The potassium channel Kv1.3 is highly expressed in the mitochondria of various cancerous cells. Here we show that direct inhibition of Kv1.3 using two mitochondria-targeted inhibitors alters mitochondrial function and leads to reactive oxygen species (ROS)-mediated death of even chemoresistant cells independently of p53 status. These inhibitors killed 98% of ex vivo primary chronic B-lymphocytic leukemia tumor cells while sparing healthy B cells. In orthotopic mouse models of melanoma and pancreatic ductal adenocarcinoma, the compounds reduced tumor size by more than 90% and 60%, respectively, while sparing immune and cardiac functions. Our work provides direct evidence that specific pharmacological targeting of a mitochondrial potassium channel can lead to ROS-mediated selective apoptosis of cancer cells in vivo, without causing significant side effects.

The size of the light-induced proton motive force (pmf) across the thylakoid membrane of chloroplasts is regulated in response to environmental stimuli. Here, we describe a component of the thylakoid membrane, the two-pore potassium (K(+)) channel TPK3, which modulates the composition of the pmf through ion counterbalancing. Recombinant TPK3 exhibited potassium-selective channel activity sensitive to Ca(2+) and H(+). In Arabidopsis plants, the channel is found in the thylakoid stromal lamellae. Arabidopsis plants silenced for the TPK3 gene display reduced growth and altered thylakoid membrane organization. This phenotype reflects an impaired capacity to generate a normal pmf, which results in reduced CO2 assimilation and deficient nonphotochemical dissipation of excess absorbed light. Thus, the TPK3 channel manages the pmf necessary to convert photochemical energy into physiological functions.

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca 2+ signaling may play a central role in this process. Free Ca 2+ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca 2+ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca 2+ uniporter machinery in mammals. MICU binds Ca 2+ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca 2+ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca 2+ in the matrix. Furthermore, Ca 2+ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca 2+ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca 2+ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca 2+ uptake by moderating influx, thereby shaping Ca 2+ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca 2+ signaling in plants.

Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Cauptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis (Arabidopsis thaliana) in which mitochondria can accumulate Ca, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology, we show that one isoform, AtMCU1, gives rise to a Ca-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red and Gd, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria, and its absence causes mild changes in Ca dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca uptake complex with differences to the mammalian MCUC, and identifies a new player of the intracellular Ca regulation network in plants.

Highlight Arabidopsis suspension-cultured cells stably expressing aequorin in the plastid stroma were used as a suitable tool to dissect differential Ca2+ responses of non-green plastids versus chloroplasts to several environmental cues.