Examining the Role of Phosphorylation on Interactions between the Cardiac Potassium Channel Alpha-Subunits hERG and KVLQT1

Sunday, February 12, 2017 542-Pos Board B307 CaV1.3 (Cacna1d) Gain-of-Function De Novo Missense Mutations are Associated with CNS Disorders Alexandra Pinggera1, Luisa Mackenroth2, Jo¨rg Striessnig1. 1 Pharmacology and Toxicology, University of Innsbruck, Innsbruck, Austria, 2 Institute for Clinical Genetics, Technical University Dresden, Dresden, Germany. Cav1.3 belongs to the family of L-type voltage gated Ca2þ channels. It is involved in many physiological functions, like hearing, sinoatrial node pacemaking and hormone secretion. Moreover, it is expressed postsynaptically in neurons where it shapes neuronal firing, mediates gene transcription, and controls synaptic morphology and pruning. Recently we described two different de novo Cav1.3 gain-of-function missense mutations in two patiens with autism spectrum disorders (ASD) suggesting that they present a strong risk for the disorder. Here we strenghten this hypothesis by reporting the discovery and characterization of a third Cav1.3 de novo missense mutation in a patient with ASD and epilepsy, localized in a highly conserved region within the channel’s activation gate. The mutation was identified by sequencing of the coding exons of 4813 genes associated with known Mendelian disorders. We introduced the mutation into two functionally distinct C-terminal long (Cav1.3L) and short (Cav1.343s) splice variants of the Cav1.3 a1-subunit. We co-expressed wild-type or mutant a1-subunits with auxiliary b3 and a2d-1 subunits in tsA-201 cells and performed whole-cell patch-clamp recordings. The mutation resulted in a pronounced gain-of-function in both splice variants, which, next to other gating changes, was evident by enhanced current densities and a negative shift of steady-state activation and inactivation leading to an increased window current at more negative voltages. Moreover, it significantly reduced Ca2þ-dependent inactivation in both splice variants whereas voltage-dependent inactivation was only affected when introduced into Cav1.343s. The observed gating changes are expected to affect neuronal signaling and excitability. Together with previous findings we identify recurrent Cav1.3 gain-of-function mutations as a strong risk factor for CNS disorders presenting as ASD, with and without epilepsy. Existing L-type Ca2þ channel blockers used as antihypertensives may provide a therapeutic option for such patients. Supported by Austrian Science Fund (FWF F44020, W11010). 543-Pos Board B308 Dual Effect of Palmitate on Voltage-Gated Calcium Channels and Insulin Secretion in Pancreatic Beta Cells of Rats Neivys Garcı´a-Delgado1, Myrian Velasco-Torres1,2, Carmen Sanchez-Soto1, Marcia Hiriart2,3. 1 Inst Fisiologia Celular, Neuroscience Division, Universidad Nacional Autonoma de Mexico, Mexico DF, Mexico, 2Centro de Ciencias de la Complejidad, Universidad Nacional Autonoma de Mexico, Mexico, Mexico, 3 Inst Fisiologia Celular, Neuroscience Division, Universidad Nacional Autonoma de Mexico, Mexico, Mexico. A variety of signaling molecules modify voltage-gated calcium channels activity in pancreatic beta cells, among them are, free fatty acids (FFA). Acute exposure to FFA increases glucose-stimulated insulin secretion, while chronic exposure increases basal insulin secretion, but decreases glucosestimulated (Olofsson et al., 2004; Zhou and Grill, 1994). Preliminary studies in our laboratory showed that pre-incubation with 1 mM palmitate during 48-72 hours reduced barium currents in beta cells. In this work, we analyze the effect of different palmitate concentrations on calcium currents and insulin secretion in beta cells of adult male Wistar rats. Electrophysiological recordings were performed using whole-cell voltage clamp technique. Reverse hemolytic plaque assay measured insulin secretion by isolated beta cells. Pre-incubation with 0.25 and 0.5 mM palmitate during 48-72 h increased the high voltage-activated (HVA) calcium current without affecting the LVA current. An acute 5 min pre-incubation with 0.5 mM palmitate also increased HVA current. In contrast, 24 h pre-incubation with 1 mM decreased both, LVA and HVA currents. A similar effect was observed during an acute incubation. Chronic pre-incubation with 0.25, 0.5 and 1 mM palmitate decreased the percentage of insulin secretory cells, immunoplaque area and insulin secretion index in a glucose-stimulated condition (15.6 mM). Chronic pre-incubation with 0.5 and 1 mM palmitate, also decreased the secretion index at basal glucose (5.6 mM). However, acute pre-incubation with 1 mM palmitate increased the immunoplaque area in 5.6 mM glucose. Together these results show that palmitate has a dual effect, depending on time and concentration on the calcium currents and insulin secretion in beta cells. Partially supported by DGAPA PAPIIT-UNAM Grants: IN213114, M Hiriart; IV100116 to A. Frank and M Hiriart, and IN211416 to M Velasco-Torres.

109a

Voltage-gated K Channels and Mechanisms of Voltage Sensing and Gating I 544-Pos Board B309 Examining the Role of Phosphorylation on Interactions between the Cardiac Potassium Channel Alpha-Subunits hERG and KVLQT1 Medeea C. Popescu1, Louise E.O. Darling2. 1 Biochemistry Program, Wellesley College, Wellesley, MA, USA, 2 Biological Sciences Department and Biochemistry Program, Wellesley College, Wellesley, MA, USA. KvLQT1 and hERG are the voltage-gated Kþ channel a-subunits of the channels which carry the cardiac repolarizing currents IKs and IKr, respectively. These currents function in vivo with some redundancy to maintain appropriate action potential durations (APDs) in cardiomyocytes. As such, protein-protein interactions between hERG and KvLQT1 may be important in normal cardiac electrophysiology, as well as in arrhythmia and sudden cardiac death. Previous phenomenological observations of functional, mutual downregulation between these complementary repolarizing currents in transgenic rabbit models and cell culture have motivated our investigations into interactions between hERG and KvLQT1. These data suggest that a dynamic physical interaction between hERG and KvLQT1 modulates the respective currents. However, the mechanism by which HERG-KvLQT1 interactions are regulated is still poorly understood. Phosphorylation is thought to play a regulatory role in this process: modifying the phosphorylation state of each the proteins has been shown to alter channel kinetics, and both hERG and KvLQT1 are targets of the Ser/Thr protein kinase PKA, activated by elevated intracellular cAMP concentration. Through classic biochemical assays and quantitative FRET approaches, we aim to characterize the effects of phosphorylation in regulating interactions between KvLQT1 and hERG in cellular model systems. We have developed ion channel fusions to fluorescent proteins, which include hERG and KvLQT1 phosphonull and phosphomimetic mutants. We hypothesize that phosphorylation abrogates protein-protein interactions, as suggested by findings that increased cAMP levels leads to decreased hERG-KvLQT1 interaction. This work potentially furthers our understanding of hERG-KvLQT1 interactions and may elucidate mechanisms that underlie many types of arrhythmia as well as characterize novel interactions between two distinct potassium channel families. 545-Pos Board B310 Monitoring Structural Reorganization of Calmodulin in Complex with the C-Terminus of KCNQ Channels Carolina Gomis-Perez1, Eider Nunez-Viadero1, Ganeko BernardoSeisdedos1, Covadonga Malo1, Pilar Areso2, Alvaro Villarroel1. 1 Instituto Biofisika (CSIC, UPV/EHU), Leioa, Spain, 2Dpt. Farmacologı´a (UPV/EHU), Leioa, Spain. Calmodulin (CaM) is an essential component of the non-inactivating voltage-dependent potassium channels conformed by Kv7 subunits, and mediates current suppression upon intracellular calcium elevation. Despite recent atomic-level information on CaM complexed to Kv7 domains, the structural consequences upon calcium binding remains elusive. To obtain insights on the structural changes caused by calcium, we have monitored FRET between the AB module tagged with the blue protein mTFP1 and CaM tagged with the yellow protein Venus using purified recombinant proteins. In addition, FRET has been monitored in CaM/AB complexes in which the AB module was tagged with both fluorescent proteins. Significant changes in energy transfer were observed in the presence of calcium, being more prominent for Kv7.1 than for Kv7.2 subunits. Thus, these data suggest that calcium causes structural changes to different extent on each Kv7 isoform. Financed by grant BFU2015-66910-R from MINECO. 546-Pos Board B311 Novel Insights from Structural Analysis of Interactions of KCNQ KD Channels with Calmodulin Crystal R. Archer1, Akash Bhattacharya1, Benjamin T. Enslow2, Alex B. Taylor1, Dmitri N. Ivanov1, Mark S. Shapiro3. 1 Biochemistry, University of Texas Health Science Center, San Antonio, San Antonio, TX, USA, 2School of Medicine, University of Texas Health Science Center, San Antonio, San Antonio, TX, USA, 3Cellular and Integrative Physiology, University of Texas Health Science Center, San Antonio, San Antonio, TX, USA. Voltage-gated M-type Kþ channels, made by KCNQ subunits, regulate excitability in nerve and muscle. The gating of these channels are modulated by