The overall focus of my laboratory is the investigation of ion channel modulation in the heart. There are two major areas of research currently underway. The first major area of investigation is on identifying a volatile anesthetic interaction site on a cardiac potassium channel, IKs, and characterizing the molecular determinants that modulate anesthetic action on this channel. The cardiac IKs is a major repolarizing current and its dysfunction results in the long QT syndrome, a clinical manifestation that can predispose an individual to fatal arrhythmias. In fact, a majority of the inherited form of LQTS (Type 1) is due to a dysfunction of IKs. Pharmacological inhibition of IKs can also potentially lead to long QT. IKs is very sensitive to inhibition by volatile anesthetics. Yet the molecular mechanism underlying the interaction between the anesthetic agent and IKs is unknown. We have previously reported that a phenylalanine, F340, located in the S6 domain of the channel’s pore-forming α-subunit is critical to the inhibitory action of isoflurane on IKs. Furthermore, some of the identified inherited mutations of IKs linked to LQTS occur in the vicinity of F340. Thus, our goals are to further characterize the molecular interaction between IKs and volatile anesthetics, and to determine the impact of inherited IKs mutations on the channel’s pharmacology. To pursue these goals, the laboratory utilizes a combination of electrophysiological (patch clamp) and cellular/molecular (western blot analysis, immunohistochemistry, site-directed mutagenesis) techniques.
The second major area of research is the investigation of the mechanism underlying cardioprotection by anesthetic-induced preconditioning. We are particularly interested in the role of various ion channels involved in cardioprotection. Our previous work has characterized the interaction of volatile anesthetics with the sarcolemmal ATP-sensitive potassium (KATP) channel, one of the key components in the preconditioning pathway. In addition, we have reported on a persistent acceleration in the inactivation of the L-type calcium channel which can potentially lead to an attenuation of calcium overload. Recently, we have started to direct our focus to the modulation of ion channels of the mitochondria. A critical event in ischemia-reperfusion injury is the opening of the mitochondrial permeability transition pore (mPTP). Evidence suggests that the cardioprotective pathways converge onto the mitochondria and increase the threshold for mPTP opening, ultimately leading to cardioprotection. The mechanism underlying this modulation of mPTP is not known. Phosphorylation of key signal transduction mediators appears to be a necessary step. However, the identities of mitochondrial proteins that are phosphorylated have not been identified. Furthermore, opening of potassium channels on the inner mitochondrial membrane appears to be a key step toward cardioprotection. What triggers the opening and how these potassium channels are regulated are some of the important questions that are yet to be answered. In the quest for understanding the role of mitochondrial ion channels in cardioprotection, we utilize two electrophysiological approaches. One is to incorporate purified mitochondrial proteins into planar lipid bilayers for biophysical characterizations. We have already successfully purified cardiac VDAC (voltage-dependent anion channel on the outer mitochondrial membrane) and ANT (adenine nucleotide translocator on the inner mitochondrial membrane), potential regulators of mPTP, and incorporated them into the lipid bilayer system. Experiments on the impact of phosphorylation on VDAC and ANT are currently underway. The other is to directly patch clamp the mitochondria and, specifically, the mitoplast (mitochondrion with a ruptured outer membrane). Patch clamp experiments are currently underway to characterize the ion channels found on the inner mitochondrial membrane.
Suzuki A, Bosnjak ZJ, Kwok WM. The effects of isoflurane on the cardiac slowly activating delayed-rectifier potassium channel in guinea pig ventricular myocytes. Anesth Analg 96:1308-1315, 2003.
Aizawa K, Turner LA, Weihrauch D, Bosnjak ZJ and Kwok WM. Protein kinase C-ε primes the sarcolemmal KATP channel to modulation by isoflurane. Anesthesiology 101: 381-389: 2004.
Kwok, W.M. and Aizawa, K. Preconditioning of the myocardium by volatile anesthetics. Current Medicinal Chemistry: Cardiovascular and Hematological Agents 2:249-255, 2004.
Turner LA, Fujimoto K, Suzuki A, Stadnicka A, Bosnjak ZJ, Kwok WM. Interaction of isoflurane and PKC activators on sarcolemmal KATP channels. Anesth Analg 100:1680-1686, 2005.
Jiang MT, Nakae Y, Ljubkovic M, Kwok WM, Stowe DF, Bosnjak ZJ. Isoflurane activates human cardiac mitochondrial ATP-sensitive K channels reconstituted in lipid bilayers. Anesth Analg 105(4):926-32, 2007.
Wan TC, Ge ZD, Tampo A, Mio Y, Bienengraeber MW, Tracey WR, Gross GJ, Kwok WM, Auchampach JA. The A3 adenosine receptor agonist CP-532,903 protects against myocardial ischemia/reperfusion injury via the Sarcolemmal ATP sensitive potassium channel. J Pharmacol Exp Ther 324(1):234-43, 2008. [PMCID: PMC2435594]
Kawano T, Zoga V, Kimura M, Liang MY, Wu HE, Gemes G, McCallum JB, Kwok WM, Hogan QH, Sarantopoulos C. Nitric oxide activates ATP-sensitive potassium channels in mammalian sensory neurons: action by direct S-nitrosylation. Mol Pain 5:12, 2009. [PMCID: PMC2673211]
Kawano T, Zoga V, McCallum JB, Wu HE, Gemes G, Liang MY, Abram S, Kwok WM, Hogan QH, Sarantopoulos CD. ATP-sensitive potassium currents in rat primary afferent neurons: biophysical, pharmacological properties, and alterations by painful nerve injury. Neuroscience 162(2):431-43, 2009.
Kawano T, Zoga V, Gemes G, McCallum JB, Wu HE, Pravdic D, Liang MY, Kwok WM, Hogan QH, Sarantopoulos C. Suppressed Ca/CaM/CaMKII activation of KATP channels in primary afferent neurons mediates hyperalgesia after painful nerve injury. PNAS 106(21):8725-30, 2009. [PMCID: PMC2681318]
Tampo A, Hogan C, Sedlic F, Bosnjak ZJ, Kwok WM. Accelerated cardiac L-type calcium channel inactivation triggered by anaesthetic-induced preconditioning. Br J Pharm 156:432-43, 2009. [PMCID: PMC2697672]
Aldakkak M, Stowe DF, Cheng Q, Kwok WM, Camara AK. Mitochondrial matrix K+ flux independent of large conductance Ca2+ activated K+ channel opening. Am J Physiol Cell Physiol. 298(3):C530-41, 2010.