Oncogenic potential of TASK3 (Kcnk9) depends on K⁺channel function

Abstract

TASK3 gene (Kcnk9) is amplified and overexpressed in several types of human carcinomas. In this report, we demonstrate that a point mutation (G95E) within the consensus K ؉ filter of TASK3 not only abolished TASK3 potassium channel activity but also abrogated its oncogenic functions, including proliferation in low serum, resistance to apoptosis, and promotion of tumor growth. Furthermore, we provide evidence that TASK3 G95E is a dominant-negative mutation, because coexpression of the wild-type and the mutant TASK3 resulted in inhibition of K ؉ current of wild-type TASK3 and its tumorigenicity in nude mice. These results establish a direct link between the potassium channel activity of TASK3 and its oncogenic functions and imply that blockers for this potassium channel may have therapeutic potential for the treatment of cancers.T ASK (TWIK-related acid-sensitive K ϩ channels) channels are members of the two-pore domain family of potassium channels, whose structure consists of two-pore forming regions flanked by four transmembrane domains (1, 2). Like other two-pore domain members, these channels show little time or voltage dependence; thus, they have characteristics of leaky K ϩ channels, generating background currents that contribute to membrane potential and the regulation of cell excitability. The activity of TASK channels is modulated by volatile anesthetics (3, 4), neurotransmitters (5, 6), as well as extracellular pH in the physiological range (7-11). TASK3 is expressed at very low levels among the normal tissues except in the brain, where high levels expression of TASK3 were detected (7-9). The physiological functions of TASK channels are largely unknown, though their roles in the regulation of breathing (12, 13), aldosterone secretion (5) and anesthetic-mediated neuronal activity (14) have been proposed. Recently, we showed that TASK3 is amplified in 10% of breast cancers and is overexpressed at a higher frequency of breast, lung, colon, and metastatic prostate cancers (15), suggesting that TASK3 may play a role in pathogenesis of some human carcinomas.Is the dysregulated expression of TASK3 in tumor cells a consequence of their abnormal growth or is this K ϩ channel involved in promoting tumor growth? To begin to answer this question, we created an inactivating mutation of TASK3. We report here that TASK3 G95E is a dominant-negative mutation that abolishes not only TASK3 K ϩ channel activity but also its oncogenic functions. These results provide molecular basis for developing specific blockers for this K ϩ channel in the treatment of cancer. Materials and MethodsPlasmids and Mutagenesis. The coding region of TASK3 was cloned at BamHI and EcoRI sites of the pLPC retroviral expression vector (15) to generate pLPC-TASK3. Site-directed mutagenesis was performed to change Gly-95 to Glu to create pLPC-TASK3 G95E , by using QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's protocol. pTracer-TASK 3 G95E was generated by excising TASK3 G95E from pLPC vector and cloned at KpnI...

KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer

“…There is mounting evidence that K þ channels play a crucial role in oncogenesis, and their potential as therapeutic targets in cancer has been recognized (Pardo et al, 1999;Pei et al, 2003;Conti, 2004;Wang, 2004). Here, we demonstrate that the large-conductance Ca 2 þ -activated potassium channel (KCNMA1) is amplified at 10q22 in 16% of late-stage, metastatic and hormonerefractory human prostate cancers.…”

The Impact of Heterozygous KCNK3 Mutations Associated With Pulmonary Arterial Hypertension on Channel Function and Pharmacological Recovery

“…G203D KCNK3 disrupts the conserved “GxG” potassium selectivity filter amino acid sequence in 1 of the channel's 2 pore‐loop domains, a region sensitive to dominant‐negative mutations 24, 25, 26. We first engineered and studied the WT‐G203D KCNK3 heterodimer (Figure 7B, left), and indeed observed severe dominant‐negative dysfunction, as the mutant channels produced small currents across pH 6.4 through 10.4 (Figure 7B and 7C; also see Figure S4).…”

Motoneurons Express Heteromeric TWIK-Related Acid-Sensitive K+(TASK) Channels Containing TASK-1 (KCNK3) and TASK-3 (KCNK9) Subunits

“…The ability of TASK subunits to form heterodimeric channels has been tested previously in other expression systems and with other approaches, but the results have not been uniformly positive Czirjak and Enyedi, 2002a;Kang et al, 2003;Lauritzen et al, 2003;Pei et al, 2003). It is clear that a tandem-linked construct, in which the two TASK subunits are concatenated into a single polypeptide, supports formation of heterodimeric channels and allows their functional and pharmacological characterization (Czirjak and Enyedi, 2002a;Talley and Bayliss, 2002;Kang et al, 2003).…”

“…This combinatorial potential provides a mechanism for additional diversity in K ϩ channel properties within individual cells (Wei et al, 1990;Krapivinsky et al, 1995;Wang et al, 1998), but evidence in support of native heterodimeric channels of the KCNK family is scarce (Kang et al, 2003). In the case of TASK-1 and TASK-3, heterodimeric channels have been detected in heterologous expression systems (Czirjak and Enyedi, 2002a;Kang et al, 2003;Lauritzen et al, 2003; but see Karschin et al, 2001;Pei et al, 2003), and a substantial overlap of subunit expression in many areas of the CNS provides a substrate for their coassembly in vivo Talley et al, 2001;Vega-Saenz de Miera et al, 2001). In cerebellar granule neurons that express TASK-1 and TASK-3, coassociation of TASK channels into functional native heterodimers was demonstrated by taking advantage of the unique single-channel properties and ruthenium red sensitivity defined for recombinant linked heterodimeric TASK-1/TASK-3 channels (Kang et al, 2003).…”