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Resumen del póster presentado al Biophysical Society 56th Annual Meeting, celebrado en San Diego-California (US) del 25 al 29 de febrero de 2012., Irvalec© (PM02734, Elisidepsin) is a marine-derived cytotoxic depsipeptide that is currently undergoing phase II clinical studies in non-small cell lung cancer. In vitro treatment of tumor cells with Irvalec© induces necrotic cell death, a process associated with rapid loss of membrane integrity and subsequent cell permeabilization. These results suggested an effect of the drug, either on an ion channel or in the plasma membrane. In order to explore this hypothesis patch-clamp experiments were performed in different tumor cell lines (HeLa, A549 and HCT116). Treated cells underwent rapid and dramatic morphological changes, including cell blebbing, severe swelling, plasma membrane permeabilization and cell lysis. Apart from the numerous small blebs, membranes from damaged cells also re-organized to form enormous bubbles surrounded by cell membrane. Using electrophysiological techniques, it was shown that Irvalec© induced an important increase in membrane conductance. The compound permeabilized the plasma membrane to ions, even when the cells were not pulsed, causing important changes in the holding current. It has been described that zinc attenuates the drastic effects of some membrane disrupting agents. Hence, to test if zinc exerted some protective effect against Irvalec© effects, cells were treated with this drug in the presence or absence of zinc salts and its membrane permeability was analyzed by using electrophysiology techniques, measuring the variations in the ion currents induced by the drug. Interestingly, in cells treated with zinc (10 mM), a decrease in the membrane permeability induced by Irvalec© was observed. Altogether, these results suggest that Irvalec© induces a rapid membrane permeabilization that lead to a necrotic cell death., Supported by CICYT SAF2010-14916 and FIS (RD06/0014/0006) Grants.

Kv1.5 channels are the primary channels contributing to the ultrarapid outward potassium current (IKur). The regulatory Kvβ1.3 subunit converts Kv1.5 channels from delayed rectifiers with a modest degree of slow inactivation to channels with both fast and slow inactivation components. Previous studies have shown that inhibition of PKC with calphostin C abolishes the fast inactivation induced by Kvβ1.3. In this study, we investigated the mechanisms underlying this phenomenon using electrophysiological, biochemical, and confocal microscopy approaches. To achieve this, we used HEK293 cells (which lack Kvβ subunits) transiently cotransfected with Kv1.5+Kvβ1.3 and also rat ventricular and atrial tissue to study native α-β subunit interactions. Immunocytochemistry assays demonstrated that these channel subunits colocalize in control conditions and after calphostin C treatment. Moreover, coimmunoprecipitation studies showed that Kv1.5 and Kvβ1.3 remain associated after PKC inhibition. After knocking down all PKC isoforms by siRNA or inhibiting PKC with calphostin C, Kvβ1.3-induced fast inactivation at +60 mV was abolished. However, depolarization to +100 mV revealed Kvβ1.3-induced inactivation, indicating that PKC inhibition causes a dramatic positive shift of the inactivation curve. Our results demonstrate that calphostin C-mediated abolishment of fast inactivation is not due to the dissociation of Kv1.5 and Kvβ1.3. Finally, immunoprecipitation and immunocytochemistry experiments revealed an association between Kv1.5, Kvβ1.3, the receptor for activated C kinase (RACK1), PKCβI, PKCβII, and PKCθ in HEK293 cells. A very similar Kv1.5 channelosome was found in rat ventricular tissue but not in atrial tissue., This work was supported by Ministerio de Ciencia e Innovación (MICINN) Grants SAF2010-14916, Ministerio de Educación y Ciencia-PR2003-0056, and Red Cooperativa de Enfermedades Cardiovasculares RECAVA FIS RD06/0014/0006 (to C. V.) and BFU2011-23268 and CSD2008-00005 (to A. F.). C. V. recipient of Grant MEC-PR2003-0056.

Kv1.5 channels are homotetramers of α-pore subunits mainly present in human atrium and pulmonary vasculature. Thus, Kv1.5 is a pharmacological target for cardiovascular diseases. Kvβ1.3 assemblies with Kvα1.5 and modifies its gating and pharmacology. A further knowledge of α-β interactions and pharmacology will lead a better design of new drugs.