Unexpected Down-regulation of the hIK1 Ca2+-activated K+ Channel by Its Opener 1-Ethyl-2-benzimidazolinone in HaCaT Keratinocytes

Abstract

We used a combination of electrophysiological and cell and molecular biological techniques to study the regulation and functional role of the intermediate conductance Ca 2؉ -activated K ؉ channel, hIK1, in HaCaT keratinocytes. When we incubated cells with the hIK1 opener, 1-ethyl-2-benzimidazolinone (1-EBIO), to investigate the cellular consequences of prolonged channel activity, an unexpected down-regulation of channels occurred within a few hours. The same effect was produced by the hIK1 openers chlorzoxazone and zoxazolamine and was also observed in a different cell line (C6 glioma cells). After 3 days of treatment with 1-EBIO, mRNA levels of hIK1 were substantially diminished and no channel activity was detected. Down-regulation of hIK1 was accompanied by a loss of mitogenic activity and a strong increase in cell size. After withdrawal of 1-EBIO, hIK1 mRNA and channel activity fully recovered and the cells resumed mitogenic activity. Our data present evidence for a novel feedback mechanism of hIK1 expression that appears to result from the paradoxical action of its pharmacological activator during prolonged application. Because the down-regulation of hIK1 bears immediate significance on the biological fate of keratinocytes, 1-EBIO and related compounds might emerge as potent tools to influence the proliferation of various non-excitable cells endowed with IK channels.Unlike small conductance and big conductance Ca 2ϩ -activated K ϩ channels (SK and BK, respectively), 1 intermediate conductance Ca 2ϩ -activated K ϩ channels (IK) are exclusively expressed in non-excitable cells, including fibroblasts, endothelial cells, secretory epithelial cells, immature smooth muscle cells, T-lymphocytes, and erythrocytes. The activity of IK channels has been implicated in the regulation of secretion, in cellular migration, and in the proliferation of mitogenically active cells (for review see Refs. 1 and 2).We recently demonstrated the expression of hIK1 (hSK4) mRNA in the human keratinocyte cell line, HaCaT (3). In perforated-patch whole-cell recordings, the extracellular mediator ATP produced a prominent and long-lasting hyperpolarization of HaCaT cells through a signaling pathway that involves IP 3 -mediated Ca 2ϩ release and subsequent activation of IK channels. Because ATP has been shown to promote proliferation of keratinocytes (4), it is likely that the mitogenic effect of ATP is at least partially mediated by the activation of hIK1. In support of this notion, we found that the levels of hIK1 mRNA declined as HaCaT cells began to differentiate (3).This finding is consistent with observations from other types of non-excitable cells, which corroborate a link between IK channel activity and cellular proliferation. For example, unstimulated T cells express a low number of IK channels, whereas stimulation of T cells with mitogens or specific antigens results in increased IK channel density (5-8). In a myogenic fibroblast cell line, the mitogenic action of basic fibroblast growth factor (bFGF) was linked to the up-regulati...

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

“…The resultant over-hyperpolarization causes an excess of Ca 2ϩ entry that favors cell differentiation rather than proliferation (43). 3) Consistent with a study in HaCaT keratinocyte and C6 glioma cell lines where EBIO also inhibited proliferation (42), an incisive general mechanism of negative feedback abolishes KCa3.1 expression, leading to lowered [Ca 2ϩ ] i and thereby attenuated proliferation during the prolonged stimulation. It is unlikely that these activators nonspecifically evoke these effects, independently of KCa3.1 stimulation, because the morphological changes induced by EBIO were suppressed by TRAM-34; SKA-16, an inactive analog of SKA-31, had no effects; and KCa3.1-deficient VSMCs exhibit an almost complete loss of proliferative response to PDGF (3).…”

Functional and molecular identification of intermediate-conductance Ca2+-activated K+ channels in breast cancer cells: association with cell cycle progression

“…However, the prolonged exposure (3 days) of MCF-7 cells to CZ or ZOX inhibited cell proliferation. The results are in agreement with the work of Koegel et al (29), who reported a downregulation of both hIK1 mRNA levels and channel activity after 3 days of treatment of HaCaT keratinocytes with hIK1 openers, i.e., 1-EBIO, CZ, and ZOX. The downregulation of hIK1 was accompanied by a loss of mitogenic activity and a strong increase in cell size (29).…”

“…The results are in agreement with the work of Koegel et al (29), who reported a downregulation of both hIK1 mRNA levels and channel activity after 3 days of treatment of HaCaT keratinocytes with hIK1 openers, i.e., 1-EBIO, CZ, and ZOX. The downregulation of hIK1 was accompanied by a loss of mitogenic activity and a strong increase in cell size (29). They suggested that 1-EBIO induces a conformational change in the channel protein that leads to more rapid internalization and/or degradation.…”

The Phosphatidylinositol 3-Phosphate Phosphatase Myotubularin- Related Protein 6 (MTMR6) Is a Negative Regulator of the Ca2+-Activated K+ Channel KCa3.1

“…Agonists of K Ca 3.1 have been proposed as therapy for cystic fibrosis, in which enhanced K ϩ secretion via K Ca 3.1 can be a means to promote Cl Ϫ secretion. However, only poor K Ca 3.1 agonists exist, and they are ineffective because they result in downregulation of K Ca 3.1 (26,35). Thus, the inhibition of MTM6 family members may provide a novel target strategy for upregulating K Ca 3.1 activity.…”