Regulation and Roles of the Sulfonylurea Receptor 2-Based KATP Channels in Ischemic PreconditioningBin Ye1, Jie-lin Pu1, Tomoyki Wada1, Jason Sims2, Stacie Kroboth1, Douglas Stoller1, Elizabeth McNally3, Jonathan Makielski1, Nian-Qing Shi1* nqsmedicine.wisc.edu1Department of Medicine, University of Wisconsin, Madison, WI USA2Department of Pharmacology, University of Wisconsin, Madison, WI USA3Department of Medicine, The University of Chicago, Chicago, IL USA The Cellular and Molecular Arrhythmia Research ProgramIon Channels Play Pivotal Roles in Cardiac Functions and Protection Situate on plasma membrane Contain multiple transmembrane helixes Opening of ion channels is within milliseconds Impact the electrophysiological state of the cell Mutations in ion channels are linked to many cardiovascular diseases (i.e. Long QT syndrome) Noma (1983): Sarcolemmal KATP Inoue et al. (1991): Mitochondrial KATPK+MitochondrionK+K+Plasma membraneKATP channels open in ischemia like metabolic sensors of the cellCardiac ATP-sensitive Potassium Channels (KATP)Reviewed by Shi, Ye, Makielski (2005) JMCC 39:51N1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17CWalker A Walker BSulfonylurea Receptor(ATP-binding cassette protein)SUR2A (Major cardiac splice variant) SUR2B (Major smooth muscle splice variant)175 kDaRKRM1 M2P loopNCRKRPore (K+ inward rectifier)Kir6.248 kDaStructure of Cardiac KATP ChannelsTMD0 TMD1 TMD2 Ischemia is a disease condition where the heart has insufficient levels of oxygen and blood supply Acute myocardial infarction is a life-threatening event that can causesudden death and heart failure Blocking KATP channels during ischemia abolishes cardioprotection(Gross & Auchampach, 1992) The opening of KATP can re-polarize the cell membrane, reduce calcium entry and decrease ATP consumption MitoKATP is more likely to confercardioprotection (Garlid et al., 1996 & 1997) but its molecular nature is not completely understoodIschemia and KATP ChannelsmitoKATPReviewed by Gross et al., 2003 IPC is a natural phenomenon where brief periods of ischemiaprovide in vivo protection from subsequent lethal ischemia that can cause infarction Acute IPC: where the protective period lasts for few hours after the preconditioning stimulusDelayed IPC (SWOP): where the protective period can last for 24-72 h after the preconditioning stimulusIschemic Preconditioning (IPC)Reviewed by Yellon & Downey, 2003Stimulus-Memory-ProtectionTo evaluate the responses of SUR2 mutant mice in ischemia and IPC To understand the regulation and roles of SUR2 variants in IPCPurpose of This StudyHypothesis:Losing the regulatory subunit of cardiac KATP leads to reduced cardiac protectionChutkow, Pu, Wada, Makielski, Burant, McNally. PNAS (2001) 98:11760 Kakkar, Ye, Shi, Makielski, McNally et al., Cir Res (2006) 98:675SUR2 Knockout Mutant MiceExon12-16Glibenclamide (a KATP blocker) action sitesThe Glibenclamide-Sensitive KATP Current is Absent in the Mutant Myocytes and VSM CellsWTMutantChutkow et al (2001) PNAS 98:11760010203040506070809010033%74% of Patches Containing IKATPA Glibenclamide-Insensitive KATP Current (IKATPn) is recorded in KO miceKO(n=15)01020304050607080Amplitude (pA)5410.012.35.4WT(n=19)0 pA0 pAPu*, Ye*, McNally, Makielski, Shi (2008) JMCC 44:188 1 s2 pASUR2 mutantIKATPn is More Sensitive to ATPKinetics of IKATPn Differ from the Conventional IKATPMean burst duration (ms)WT SUR2 Mutant 44.29.520.61.8WT SUR2 Mutant pSWT SUR2 Mutant PO23.71.123.11.20.540.13 0.490.12Duration Time (ms)Total number of burstsWTSUR2 MutantIKATPn is Glibenclamide-InsensitiveSummary of Part I The conventional glibenclamide-sensitive KATP activity is absent in SUR2 mutant mice A novel ATP-sensitive, glibenclamide-insensitive KATP activity is recorded in SUR2 mutant mice This IKATPn has distinct channel kinetics, ATP sensitivity, and pharmacological properties from the conventional IKATPNC1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17* *BNJ-1T1BNJ-UBNJ-2BNJ-39 (2A-specific)BNJ-40 (2B-specific)New SUR2 AntibodiesTMD0 TMD1 TMD2SUR1NBD1NBD2NBD1Walker A Walker BBNJ-17BNJ-14155kDa220110Na/K VDAC1 COXIV ATPaseNav1.5 HCN4150T1 BNJ-2 BNJ-39 BNJ-40Cut off at 100-kDa6828kDaBNJ-39BNJ-40T1 BNJ-2 BNJ-39 BNJ-406828Cut off at 100-kDakDa150WTSUR2 MutantNovel SUR2 Short Variants are Detected in Cardiac Sarcolemmal Fractions1406828Na/K VDAC1 COXIV ATPase1730kDa110Na/K VDAC1 COXIV ATPase1730kDa1102855kDa68Heart Brain 120BNJ-2 BNJ-39 BNJ-4055BNJ-2 BNJ-39 BNJ-40kDaNovel SUR2 Short Variants are Detected in Mitochondrial FractionsYe, Kroboth, Wada, McNally, Makielski, Shi. 2008Summary of Part II A panel of new SUR2 antibodies are generated with testedspecificity against SUR2 splice variants