Redox Properties of the Adenoside Triphosphate-Sensitive K 1 Channel in Brain Mitochondria Maynara Fornazari, 1 Juliana G. de Paula, 1 Roger F. Castilho, 2 and Alicia J. Kowaltowski 1 * 1 Departamento de Bioquı ´mica, Instituto de Quı ´mica, Universidade de Sa ˜o Paulo, Sa ˜o Paulo, Brazil 2 Departamento de Patologia Clı ´nica, Faculdade de Cie ˆncias Me ´dicas, Universidade Estadual de Campinas, Campinas, Brazil Brain mitochondrial ATP-sensitive K 1 channel (mito- K ATP ) opening by diazoxide protects against ischemic damage and excitotoxic cell death. Here we studied the redox properties of brain mitoK ATP . MitoK ATP activa- tion during excitotoxicity in cultured cerebellar granule neurons prevented the accumulation of reactive oxygen species (ROS) and cell death. Furthermore, mitoK ATP activation in isolated brain mitochondria significantly prevented H 2 O 2 release by these organelles but did not change Ca 21 accumulation capacity. Interestingly, the activity of mitoK ATP was highly dependent on redox state. The thiol reductant mercaptopropionylglycine prevented mitoK ATP activity, whereas exogenous ROS activated the channel. In addition, the use of mitochon- drial substrates that led to higher levels of endogenous mitochondrial ROS release closely correlated with enhanced K 1 transport activity through mitoK ATP . Alto- gether, our results indicate that brain mitoK ATP is a redox-sensitive channel that controls mitochondrial ROS release. V V C 2008 Wiley-Liss, Inc. Key words: brain mitochondria; ATP-sensitive K 1 channel; reactive oxygen species; excitotoxicity; calcium As organelles that present inner membrane poten- tials (DC), mitochondria must exhibit highly controlled ion transport. In particular, transport of K 1 , the main intracellular cation, is a richly regulated process. K 1 ions constantly leak across the inner membrane in a manner stimulated by DC. To compensate for this leak, mito- chondria possess a K 1 /H 1 exchanger, which is activated by increases in matrix volumes promoted by K 1 trans- port and removes this cation in exchange for protons. This exchange activity prevents excessive volume increases resulting from K 1 uptake under high DC con- ditions, which could lead to large-amplitude mitochon- drial swelling and membrane rupture (for review see Garlid et al., 2003). In addition to the K 1 leak, mitochondria present a regulated pathway for K 1 uptake: ATP-sensitive K 1 channels (mitoK ATP ), located in their inner membrane (Garlid et al., 2003; Facundo et al., 2006b). These chan- nels promote controlled entry of K 1 ions into the ma- trix, at rates sufficient to increase matrix volumes but not to rupture the organelle (Kowaltowski et al., 2001). Transport of K 1 through mitoK ATP is physiologically regulated by inhibitors such as ATP and ADP, whereas UDP, GTP, and GDP act as intracellular activators (Paucek et al., 1996; Mironova et al., 1999, 2004). A wealth of pharmacological mitoK ATP agonists and antag- onists also exists. Diazoxide (DZX) and 5-hydroxydeca- noate (5-HD) are particularly useful as agonist and antag- onist, respectively, of this channel because of their selec- tivity toward mitochondrial, and not plasma membrane, ATP-sensitive K 1 channels (Garlid et al., 1997; Jaburek et al., 1998). The use of selective agonists and antagonists of mitoK ATP has identified opening of this channel as a protec- tive measure during ischemia/reperfusion in many tissues, including heart (Garlid et al., 1997; Facundo et al., 2006b), skeletal muscle (Grover et al., 2003), and brain (Liu et al., 2002; Shimizu et al., 2002). The mechanism through which protection occurs has been studied more thoroughly in heart (Garlid et al., 2003) and is related to changes in mitochondrial redox state (Facundo et al., 2006b). Heart mitoK ATP opening, which is stimulated by mitochondrially generated ROS (Zhang et al., 2001; Facundo et al., 2007), decreases mitochondrial ROS release during reperfusion (Vanden Hoek et al., 2000; Facundo et al., 2006a). The decrease in ROS release is Contract grant sponsor: National Institutes of Health; Contract grant number: HD047388-01A1; Contract grant sponsor: Fundac ¸a ˜o de Amparo a ` Pesquisa no Estado de Sa ˜o Paulo (FAPESP); Contract grant sponsor: Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´gico; Con- tract grant sponsor: Instituto do Mile ˆnio Redoxoma; Contract grant sponsor: John Simon Guggenheim Memorial Foundation. *Correspondence to: Alicia J. Kowaltowski, Av. Prof. Lineu Prestes, 748, Cidade Universita ´ria, 05508-900 Sa ˜o Paulo, Brazil. E-mail: alicia@iq.usp.br Received 28 August 2007; Revised 26 October 2007; Accepted 29 October 2007 Published online 11 January 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jnr.21614 Journal of Neuroscience Research 86:1548–1556 (2008) ' 2008 Wiley-Liss, Inc.