The Role Of Potassium Channels In The B-16 Melanocyte Tumor Cells And Rat Cerebellar Granule Cell Migration

Cell migration is involved in a wide variety of physiological and pathophysiological processes, such as embryogenesis, nerve system development, immune defense, wound healing, and tumor metastasis. Until now, the cytoskeletal mechanism of migration has been shown to be modulated by activation of transporters and ion channels including a wide variety of K⁺ channels, Ca²⁺ channels, Cl^- channels and NMDA ligand-gated ion channels. Voltage-dependent K⁺ currents have been associated with migration of lymphocytes and embryonic nerve cells, and migration of granule cells from the cerebellum requires the activity of N-type Ca²⁺ channels and NMDA receptors. Although ion channel blockers have been used to study ion channels and cell migration, few reports have provided direct evidence for the link between ion channels and cell migration. In order to further understand the mechanism of the potassium channels in cell migration, we chose two different cell models to investigate the relationship between potassium channels and cell migration by whole-cell patch clamp, Transwell migration analysis, etc. The research is divided into two parts as following, and these results indicate that potassium channels participate in the cell migration:Part One: Cell migration is mediated by ion channels and transporters, and plays crucial roles in a variety of physiological and pathological processes. Previously, our studies have shown that a Ca²⁺-regulated K⁺ current exists in B-16 murine melanoma cells, and that endothelin-1 (ET-1) inhibits the K⁺ current via a PKC-dependent pathway. In the present study, patch-clamp whole-cell recording and Transwell migration assays were used to examine the effects of ET-1 on B-16 murine melanoma cell migration. ET-1 (100 nM in the injection pipette and 10 nM in the incubation medium) decreased the K⁺ current amplitude and inhibited migration of B-16 cells. Similarly, the Ca²⁺-regulated K⁺ channel blockers, BaCl₂ and quinidine decreased the K⁺ current and slowed migration of B-16 melanoma cells. The effect of ET-1 on the K⁺ current and cells migration was simulated by ET-3. In contrast, the K⁺ channel opener, diclofenac, increased the K⁺ current. Likewise, the migration of B-16 murine melanoma cells dramatically increased in the presence of diclofenac in incubation medium. Furthermore, the ET-1 and ET-3-induced inhibition of K⁺ current and migration was abrogated by diclofenac. In the presence of diclofenac, ET-1 only reduced the K⁺ current amplitude a little and slowed B-16 cell migration a little. The results suggest that the K⁺ channel-dependent migration of B-16 melanoma cells is modulated by ET-1.Part Two: Melatonin may work as a neuromodulator through the associated melatonin receptors in the central nervous system. Previously, our studies have shown that melatonin increased the I_K current via a G-protein-related pathway. In the present study, patch-clamp whole-cell recording, transwell migration assays and organotypic cerebellar slice cultures were used to examine the effect of melatonin on granule cell migration. Melatonin increased the I_K current amplitude and migration of granule cells. Meanwhile, TEA, the I_K channel blocker, decreased the I_K current and slowed migration of granule cells. Furthermore, the effects of melatonin on the I_K current and cell migration were not abolished by pre-incubation with P7791, a specific antagonist of MT3R, but were eliminated by application of the MT2R antagonists K185 and 4-P-PDOT. I_K current and cell migration were decreased by application of dibutyryl cyclic AMP (dbcAMP), which is in contrast to the melatonin effect on the I_K current and cell migration. Incubation with dbcAMP essentially blocked the melatonin-induced increasing effect. Moreover, incubation of isolated cell cultures in the melatonin-containing medium also decreased cAMP immunoreactivity in the granule cells. It is concluded, therefore, that I_K current, downstream of a cAMP transduction pathway, mediates the migration of rat cerebellar granule cells stimulated by melatonin.