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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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An absence of RET between β1 integrins (CD29) and Kv1.3 potassium channels on LOX cells in suspension as determined by RET imaging and microspectrophotometry. (A–D) Representative immunofluorescence microscopy experiments of nonadherent cells labeled with anti-CD29 (B) and anti-Kv1.3 (C) are shown. The corresponding DIC image is shown in A. Although cells are labeled with anti-CD29 and anti-Kv1.3 channel reagents, no RET is observed between these labels (D). (E–H) LOX cells in suspension were examined by fluorescence emission microspectrophotometry. The data shown here and elsewhere are plotted as intensity (photon counts) vs. wavelength (nm). LOX cells in suspension were labeled with FITC-conjugated anti-CD29 mAb only (E), first step rabbit anti-Kv1.3 Ab and second step goat anti–rabbit TRITC-conjugated mAb only (F), or both FITC-conjugated anti-CD29 mAb and rabbit anti-Kv1.3 Ab followed with TRITC-conjugated goat anti–rabbit Ab (G). The difference spectrum obtained by mathematical subtraction of anti–β1-integrin FITC (E) from RET spectrum (G) is shown in H. (See text for additional controls.) LOX cells in suspension revealed no RET between β1 integrins and Kv1.3 channels.
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fig1: An absence of RET between β1 integrins (CD29) and Kv1.3 potassium channels on LOX cells in suspension as determined by RET imaging and microspectrophotometry. (A–D) Representative immunofluorescence microscopy experiments of nonadherent cells labeled with anti-CD29 (B) and anti-Kv1.3 (C) are shown. The corresponding DIC image is shown in A. Although cells are labeled with anti-CD29 and anti-Kv1.3 channel reagents, no RET is observed between these labels (D). (E–H) LOX cells in suspension were examined by fluorescence emission microspectrophotometry. The data shown here and elsewhere are plotted as intensity (photon counts) vs. wavelength (nm). LOX cells in suspension were labeled with FITC-conjugated anti-CD29 mAb only (E), first step rabbit anti-Kv1.3 Ab and second step goat anti–rabbit TRITC-conjugated mAb only (F), or both FITC-conjugated anti-CD29 mAb and rabbit anti-Kv1.3 Ab followed with TRITC-conjugated goat anti–rabbit Ab (G). The difference spectrum obtained by mathematical subtraction of anti–β1-integrin FITC (E) from RET spectrum (G) is shown in H. (See text for additional controls.) LOX cells in suspension revealed no RET between β1 integrins and Kv1.3 channels.

Mentions: To assess the physical proximity of Kv1.3 channels and β1-integrins on LOX melanoma cells, RET experiments were conducted on cells labeled with donor- and acceptor-conjugated antibodies directed against Kv1.3 and the common chain of β1-integrins. Experiments were first performed using cells in suspension. Cells were detached from tissue culture plates, fixed with paraformaldehyde, washed extensively, and then labeled with fluorescent antibodies directed against the Kv1.3 channel and β1-integrin molecules. Immunofluorescence microscopy showed uniform distributions of β1-integrins and Kv1.3 channels on the LOX cell surface (Fig. 1, A–D). RET imaging experiments did not demonstrate energy transfer (Fig. 1 D). Moreover, single cell emission spectrophotometry did not reveal energy transfer between these two labels on LOX cells Fig. 1, E–H. Thus, these two molecules are expressed on LOX cells, but are not in the physical proximity of one another on nonadherent cells.


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)

An absence of RET between β1 integrins (CD29) and Kv1.3 potassium channels on LOX cells in suspension as determined by RET imaging and microspectrophotometry. (A–D) Representative immunofluorescence microscopy experiments of nonadherent cells labeled with anti-CD29 (B) and anti-Kv1.3 (C) are shown. The corresponding DIC image is shown in A. Although cells are labeled with anti-CD29 and anti-Kv1.3 channel reagents, no RET is observed between these labels (D). (E–H) LOX cells in suspension were examined by fluorescence emission microspectrophotometry. The data shown here and elsewhere are plotted as intensity (photon counts) vs. wavelength (nm). LOX cells in suspension were labeled with FITC-conjugated anti-CD29 mAb only (E), first step rabbit anti-Kv1.3 Ab and second step goat anti–rabbit TRITC-conjugated mAb only (F), or both FITC-conjugated anti-CD29 mAb and rabbit anti-Kv1.3 Ab followed with TRITC-conjugated goat anti–rabbit Ab (G). The difference spectrum obtained by mathematical subtraction of anti–β1-integrin FITC (E) from RET spectrum (G) is shown in H. (See text for additional controls.) LOX cells in suspension revealed no RET between β1 integrins and Kv1.3 channels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2311400&req=5

fig1: An absence of RET between β1 integrins (CD29) and Kv1.3 potassium channels on LOX cells in suspension as determined by RET imaging and microspectrophotometry. (A–D) Representative immunofluorescence microscopy experiments of nonadherent cells labeled with anti-CD29 (B) and anti-Kv1.3 (C) are shown. The corresponding DIC image is shown in A. Although cells are labeled with anti-CD29 and anti-Kv1.3 channel reagents, no RET is observed between these labels (D). (E–H) LOX cells in suspension were examined by fluorescence emission microspectrophotometry. The data shown here and elsewhere are plotted as intensity (photon counts) vs. wavelength (nm). LOX cells in suspension were labeled with FITC-conjugated anti-CD29 mAb only (E), first step rabbit anti-Kv1.3 Ab and second step goat anti–rabbit TRITC-conjugated mAb only (F), or both FITC-conjugated anti-CD29 mAb and rabbit anti-Kv1.3 Ab followed with TRITC-conjugated goat anti–rabbit Ab (G). The difference spectrum obtained by mathematical subtraction of anti–β1-integrin FITC (E) from RET spectrum (G) is shown in H. (See text for additional controls.) LOX cells in suspension revealed no RET between β1 integrins and Kv1.3 channels.
Mentions: To assess the physical proximity of Kv1.3 channels and β1-integrins on LOX melanoma cells, RET experiments were conducted on cells labeled with donor- and acceptor-conjugated antibodies directed against Kv1.3 and the common chain of β1-integrins. Experiments were first performed using cells in suspension. Cells were detached from tissue culture plates, fixed with paraformaldehyde, washed extensively, and then labeled with fluorescent antibodies directed against the Kv1.3 channel and β1-integrin molecules. Immunofluorescence microscopy showed uniform distributions of β1-integrins and Kv1.3 channels on the LOX cell surface (Fig. 1, A–D). RET imaging experiments did not demonstrate energy transfer (Fig. 1 D). Moreover, single cell emission spectrophotometry did not reveal energy transfer between these two labels on LOX cells Fig. 1, E–H. Thus, these two molecules are expressed on LOX cells, but are not in the physical proximity of one another on nonadherent cells.

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