Integrating genetically encoded fluorescent sensors to elucidate the spatiotemporal choreography of necrosis by sodium overload

Abstract

Necrosis by sodium overload (NECSO) is a distinct cell death modality induced by the chemical regulator necrocide 1 (NC1), which targets the transient receptor potential melastatin 4 (TRPM4) channel to drive excessive sodium influx and potassium efflux. This unique necrosis is characterized by the exchange of monovalent ions, a restrained ATP supply, redox disorder and a final membrane rupture as main features. Real-time monitoring of metabolic and redox causality during the whole process in living cells has been technically challenging. To bridge this gap, we integrate an advanced toolset of genetically encoded fluorescent sensors to monitor monovalent ions, energy metabolites, and redox equivalents with high spatiotemporal resolution. By directing these sensors to specific subcellular compartments, we successfully capture the real-time choreography of potassium loss specifically induced by NC1 via TRPM4. Furthermore, we establish a precise detection paradigm for evaluating energy currency by integrating sensors for NADH and ATP that are capable of subcellular imaging. We observe general and rapid NADH accumulation along with an ATP shortage in the mitochondria and cytosol. A concomitant reduction in mitochondrial oxidative stress is observed. This study not only elucidates the metabolic progression of a peculiar type of necrosis but also establishes a robust methodological framework for applying genetically encoded sensors to broader physiological and toxicological research.

1. Introduction

Sodium (Na+) is the most abundant extracellular cation, and its steep transmembrane gradient is essential for cellular volume regulation, nutrient transport, and electrical excitability [1–4]. While sodium influx is a hallmark of tissue injury in conditions such as stroke and myocardial infarction [5,6], whether it serves as a primary executioner of regulated cell death (RCD) remains unclear compared with well-characterized pathways such as apoptosis [7], ferroptosis [8,9], or necroptosis [10]. This gap was recently bridged by the discovery of necrocide 1 (NC1), which targets the TRPM4 channel, a Ca2+-activated nonselective cation channel [11–14]. Recent cryo-EM studies have mapped the vanilloid binding pocket (VBP) of TRPM4, where NC1 acts as a molecular wedge to lock the channel in a persistent open state [15–18]. This leads to massive sodium overload and a novel necrotic pathway termed necrosis by sodium overload (NECSO) [15,19,20]. The execution of NECSO involves not only osmotic swelling via aquaporin-4 (AQP4) [21] but also catastrophic metabolic collapse, as excessive amounts of sodium enter mitochondria via the NCLX exchanger [22], disrupting the TCA cycle and ATP production [23,25].

Deciphering the spatiotemporal orchestration of NECSO requires robust tools to monitor these rapid ionic and metabolic shifts [26]. However, a significant technological challenge persists: while chemical dyes such as CoroNa Green can detect sodium overload, a reliable genetically encoded sodium indicator is still lacking. This lack of organelle-specific sodium sensors limits our ability to track mitochondrial sodium dynamics with high precision [26,27]. Sodium and potassium are closely associated and, in most cases, are transported via mutual displacement. In contrast to sodium sensing, potassium sensing has entered a sophisticated era of genetic engineering. By utilizing bacterial K+-binding protein (Kbp), researchers have developed FRET-based ratiometric indicators such as GEPIIs and KIRIN1, as well as single-FP sensors such as GINKO1 [28,29]. Furthermore, the BRIPO system has introduced bioluminescence imaging for potassium, enabling high-contrast monitoring in vivo [30].