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  • Biophysical characterization and expression analysis of Kv1.3 potassium channel in primary human leukemic B cells.

Biophysical characterization and expression analysis of Kv1.3 potassium channel in primary human leukemic B cells.

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology (2015-09-24)
Ildiko Szabo, Livio Trentin, Valentina Trimarco, Gianpietro Semenzato, Luigi Leanza
ABSTRACT

Pharmacological inhibition of the potassium channel Kv1.3 has been shown to selectively kill B cells from patients with chronic lymphocytic leukemia (B-CLL). Here we aimed to biophysically characterize and compare Kv1.3 channel activity in B cells isolated either from healthy subjects or patients and investigated the mechanism accounting for the increased protein expression in B-CLL cells. Kv1.3 activity was measured by patch clamp, while expression of the channel protein was assessed by Western blot and FACS analysis. B-CLL cells were co-cultured with mesenchymal stromal cells (MSC) and Kv1.3 inhibitor-induced apoptosis was assessed. We demonstrate that Kv1.3 is highly expressed and is more active at resting membrane potential in human B-CLL cells than in healthy cells. Channel expression in pathologic cells decreased by the B-RAF kinase inhibitor PLX-4720, while it increased with Doxazosin, an α1-adrenoceptor antagonist. Kv1.3 inhibitors induced death in B-CLL cells also when co-cultured with MSC. Our results contribute to the characterization of B-CLL cells, as it shows that upregulation of Kv1.3 in pathologic B lymphocytes is linked to the oncogenic B-RAF signalling. We also conclude that Kv1.3 inhibitors represent a valuable tool to induce apoptosis of B-CLL cells even in the presence of MSC.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5, clone 6C5, Chemicon®, from mouse
Sigma-Aldrich
Anti-Potassium Channel Kv1.3 (extracellular)−FITC antibody produced in rabbit, affinity isolated antibody, lyophilized powder