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Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers.

Artym VV, Petty HR - J. Gen. Physiol. (2002)

Bottom Line: Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels.However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels.Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA.

ABSTRACT
Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.

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Inhibitory effect of potassium channel blockers on RET between β1-integrins and Kv1.3 channels. The effect of K+ channel blockers on RET between β1-integrins and Kv1.3 channels was investigated using fluorescence microscopy. LOX cells adherent to the fibronectin-coated coverslips in the presence of 10−3 M TEA (A–D), 10−4 M 4-AP (E–H), or 10−9 M apamin (I–L) were labeled with anti–β1-integrin and anti-Kv1.3 channel reagents. Columns 1–4 show (a) DIC, (b) FITC fluorescence of anti-β1-integrin, (c) TRITC fluorescence of anti-Kv1.3 channel, and (d) RET between these two reagents. Note that although all of the cells were labeled with both reagents, RET was only observed in the presence of apamin.
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fig4: Inhibitory effect of potassium channel blockers on RET between β1-integrins and Kv1.3 channels. The effect of K+ channel blockers on RET between β1-integrins and Kv1.3 channels was investigated using fluorescence microscopy. LOX cells adherent to the fibronectin-coated coverslips in the presence of 10−3 M TEA (A–D), 10−4 M 4-AP (E–H), or 10−9 M apamin (I–L) were labeled with anti–β1-integrin and anti-Kv1.3 channel reagents. Columns 1–4 show (a) DIC, (b) FITC fluorescence of anti-β1-integrin, (c) TRITC fluorescence of anti-Kv1.3 channel, and (d) RET between these two reagents. Note that although all of the cells were labeled with both reagents, RET was only observed in the presence of apamin.

Mentions: We investigated the effect of K+ channel blockers on RET between Kv1.3 channels and β1-integrins. The inhibitors TEA, which blocks voltage-gated K+ channels and large conductance Ca2+-dependent K+ channels, and 4-AP, which blocks only voltage-gated K+ channels, were tested. These “classical” K+ channel blockers are known to inhibit Kv1.3 K+ channel currents (Grissmer et al., 1994). Treatment of LOX cells with TEA or 4-AP blocked RET between β1-integrins and Kv1.3 channels as shown by both emission spectrophotometry and fluorescence imaging (Figs. 3 and 4). Emission spectrophotometry data show that treatment of LOX cells with 10−3 M TEA or 10−4 M 4-AP reduced the number of RET positive cells by 77% and 93%, respectively (Table II). Apamin, an irrelevant small conductance Ca2+-dependent K+ channel blocker, was used as a negative control. Adherence of cells to fibronectin-coated coverslips in the presence of 10−9 M apamin had no effect on RET between β1-integrins and Kv1.3 channels (Figs. 3 and 4). Thus, certain K+ channel blockers inhibit β1-integrin-to-Kv1.3 channel proximity.


Molecular proximity of Kv1.3 voltage-gated potassium channels and beta(1)-integrins on the plasma membrane of melanoma cells: effects of cell adherence and channel blockers.

Artym VV, Petty HR - J. Gen. Physiol. (2002)

Inhibitory effect of potassium channel blockers on RET between β1-integrins and Kv1.3 channels. The effect of K+ channel blockers on RET between β1-integrins and Kv1.3 channels was investigated using fluorescence microscopy. LOX cells adherent to the fibronectin-coated coverslips in the presence of 10−3 M TEA (A–D), 10−4 M 4-AP (E–H), or 10−9 M apamin (I–L) were labeled with anti–β1-integrin and anti-Kv1.3 channel reagents. Columns 1–4 show (a) DIC, (b) FITC fluorescence of anti-β1-integrin, (c) TRITC fluorescence of anti-Kv1.3 channel, and (d) RET between these two reagents. Note that although all of the cells were labeled with both reagents, RET was only observed in the presence of apamin.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2311400&req=5

fig4: Inhibitory effect of potassium channel blockers on RET between β1-integrins and Kv1.3 channels. The effect of K+ channel blockers on RET between β1-integrins and Kv1.3 channels was investigated using fluorescence microscopy. LOX cells adherent to the fibronectin-coated coverslips in the presence of 10−3 M TEA (A–D), 10−4 M 4-AP (E–H), or 10−9 M apamin (I–L) were labeled with anti–β1-integrin and anti-Kv1.3 channel reagents. Columns 1–4 show (a) DIC, (b) FITC fluorescence of anti-β1-integrin, (c) TRITC fluorescence of anti-Kv1.3 channel, and (d) RET between these two reagents. Note that although all of the cells were labeled with both reagents, RET was only observed in the presence of apamin.
Mentions: We investigated the effect of K+ channel blockers on RET between Kv1.3 channels and β1-integrins. The inhibitors TEA, which blocks voltage-gated K+ channels and large conductance Ca2+-dependent K+ channels, and 4-AP, which blocks only voltage-gated K+ channels, were tested. These “classical” K+ channel blockers are known to inhibit Kv1.3 K+ channel currents (Grissmer et al., 1994). Treatment of LOX cells with TEA or 4-AP blocked RET between β1-integrins and Kv1.3 channels as shown by both emission spectrophotometry and fluorescence imaging (Figs. 3 and 4). Emission spectrophotometry data show that treatment of LOX cells with 10−3 M TEA or 10−4 M 4-AP reduced the number of RET positive cells by 77% and 93%, respectively (Table II). Apamin, an irrelevant small conductance Ca2+-dependent K+ channel blocker, was used as a negative control. Adherence of cells to fibronectin-coated coverslips in the presence of 10−9 M apamin had no effect on RET between β1-integrins and Kv1.3 channels (Figs. 3 and 4). Thus, certain K+ channel blockers inhibit β1-integrin-to-Kv1.3 channel proximity.

Bottom Line: Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels.However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels.Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA.

ABSTRACT
Tumor cell membranes have multiple components that participate in the process of metastasis. The present study investigates the physical association of beta1-integrins and Kv1.3 voltage-gated potassium channels in melanoma cell membranes using resonance energy transfer (RET) techniques. RET between donor-labeled anti-beta1-integrin and acceptor-labeled anti-Kv1.3 channels was detected on LOX cells adherent to glass and fibronectin-coated coverslips. However, RET was not observed on LOX cells in suspension, indicating that molecular proximity of these membrane molecules is adherence-related. Several K(+) channel blockers, including tetraethylammonium, 4-aminopyridine, and verapamil, inhibited RET between beta1-integrins and Kv1.3 channels. However, the irrelevant K(+) channel blocker apamin had no effect on RET between beta1-integrins and Kv1.3 channels. Based on these findings, we speculate that the lateral association of Kv1.3 channels with beta1-integrins contributes to the regulation of integrin function and that channel blockers might affect tumor cell behavior by influencing the assembly of supramolecular structures containing integrins.

Show MeSH
Related in: MedlinePlus