Feb 12, 2017 - Panpan Hou, Ling Zhong, Powei Kang, Zachary Beller, Kelli McFarland,. Jingyi Shi, Jianmin ... Hui Huang, Keenan Taylor, Charles Sanders.
Sunday, February 12, 2017 fatty acid analogues, which may inspire development of future KV7.1/KCNE1 channel activators to treat congenital Long QT syndrome caused by mutations in KV7.1/KCNE1. 551-Pos Board B316 ML277 Opens KCNQ1 Channels by Selectively Enhancing the AO State Panpan Hou, Ling Zhong, Powei Kang, Zachary Beller, Kelli McFarland, Jingyi Shi, Jianmin Cui. Biomedical engineering, Washington University in St Louis, St. Louis, MO, USA. The KCNQ1 Kþ channel a subunit and the auxiliary subunit KCNE1 form the IKs channel in the heart that is important in controlling heart rhythm. In response to membrane depolarization, the KCNQ1 channel undergoes intermediate open (IO) and activated open (AO) states that correspond to the stepwise movement of the voltage sensor to the intermediate and full activation. IO and AO states showed different properties in voltage dependence of gating, permeation, and pharmacology. The association of KCNE1 suppresses IO but enhances AO, thereby radically alters properties of the channel to suit the physiological role of IKs in terminating the action potential. In this study, we found that a recently identified KCNQ1 opener, ML277, increased the KCNQ1 current by selectively enhancing the AO state without affecting the IO state. ML277 effects on KCNQ1 channels showed a time and voltage dependence that coincide with that of the AO state. Voltage clamp fluorometry studies revealed that ML277 did not change the voltage sensor movement but enhanced channel opening in the AO state, indicating an enhanced VSDpore coupling selectively in the AO state. Consistently, ML277 was found to increase the sensitivity of channel activation to PIP2, which is required for mediating the VSD-pore coupling as shown in our previous studies. These results suggest that ML277 enhances the VSD-pore coupling specifically when the VSD is at the fully activated state to increase the AO state occupancy, thereby increasing the KCNQ1 current. Given that the IKs channel primarily opens at the AO state, ML277 provides an example of drugs that can modulate IKs channels with a high specificity. ML277 may also be used as an excellent tool to study the molecular mechanisms of how the IO and AO states are determined in KCNQ1 channels. 552-Pos Board B317 Comprehensive Assessment of Disease Mutant Forms of the Human KCNQ1 Potassium Channel Hui Huang, Keenan Taylor, Charles Sanders. Biochemistry, Vanderbilt University, Nashville, TN, USA. The voltage-gated potassium channel KCNQ1 is critical for the cardiac action potential. Mutations in KCNQ1 and its accessory protein KCNE1 are the most common cause of congenital long-QT syndrome (LQTS). There are a variety of mechanisms by which a given mutation may cause KCNQ1 channel dysfunction and prolonged activation potentials. The ideal treatment of patients harboring a KCNQ1 mutation is dependent on which specific mechanisms cause loss of function. In the present study, we have employed a multidisciplinary approach to systematically investigate the specific effects of 51 KCNQ1 mutations on the channel structure, stability, trafficking, and (i.e. through collaboration) electrophysiological properties. The 51 mutations, located in the voltage sensor domain (VSD), are disease causing, benign, or of unknown significance. High quality NMR spectra of the isolated wild type VSD and of its mutant forms that are locked in fully activated state or in resting state serve as reference spectra. The 1H-15N TROSY spectrum of each mutant was collected and compared with the reference spectra to determine whether the mutation destabilizes the protein, or shifts the basal activated vs. resting state equilibrium. We have also expressed each mutant full length KCNQ1 in HEK293 cells and quantitatively assessed its total protein expression and cell surface expression using flow cytometry. These results will help elucidate the exact defects of each mutant associated with LQTS, potentially providing information that can be used to inform personalized treatment of LQTS subjects harboring KCNQ1 mutations. This work was supported by NIH Grant RO1 HL122010. We also thank the lab of Prof. Alfred George at Northwestern University for providing the cDNA for the KCNQ1-VSD mutants. 553-Pos Board B318 A Voltage- to Ligand- Gated Switch in Voltage-Gated Potassium Channels Xiaoping Pi, Qiang Ding, Zhaobing Gao. SIMM, Shanghai, China. To open voltage-gated ion channels (VGIC), the changes of membrane potential, usually depolarization, is prerequisite. In contrast, ligand-gated ion channels (LGIC) open when ligands bind. VGIC often stay in the close state under hyperpolarize membrane potentials. Small chemical ligands have been found to be able to allosterically modulate VGIC,
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but none of them can open VGIC in spite of membrane potential. Here, we report a cysteine is a ‘voltage- to ligand- gated’ switch for KCNQ2 channel. Substitutions with alanine (A) or other smaller volume amino acids in this position endow a ligand-gating to KCNQ2 channel in a measurable range of membrane potential. Under constant 120 mV or even lower membrane potentials, the mutant channels can be opened by small chemical ligands, such as ztz240 and ZnPy . Whereas, the wild type channel kept in close under the identical conditions. Further mechanism investigations revealed that the volumn rather than other properties in this position is essential for the ligand induced opening, inferring that this position may function as a baffle plate that restricts the upward movement of the S4 segment. Our study reports a ligand-gated opening of KCNQ2 channel for the first time. The identification of the ‘voltage- to ligand-gated’ switch in KCNQ2 channels breaks through the traditional barrier between VGIC and LGIC. 554-Pos Board B319 Voltage and Ca2D Sensor Coupling Modulation by b Subunits in the BK Channel Yenisleidy Lorenzo1,2, Karen Castillo1, Gustavo Contreras1, Willy Carrasquel-Ursulaez1, Carlos Gonzalez1, Ramon Latorre1. 1 Centro Interdisciplinario de Neurociencia de Valparaiso, Facultad de Ciencias, Universidad de Valparaiso, Valparaiso, Chile, 2Doctorado en Ciencias Mencio´n Neurociencia, Facultad de Ciencias, Universidad de Valparaiso, Valparaiso, Chile. BK channels are modulated by b-subunits (b1-b4) in a tissue-specific manner. The b1- and b2-subunits increase in the apparent Ca2þ sensitivity of BK can be explained by a stabilization of the voltage sensor domain (VSD) in its active configuration. However, whether b1 modifies the number of charges associated with the voltage sensor activation is still a matter of controversy. The purpose of our study is to determine the effects of the presence of b-subunits on the gating charge in the presence of Ca2þ, and to evaluate the coupling between Ca2þ-binding and VSD activation. The number of gating charges per channel was measured in BK channels formed by a-subunit alone and with the different b-subunits in Ca2þ-free internal solutions. The maximum gating charge displaced was obtained from the charge-voltage (Q-V) curve and the total number of channels in the patch was determined using noise analysis. Furthermore, we evaluated the effect of b-subunits on the interaction between Ca2þ sensors and VSD in different Ca2þ concentrations. We found that the total number of charges per channel was 4.4, 3.0 and 4.2 e0 for BKa, BKa/b1 and BKa/b3b channels respectively. Increasing intracellular [Ca2þ] in BKa (100 mM) promotes a significant leftward shift (~-140 mV) of the Q-V curve. The calcium effect on voltage sensor in BKa becomes apparent at [Ca2þ] R 1 mM (~V 30 mV). However, the leftward shift of the Q-V curve in BKa/b1 channels becomes evident in the nanomolar [Ca2þ] range (100-500 nM). BKa/b3b channels behave as BKa channels. We conclude that: a) b1-subunit not only modifies the resting-active equilibrium of the voltage sensor but also decreases the total number of apparent gating charges; b) there is a strong coupling between voltage and Ca2þ sensors, this coupling is increased in the presence of b1-subunit. 555-Pos Board B320 Alpha-B Helix of RCK1 is a Major Transduction Pathway for Ca2D Activation of BK Channels Yanyan Geng1, Zengqin Deng2, Gonzalo Budelli3, Alice Butler Butler2, Jianmin Cui2, Peng Yuan2, Lawrence Salkoff2, Karl Magleby1. 1 University of Miami, Miami, FL, USA, 2Washington University, St. Louis, MO, USA, 3Brandeis University, Waltham, MA, USA. BK (Slo1) type Kþ selective channels are activated by both depolarization and intracellular Ca2þ. BK channels are comprised of an integralmembrane core consisting of four lateral voltage sensor domains surrounding a central pore-gate domain (S5-S6), and a large cytosolic domain (CTD) assembled from 8 RCK domains. The CTD (gating ring) is attached to the core through four short S6-RCK1 peptide linkers. Ca2þ binding to the CTD activates the channel. Crystal structures (Yuan et al. 2010, 2012) suggest that Ca2þ binding elevates the alpha-B helix in each RCK1 domain where it could push upwards against the core while simultaneously moving a rigid protein arm in RCK1 laterally and downward to pull on S6 through the S6-RCK1 linkers. Ca2þ activation thus may involve a synergistic pushpull mechanism. We examined the contribution of the alpha-B helix to this hypothetical mechanism by introducing several discrete mutations into the alpha-B helix. The mutation L390P in the alpha-B helix of mSlo1 greatly decreased Ca2þ activation. The crystal structure of the hSlo1 CTD with the L390P mutation indicated a discrete alteration of the alpha-B helix
