A Voltage Dependent Non-Inactivating Na + Channel Activated during Apoptosis in Xenopus Oocytes Ulrika H. Englund, Jens Gertow, Katarina Ka ˚gedal, Fredrik Elinder* Department of Clinical and Experimental Medicine, Linko ¨ ping University, Linko ¨ ping, Sweden Abstract Ion channels in the plasma membrane are important for the apoptotic process. Different types of voltage-gated ion channels are up-regulated early in the apoptotic process and block of these channels prevents or delays apoptosis. In the present investigation we examined whether ion channels are up-regulated in oocytes from the frog Xenopus laevis during apoptosis. The two-electrode voltage-clamp technique was used to record endogenous ion currents in the oocytes. During staurosporine-induced apoptosis a voltage-dependent Na + current increased three-fold. This current was activated at voltages more positive than 0 mV (midpoint of the open-probability curve was +55 mV) and showed almost no sign of inactivation during a 1-s pulse. The current was resistant to the Na + -channel blockers tetrodotoxin (1 mM) and amiloride (10 mM), while the Ca 2+ -channel blocker verapamil (50 mM) in the bath solution completely blocked the current. The intracellular Na + concentration increased in staurosporine-treated oocytes, but could be prevented by replacing extracellular Na + whith either K + or Choline + . Prevention of this influx of Na + also prevented the STS-induced up-regulation of the caspase-3 activity, suggesting that the intracellular Na + increase is required to induce apoptosis. Taken together, we have found that a voltage dependent Na + channel is up-regulated during apoptosis and that influx of Na + is a crucial step in the apoptotic process in Xenopus oocytes. Citation: Englund UH, Gertow J, Ka ˚gedal K, Elinder F (2014) A Voltage Dependent Non-Inactivating Na + Channel Activated during Apoptosis in Xenopus Oocytes. PLoS ONE 9(2): e88381. doi:10.1371/journal.pone.0088381 Editor: Valentin Cen ˜ a, Universidad de Castilla-La Mancha, Spain Received October 2, 2013; Accepted January 6, 2014; Published February 28, 2014 Copyright: ß 2014 Englund et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Swedish Research Council (www.vr.se), the Swedish Heart-Lung Foundation (www.hjart-lungfonden.se), the Swedish Brain Foundation (www.hjarnfonden.se), the County Council of O ¨ stergo ¨ tland (www.lio.se), and the King Gustaf V and Queen Victoria’s Freemasons Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: fredrik.elinder@liu.se Introduction Programmed cell death, apoptosis, is an essential process in the development and the sculpturing of the growing organism. Apoptosis is a cascade process involving a sequence of biochemical reactions. Altered functions in the apoptotic cascade, for example caused by mutations, can lead to diseases. Much research carried out in the field of apoptosis has focused on biochemical alteration in intracellular signalling cascades, where pro-apoptotic proteins and proteases are activated, mitochondrial proteins released, and DNA degradation occurs [1]. However, the initial steps triggering the apoptotic process are less known. Alterations in the intracel- lular ion concentrations have been one such suggested initial event [2,3]. A number of ion channels open during the early steps of the apoptotic process and block of the channels also prevents or delays the apoptotic process. Apoptotic stimuli, such as staurosporine (STS) or serum deprivation has been shown to activate K + channels [4]. Opening of K + channels leads to an efflux of K + ions and a decrease in the intracellular K + concentration [4–7]. The decreased K + concentration has been shown to facilitate activation of caspases and apoptosis-associated nucleases. Consequently, block of K + channels prevents apoptosis [4,8,9]. Opening of Na + channels, leading to an increased Na + influx and intracellular Na + concentration also triggers apoptosis. Consequently block of Na + channels prevents apoptosis [2,3,10–14]. Finally, also Cl 2 channels have been linked to apoptosis. STS opens of a large- conductance Cl 2 -conducting anion channel [10,15,16], which has been suggested to be identical to the mitochondrial voltage- dependent anion channel, VDAC. Because the Cl 2 equilibrium potential and consequently the intracellular Cl 2 concentration normally follows the resting potential [17], opening of the channel is not expected to alter the Cl 2 concentrations. However, opening of Cl 2 channels is possibly an essential step in combination with opening of K + channels [4,15,18]. Block of these Cl 2 channels has also been shown to prevent apoptosis. Thus, alterations in intracellular ion concentrations are early steps in the apoptotic process. Despite this crucial role, still very little is known about the details of the role of ion channels and intracellular concentrations in apoptosis. Most cells are small and less suitable for biophysical investigations. Therefore, in the present investigation, we utilized large oocytes from the clawed toad Xenopus laevis as a model cell. These oocytes are easy to investigate with electrophysiological techniques, they have been shown to undergo classical apoptosis [19,20], and was recently shown that intracellular ion concentrations can be measured by nanoprobes [21]. STS activates caspase-3 and decreases the intracellular K + concentration, but this K + concentration alter- ation was shown not to be obligatory for the apoptotic process [22]. The specific purpose of the present investigation was to explore if the Xenopus oocyte alters its electrophysiological characteristics during STS-induced apoptosis, and if so, if the apoptotic process could be affected by preventing ion fluxes. PLOS ONE | www.plosone.org 1 February 2014 | Volume 9 | Issue 2 | e88381