Small Molecule Modulation of Voltage-Gated Ion Channels

Monday, February 9, 2015 Symposium: Probing Ion Channel Structure/ Function Using Novel Tools 889-Symp Conformational Changes in Voltage-Sensing Domains: Concerted Simula- tion and Scattering Studies Douglas J. Tobias. Chemistry, University California, Irvine, Irvine, CA, USA. Electrical signals in excitable tissues such as nerves and muscles are generated by the synchronized opening and closing of ion channels in cell membranes, which generate transient transmembrane gradients in the concentrations of sodium and potassium ions. These channels open and close in response to local changes in transmembrane potential as the signal propagates, and it is well established that their voltage sensitivity is conferred by charged structural ele- ments, referred to as voltage-sensing domains. The details of how conforma- tional changes in the voltage-sensing domains lead to opening/closing of the ion-conducting pores of the channels are still being worked out. Crystal struc- tures of the open states of voltage-gated potassium channels are available, but the structure of the closed/resting state has not been determined to high resolu- tion experimentally. In this talk I will report our efforts to generate a model of the resting state of the archaeal KvAP potassium channel based on atomistic molecular dynamics (MD) simulations in explicit membrane environments, with restraints derived from experimental functional data. I will also present re- sults from simulation studies of an isolated voltage-sensing domain in a hydrat- ed membrane, which include validation by neutron diffraction measurements, and direct observations of elementary gating charge displacement events during a 30-microsecond MD simulation under applied transmembrane potential. Finally, I will discuss prospects for and present preliminary results on using x-ray and neutron interferometry measurements, carried out by our collabora- tors, to validate and refine simulation-based models of the resting state and the voltage-sensing mechanism. 890-Symp Tricking Out the Toolbox: Use of Genetic Code Expansion for the Study of Ion Channels Chris Ahern. Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, USA. The application of nonsense suppression strategies to membrane proteins has great potential for the study and manipulation of ion channels and receptors. The technique relies on the incorporation of a stop codon that is subsequently ‘suppressed’ by an orthogonal misacylated tRNA. Ion channels in particular are especially amenable to these approaches due to the built-in signal gain from the high conductance of recorded ions. On one end of the scale of possibility, ‘small mutagenesis’ provides the means for single atom replacement within an amino acid or sub-atomic redistribution of pi electrons of aromatic side- chains. Such approaches provide an atomic resolution functional correlates to match high-resolution structural data, and have been key in decoding the inter- action energetics of ligands, therapeutics, backbone contributions, salt-bridges and H-bonds in gating of voltage- and ligand-gated channels and receptors. More unconventional side-chains, such as light activated cross-linkers, hold promise for their ability to characterize transient noncovalent protein contacts. Lastly, encoded amino acid fluorophores, while highly valued, have been tech- nically resistant to ion channel application and largely remain on the experi- mental fringe. Data will be presented on the use of genetic code expansion as a tool to reveal the basis for inactivation mechanisms employed by voltage-gated sodium and potassium channels. 891-Symp Small Molecule Modulation of Voltage-Gated Ion Channels Heike Wulff 1 , Vladimir Yarov-Yarovoy 2 . Pharmacology, University of California, Davis, Davis, CA, USA, Physiology and Membrane Biology, University of California, Davis, Davis, CA, USA. Pharmacological modulation of voltage-gated-like ion channels offers tremen- dous opportunities for the discovery of new drugs for neurological, cardiovas- cular and metabolic disorders, as well as for immunosuppression and cancer. However, in contrast to other therapeutic targets such as protein kinases, voltage-gated ion channels have not been popular with medicinal chemists in the pharmaceutical industry. One reason for this unpopularity is the absence of co-crystals of medically relevant channels with drug molecules bound, a pre- requisite for structure based drug design. To address this challenge, molecular modeling, which recently made significant advances due to improved compu- tational methods, and the availability of more high-resolution ion channel struc- tures, is increasingly being used to interpret the results of mutational and 177a electrophysiological experiments in structural terms. Using the Kv1.3 blocker PAP-1 and several KCa2/3 channel modulators such as TRAM-34, NS8593, and nifedipine as examples we here demonstrate that small molecule drugs can ‘‘snuggle’’ into every available nook and cranny of voltage-gated ion chan- nels: the central cavity, the side-pockets between the pore-domain and voltage- sensor, the S5-S6 interface or the lateral fenestrations. We will further discuss the recent design of the KCa3.1 selective positive gating modulator SKA-121 (5-methylnaphtho[2,1-d]oxazol-2-amine), a compound that binds at the inter- face of the calmodulin N-lobe and the calmodulin binding domain of KCa3.1. Overall these examples illustrate that voltage-gated ion channels are highly accessible to small molecule drugs and that virtually any domain of these large, multi-subunit proteins can be targeted for the design of channel subtype-specific drugs, a major challenge remaining in the ion channel field. Supported by RO1 GM076063 (to H.W.) and UC Davis startup (to V.Y.). 892-Symp Calcium Channel Engineering Henry M. Colecraft. Physiology and Cellular Biophysics, Columbia University, New York, NY, USA. High-voltage-activated calcium (Ca V ) channels convert electrical signals into Ca 2þ ion influx to trigger biological responses in excitable cells. Ca V channel dysfunction underlies many cardiovascular and neurological diseases, from cardiac arrhythmias to autism. Modulation of Ca V channels by intracellular signaling proteins, Ca 2þ ions and various small molecules is a powerful method to regulate physiology and is therapeutic for myriad disease conditions. I will discuss Ca V channel engineering approaches that we have used to gain insights into the structure-function mechanisms of these vital proteins, to probe their physiological roles, and to develop novel genetically-encoded molecules that modulate their activity. Platform: Calcium Signaling 893-Plat Decreased Polycystin 2 Expression Alters Calcium-Contraction Coupling and Changes Beta-Adrenergic Signaling Pathways Ivana Y. Kuo 1 , Andrea T. Kwaczala 2 , Lily Nguyen 3 , Stuart G. Campbell 2 , Barbara E. Ehrlich 1 . Pharmacology, Yale University, New Haven, CT, USA, 2 Biomedical Engineering, Yale University, New Haven, CT, USA, 3 Cardiology, Yale University, New Haven, CT, USA. Cardiac disorders are the main cause of mortality in autosomal dominant poly- cystic kidney disease (ADPKD). However, how mutated polycystins predis- pose ADPKD patients to cardiac pathologies before development of renal dysfunction is unknown. We investigate the effect of decreased polycystin 2 (PC2) levels, a calcium channel that interacts with the ryanodine receptor, on myocardial function. We hypothesize that heterozygous PC2 mice (Pkd2þ/) undergo cardiac remodeling due to changes in calcium handling, separate to renal complications. We found that Pkd2þ/ cardiomyocytes have altered cal- cium handling, independent of desensitized calcium-contraction coupling. Paradoxically, in Pkd2þ/ mice, PKA phosphorylation of phospholamban was decreased, whereas PKA phosphorylation of troponin I was increased, ex- plaining the decoupling between calcium signaling and contractility. In silico modeling supported this relationship. Echocardiography measurements showed that Pkd2þ/ mice have increased left ventricular ejection fraction after stim- ulation with isoproterenol (ISO), a b-adrenergic receptor (bAR) agonist. Blockers of bAR-1 and 2 inhibited the ISO response in Pkd2þ/ mice, sug- gesting that the dephosphorylated state of phospholamban is primarily by bAR-2 signaling. Importantly, the Pkd2þ/ mice were normotensive and had no evidence of renal cysts. Our results showed that decreased PC2 levels shifted the bAR pathway balance and changed expression of calcium handling proteins, which resulted in altered cardiac contractility. We propose that PC2 levels in the heart directly contribute to cardiac remodeling in ADPKD patients in the absence of renal dysfunction. 894-Plat In vivo Reconstitution of the Mitochondrial Uniporter Erika Kovacs-Bogdan 1 , Yasemin Sancak 1 , Kimberli J. Kamer 1 , Molly Plovanich 1 , Ashwini Jambhekar 1 , Robert J. Huber 2 , Michael A. Myre 2 , Michael D. Blower 1 , Vamsi K. Mootha 1 . Molecular Biology, Massachusetts General Hospital, Boston, MA, USA, Department of Neurology, Massachusetts General Hospital, Boston, MA, USA. The mitochondrial uniporter is a highly selective calcium channel present broadly in eukaryotes, but absent in Saccharomyces cerevisiae. Therefore,