indeed all variants of GIRK1 comprise the integral transmembrane part, including permeation pathway and ion selectivity filter required to catalyze K
+ permeation across the plasma membrane. Hence, truncated splice variants of GIRK1 may, under our conditions, act as K
+ channels, although this has previously never been observed. At present, biological activities, other than K
+ permeation across the plasma membrane, might mediate the biological effects observed. By alternative splicing, full length mRNA encoding GIRK1a is composed of three different exons, i.e. exons 1–3 [
26]. GIRK1c mRNA comprises exons 1 and 2, while the one encoding GIRK1d is assembled from exons 1 and 3 (exon 2 is missing) [
12]. At the protein level, GIRK1a contains 501 amino acids. All three GIRK1 variants share amino acid positions 1–234 at the N-terminus. Due to a frameshift that prevents translation of exon 3, GIRK1d has one single additional C-terminal amino acid (glycine; position 235). In contrast and due to exon 2, GIRK1c shares amino acids positions 235–402 with GIRK1a. To sum up, the difference between GIRK1c and GIRK1d are 167 additional amino acids at the C-terminal of GIRK1c, when compared to the single additional amino acid 235 of GIRK1d. Thus, the key to the tumor promoting activity of GIRK1 must conceivably be located in the amino acid segment 235–402. It must be mentioned that the subcellular distribution observed, i.e. the major fraction of GIRK1 protein remaining within intracellular membranes rather than in the plasma membrane, is, at the first glance surprising. It is, however, identical to that reported previously upon
transient transfection of MCF-7 cells with GIRK1 splice variants [
12]. It has been frequently observed in studies dealing with GIRK1 synthesis, trafficking, and plasma membrane insertion that homooligomeric GIRK1 tetrameric protein remains mostly located in intracellular membranes whereas heteromeric assembly with other GIRK isoforms results in partial plasma membrane insertion and glycosylation of the GIRK1 subunit [
27‐
29]. It was, however, observed that even in native cells and in the presence of additional GIRK isoforms as heterooligomerization partners [
30‐
33] at least 64 % of GIRK1 protein remain permanently confined to intracellular membranes [
32,
34]. Although the (patho)physiological role of intracellular GIRK1 repositories within the ER of malignant MECs described here remains obscured, their existence is in line with the one generally observed and we can, at present, not decide whether intracellular or plasma membrane located GIRK protein is responsible for the effects observed by us. Also worth mentioning at this point are long noncoding RNAs (lnRNAs), sometimes even mRNAs, that do not require protein to be synthesized at all and have been found to shift the phenotype of cancer cells towards malignancy [
35]. In the current study, however, the overexpressed mRNAs were devoid of their 3′- and 5′-untranslated regions (UTRs) which presumably are crucial for such activities. Also the fact that IHC gives negative results for MCF-7
WT cells does not allow to rule out GIRK1 protein(s) as being responsible for the biological effects observed in wild-type and control MCF-7 cells and to favor the InRNA hypothesis. As signal transduction molecules such as GIRK complexes exert their biological activities usually at very low abundancies, immunoreactivity below detection threshold cannot be regarded as proof for the absence of protein. Therefore we favor the hypothesis that the tumor promoting effect of
KCNJ3 overexpression is provoked by the corresponding protein(s). Searching for a potential liaison between GIRK complexes in the plasma membrane, cancerogenesis and cancer progression, two major connections are obvious: First, K
+ channel proteins have been found to promote pathophysiological phenotypes responsible for malignant growth of cancer cells in a vast amount of reports (see [
36‐
39] for review). While some of these studies have identified K
+ channels to enhance proliferation, others reported on reinforcement of angiogenesis and cellular motility, as described in the present study [
20]. K
+ permeation as well as other hitherto unknown functions of K
+ channel proteins (called
“moonlighting” functions) had been found to promote the malignant phenotype [
40‐
43]. The second potential relation of GIRK signalling to cancer is exclusive amongst K
+ channel proteins as GIRK complexes act as direct G-protein effectors. For example, GPCR/G-protein mediated signalling guides the migration of metastatic breast tumor cells towards bone tissue that, in turn, forms spatial and environmental niches promoting tumor grow in response to factors released by the invaders [
44,
45]. In general terms, pathological GPCR signaling has long-since been identified as a major target in the development of novel therapeutic approaches [
46,
47]. As shown in the prevailing study, GIRK complexes are able to function as K
+ channels, but occur at extremely low abundancy in MCF-7 cells. Moreover, GIRK activation depends to a substantial extent on freely available G-protein β/γ dimers, i.e. GPCR activation. We conclude that the oncogenic potential of GIRK1 overexpression is closely linked to GPCR signaling. At present we cannot discriminate between an impact of K
+ permeation itself or another, hitherto unknown biological function. Since also GIRK1c, the subunit that so far has not been observed to function as an ion channel, exerts biological activity similar to the one of GIRK1a, one may favor the latter hypothesis. We can, however, not rule out the possibility that the GIRK1c subunit is functional as an ion channel in MCF-7 cells. More experimentation is required to arrive to definite conclusions concerning this aspect.