Rapid Na⁺ accumulation by a sustained action potential impairs mitochondria function and induces apoptosis in HEK293 cells expressing non-inactivating Na⁺ channels

Highlights

• Excess Na⁺ influx through mutated Na⁺ channels causes apoptosis in HEK293 cells.

• Na⁺-induced apoptosis occurs independently of intracellular Ca²⁺ signaling.

• Accumulation of Na⁺ or/and concomitant K⁺ loss impairs mitochondria functions.

• Mitochondrial dysfunction and the decrease in ATP production may induce apoptosis.

• Na⁺ accumulation by hyper-excitability can be involved in neuronal cell death.

Abstract

The mechanisms underlying neuronal cell death induced by the rise of intracellular Na⁺ concentration ([Na⁺]_i) following abnormal hyperexcitation are not fully understood. Previously, we have established a recombinant cell line derived from HEK293 cells, in which the occurrence of a sustained action potential (AP) induces cell death. Mutated voltage-gated Nav1.5 channel (IFM/QQQ) lacking inactivation, and inward rectifying K⁺ channel (Kir2.1) were co-expressed in HEK293 cells (IFM/QQQ + Kir2.1 cells). In this cell line, the rise of [Na⁺]_i due to a sustained AP reached maximum within 15 min without concomitant [Ca²⁺]_i rise, and then elicited significant externalization of phosphatidylserine and enhancement of caspase activity. Marked decreases in mitochondrial transmembrane potential and ATP concentration were also detected. The significant cell death occurred at 3 h from the AP onset and reached a steady state at around 12 h. The significant release of lactate dehydrogenase was not detected even after 12 h. These results provide novel findings that Na⁺ accumulation or/and possibly concomitant K⁺ loss elicits apoptosis presumably due to the mitochondrial dysfunction, which is attributable to neither the membrane depolarization nor [Ca²⁺]_i change. This apoptotic mechanism may be involved, at least in part, in neuronal cell death under pathophysiological settings with abnormal hyperexcitability.

Introduction

Apoptosis is well controlled cell death to maintain the homeostasis of the living body [1]. It has been established that the trans-cell-membrane movement of ions through channels and transporters, and the subsequent alteration of their concentrations in cytosol and/or organelle is one of the substantial components initiating apoptosis [2,3]. The roles of intracellular Ca²⁺, a ubiquitous second messenger of cell signaling, in cell death are extensively investigated to date. The mitochondrial Ca²⁺ overload mainly due to the excess Ca²⁺ influx causes a release of cytochrome c through mitochondria permeability transition pore (MPTP) and thus results in the induction of apoptosis [4].

On the other hand, the impact of changes in intracellular Na⁺ concentration ([Na⁺]_i) on the initiation of apoptosis is not well understood, while the relation has been reported in some type of cells [5]. For example, the neuronal cell death by veratridine, an alkaloid which prevents the inactivation of voltage-gated Na⁺ channels, is considered to be due to the excess influx and resulting accumulation of Na⁺ [6,7]. However, the property of veratridine-induced cell death has not been well clarified yet, including whether it is necrosis or apoptosis. More importantly, in excitable cells such as neurons and myocytes, the activation of voltage-gated Na⁺ channel elicits an action potential, which subsequently triggers the opening of voltage-gated Ca²⁺ channels and induces the rise of [Ca²⁺]_i. Thus, there is a terrific difficulty for researchers to separate the contribution of Na⁺ accumulation per se from that of Ca²⁺ overload to cell-death in the native cells.

In order to develop a new screening technology, we have established a recombinant cell line derived from HEK293 cells (IFM/QQQ + Kir2.1 cells), in which a single action potential induces cell death [[8], [9], [10]] (Supplementary Fig. 1A). Mutated voltage-gated Nav1.5 channel (IFM/QQQ), which lacked a large part of inactivation [11], was stably expressed together with an inward rectifying K⁺ channel (Kir2.1) in HEK293 cells. In this cell line, Kir2.1 activity, that keeps the well negative resting membrane potential, prevents IFM/QQQ from its activation and protects the cells from death. The application of electrical stimulation (ES) activates IFM/QQQ, and then induces a sustained action potential lasting over 1 min (Supplementary Fig. 1B). Alternatively, the block of Kir2.1 by 200 μM Ba²⁺ also induced a sustained action potential-like depolarization [10]. We demonstrated that a single sustained action potential caused cell death in IFM/QQQ + Kir2.1 cells [[8], [9], [10]].

This cell system is potentially useful for a high throughput screening (HTS) of drug discovery targeting on ion channels. The third ion channel, which is a target of drug development, for example serotonin receptor type 3A (5-HT_3A) as an ion channel type receptor [12], is expressed in IFM/QQQ + Kir2.1 (IFM/QQQ + Kir2.1+5-HT_3AR) cells for HTS. The stimulation of 5-HT_3A by an agonist depolarizes the cell and activates IFM/QQQ. The occurrence of a long action potential results in cell death, which can be prohibited by the presence of 5-TH_3A antagonist.

In the previous studies, we recognized that this cell system is an excellent model to clarify the relation between Na⁺-accumulation and cell death. Based on these scientific backgrounds, the present study was undertaken to reveal the mechanisms underlying the cell death induced by rapid Na⁺ influx due to a sustained action potential occurrence in IFM/QQQ + Kir2.1 cells. The present results strongly suggest that the cell death shows mainly apoptotic features.