Chromophore-Assisted Light Inactivation of Mitochondrial Electron Transport Chain Complex II in Caenorhabditis elegans.

Mitochondria play critical roles in meeting cellular energy demand, in cell death, and in reactive oxygen species (ROS) and stress signaling. Most Caenorhabditis elegans loss-of-function (lf) mutants in nuclear-encoded components of the respiratory chain are non-viable, emphasizing the importance of respiratory function. Chromophore-Assisted Light Inactivation (CALI) using genetically-encoded photosensitizers provides an opportunity to determine how individual respiratory chain components contribute to physiology following acute lf. As proof-of-concept, we expressed the 'singlet oxygen generator' miniSOG as a fusion with the SDHC subunit of respiratory complex II, encoded by mev-1 in C. elegans, using Mos1-mediated Single Copy Insertion. The resulting mev-1::miniSOG transgene complemented mev-1 mutant phenotypes in kn1 missense and tm1081(lf) deletion mutants. Complex II activity was inactivated by blue light in mitochondria from strains expressing active miniSOG fusions, but not those from inactive fusions. Moreover, light-inducible phenotypes in vivo demonstrated that complex II activity is important under conditions of high energy demand, and that specific cell types are uniquely susceptible to loss of complex II. In conclusion, miniSOG-mediated CALI is a novel genetic platform for acute inactivation of respiratory chain components. Spatio-temporally controlled ROS generation will expand our understanding of how the respiratory chain and mitochondrial ROS influence whole organism physiology.

Identification of a nuclear carbonic anhydrase in Caenorhabditis elegans.

BACKGROUND: Carbonic anhydrases (CA) catalyze the inter-conversion of CO(2) with HCO(3) and H(+), and are involved in a wide variety of physiologic processes such as anion transport, pH regulation, and water balance. In mammals there are sixteen members of the classical α-type CA family, while the simple genetic model organism Caenorhabditis elegans codes for six αCA isoforms (cah-1 through cah-6). METHODS: Fluorescent reporter constructs were used to analyze gene promoter usage, splice variation, and protein localization in transgenic worms. Catalytic activity of recombinant CA proteins was assessed using Hansson's histochemistry. CA's ability to regulate pH as a function of CO(2) and HCO(3) was measured using dynamic fluorescent imaging of genetically-targeted biosensors. RESULTS: Each of the six CA genes was found to be expressed in a distinct repertoire of cell types. Surprisingly, worms also expressed a catalytically-active CA splice variant, cah-4a, in which an alternative first exon targeted the protein to the nucleus. Cah-4a expression was restricted mainly to the nervous system, where it was found in nearly all neurons, and recombinant CAH-4A protein could regulate pH in the nucleus. CONCLUSIONS: In addition to establishing C. elegans as a platform for studying αCA function, this is the first example of a nuclear-targeted αCA in any organism to date. GENERAL SIGNIFICANCE: A classical αCA isoform is targeted exclusively to the nucleus where its activity may impact nuclear physiologic and pathophysiologic responses.

SLO-2 is cytoprotective and contributes to mitochondrial potassium transport.

Mitochondrial potassium channels are important mediators of cell protection against stress. The mitochondrial large-conductance "big" K(+) channel (mBK) mediates the evolutionarily-conserved process of anesthetic preconditioning (APC), wherein exposure to volatile anesthetics initiates protection against ischemic injury. Despite the role of the mBK in cardioprotection, the molecular identity of the channel remains unknown. We investigated the attributes of the mBK using C. elegans and mouse genetic models coupled with measurements of mitochondrial K(+) transport and APC. The canonical Ca(2+)-activated BK (or "maxi-K") channel SLO1 was dispensable for both mitochondrial K(+) transport and APC in both organisms. Instead, we found that the related but physiologically-distinct K(+) channel SLO2 was required, and that SLO2-dependent mitochondrial K(+) transport was triggered directly by volatile anesthetics. In addition, a SLO2 channel activator mimicked the protective effects of volatile anesthetics. These findings suggest that SLO2 contributes to protection from hypoxic injury by increasing the permeability of the mitochondrial inner membrane to K(+).

The C. elegans mitochondrial K+(ATP) channel: a potential target for preconditioning.

Ischemic preconditioning (IPC) is an evolutionarily conserved endogenous mechanism whereby short periods of non-lethal exposure to hypoxia alleviate damage caused by subsequent ischemia reperfusion (IR). Pharmacologic targeting has suggested that the mitochondrial ATP-sensitive potassium channel (mK(ATP)) is central to IPC signaling, despite its lack of molecular identity. Here, we report that isolated Caenorhabditis elegans mitochondria have a K(ATP) channel with the same physiologic and pharmacologic characteristics as the vertebrate channel. Since C. elegans also exhibit IPC, our observations provide a framework to study the role of mK(ATP) in IR injury in a genetic model organism.