Article Figures & Data
Figures
- Figure 1.
Involvement of SK channels in MDA-MB-435s cell migration. Effect of apamin (A), Lei-Dab7, TEA, 4-AP (B), and increasing concentration of external K+ (C) on cell migration. Cells were seeded at 40,000 in a cell culture insert in DMEM with 5% fetal bovine serum ± drugs or [K+] 60 mmol/L. The lower compartment of the insert contained DMEM with 10% fetal bovine serum as a chemoattractant ± drugs or [K+] 60 mmol/L. After 24 h, cells of the lower compartment were stained with hematoxylin (A, bottom) and counted. The normalized cell number corresponded to the ratio of total number of migrating cells in presence of drug or [K+] 60 mmol/L/total number of migrating cells in control experiments. The drug concentrations selected have no effect on cell proliferation and viability (example with apamin in A, inset). Bar, 10 μm for the two panels. From two separate experiments done in triplicate. Columns, mean; bars, SE. *, P < 0.05, significantly different from control.
- Figure 2.
Regulation of resting membrane potential by SK channels in MDA-MB-435s cells. A, example of whole-cell macroscopic K+ currents recorded in one cell without (control) or with apamin in the external medium. Currents were generated by stepwise 8-mV depolarizing pulses (400-ms duration; 5-sec intervals) from a constant holding potential of −70 mV up to +58 mV. Signals were filtered at 1 kHz and digitized at 10 kHz. B and C, current density was obtained by dividing the averaged steady-state current elicited at +26 mV (recorded during the latest 50 ms of the pulse) by the respective cell capacitance. Membrane capacitance was calculated by integrating the capacitive current measured during a 10-mV voltage step. Columns, mean of the inhibitory effects of apamin (n = 4), TEA (n = 7), 4-AP (n = 8), and TEA plus 4-AP (n = 3); bars, SE. D, variations of membrane potential recorded in control conditions (physiologic saline solution without drugs, n = 11) and in presence of TEA (n = 7), 4-AP (n = 8), and TEA plus 4-AP (n = 3). Membrane potential was measured in current-clamp mode (I = 0) just after the disruption of the patch membrane. Columns, mean; bars, SE *, P < 0.05, significantly different from control.
- Figure 3.
SK3 protein is expressed in MDA-MB-435s and in tumor breast tissue. A, detection of SK channels (SK1, SK2, SK3, and SK4) and of the housekeeping ribosomal S14 mRNA in MDA-MB-435s. Reverse transcription-PCR was done in MDA-MB-435s cells and in human central nervous system cDNA as a positive control. Primers used for the reverse transcription-PCR experiments are listed in Materials and Methods. Representative examples of three separate experiments. B, representative Western blot pattern of SK2 and SK3 protein expression in cancerous and noncancerous mammary epithelial cell lines. Lysates of human mammary cancer cell lines (MDA-MB-435s, MDA-MB-231, MCF-7, T47D, and SKBR3), of noncancerous mammary epithelial cell lines (184A1, MCF-10A), and of rat hippocampus tissue (used as positive control) were prepared in lysis buffer (SDS 5%, protease inhibitors 1%, phenylmethylsulfonyl fluoride 200 mmol/L). Cell extracts were subjected to electrophoresis on SDS-polyacrylamide gel under reducing conditions and the signal was detected by enhanced chemiluminescence. Results were provided in triplicate. The numbers represent the SK2 and SK3 band absorbance normalized to those obtained with actin. C, confocal pictures showing the cellular location of SK3 channel in MDA-MB-435s cells. Cells were fixed and permeabilized with 100% methanol before antibody staining. D, Western blot pattern showing the expression of SK2 and SK3 proteins in tumor (n = 4) and nontumor (n = 4) breast biopsies. The numbers represent the SK2 and SK3 band absorbance normalized to those obtained with actin.
- Figure 4.
SK3 gene transcript destruction decreases migration of MDA-MB-435s cells, and SK3 gene expression increases migration of 184A1 cells. A, top, Western blot patterns showing the silencing effect on the expression of SK3 protein of two siRNAs designed against SK3 mRNA. Cells were transfected with siRNA-LipofectAMINE complexes for 24, 48, and 72 h. A scrambled siRNA was used as negative control. siRNA oligonucleotide sequences are listed in Materials and Methods. Bottom, histograms showing the inhibitory effect on MDA-MB-4345s cell migration after 24, 48, and 72 h of siRNA transfection with or without 10 nmol/L apamin. Results from two separate experiments done in triplicate. Columns, mean; bars, SE. Normalization of cell number done as described in Fig. 1. Note that the cells have lost their sensitivity for apamin after siRNA transfection, indicating a specific effect on SK3 protein. B, top, Western blot patterns showing the expression of the SK3 protein channel after transient transfection of SK3-pTracer-CMV2 plasmid (SK3 transfection) or empty vector (control transfection) in cancerous (MCF-7) and noncancerous (184A1) mammary epithelial cell lines. Bottom, histograms showing the number of migrating cells after transient transfection, with or without 10 nmol/L apamin. Results from two separate experiments done in triplicate. Columns, mean; bars, SE Note that SK3-transfected cells have gained a sensitivity to apamin, indicating a specific expression of SK3 channel.
- Figure 5.
Proposed model to explain how SK3 channel promotes epithelial cell migration. Top, when SK3 channel is not present, plasma membrane is depolarized (−30 to −20 mV) and Ca2+ influx through voltage-independent Ca2+ channels is low. As a consequence, intracellular Ca2+ concentration is low and epithelial cell have a low migration capacity. Bottom, expression of SK3 leading to SK3 channel activity would result in an increase of K+ efflux. This leads to a shift of membrane potential of epithelial cells to more negative values (hyperpolarization to −50 mV), which is equivalent to a stronger electrochemical driving force supporting Ca2+ entry through voltage-independent Ca2+ channels. This Ca2+ entry would increase intracellular Ca2+ concentration that promotes epithelial cell migration. Like a positive feedback loop, Ca2+ entry would also increase the activity of SK3 channel, leading to a more negative membrane potential.
Volume 5, Issue 11
