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Role of an Intersubunit Ca2+ Bridge in BK Channel Gating
Monday, February 13, 2017 1242-Pos Board B310 Role of an Intersubunit Ca2D Bridge in BK Channel Gating Alexandre G. Vouga, Brad Rothberg. Biochemistry, Temple University, Philadelphia, PA, USA. Large conductance Ca2þ-activated Kþ (BK) channels are expressed in a wide variety of cell types and serve a range of functions, including control of neuronal action potential duration and smooth muscle contractility. Ca2þ-dependent opening of BK channels is mediated by conformational changes of the ‘‘gating ring,’’ formed by the C-terminal domains (CTD) of its four pore-forming subunits, each comprised of two regulators of conductance of Kþ (RCK) domains, RCK1 and RCK2. Residues D895-D897 comprise a well-established high-affinity Ca2þ binding site within RCK2 known as the ‘‘Ca2þ bowl’’, responsible for Ca2þ-activation of BK. The structure of the Ca2þ-bound BK gating ring has suggested that Ca2þ coordination at the Ca2þ bowl may involve formation of a Ca2þ bridge to residue N449 in the RCK1 domain of the adjacent subunit. Here we tested the contribution of N449 to BK channel gating using electrophysiology. In excised inside-out patches from transfected HEK cells, N449A mutant channels displayed voltage-dependent activation in the nominal absence of Ca2þ that was not statistically different from WT (V1/2 at 0 Ca2þ for WT = 159 5 4 mV; N449A= 167 5 3 mV). However, the N449A mutation blunted the shifts in V1/2 in response to 1, 3, and 10 mM Ca2þ (V1/2 at 1 mM Ca2þ: WT = 90 5 6 mV, N449A = 132 5 2 mV; V1/2 at 3 mM Ca2þ: WT = 79 5 1 mV, N449A = 96 5 2 mV; V1/2 at 10 mM Ca2þ: WT = 33 5 3 mV, N449A = 68 5 4 mV). In contrast, the mutation N449D, which preserved favorable electrostatic interactions with Ca2þ, showed no significant differences in Ca2þ responses compared to WT. Together, these results suggest that an intersubunit interaction with the N449 side chain is a key determinant of Ca2þ-activation through the Ca2þ bowl. 1243-Pos Board B311 Probing the Composition of TMEM16A-Containing Signaling Complexes in Sensory Neurons Shihab Shah1, Chase M. Carver2, Mark S. Shapiro2, Nikita Gamper1. 1 Faculty of Biological Sciences, University of Leeds, Leeds, United Kingdom, 2Department of Cellular and Integrative Physiology, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA. TMEM16A (ANO1) is a Ca2þ activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons responding to painful (noxious) stimuli; these neurons also express the noxious heat sensor TRPV1. Our previous findings indicate that in these neurons, TMEM16A is specifically coupled to Ca2þ release from intracellular stores due to close association of TMEM16A channels, G protein coupled receptors, and IP3 receptors in multiprotein complexes assembled at plasma membrane-endoplasmic reticulum (PM-ER) junctions. Like TRPV1, TMEM16A is activated by noxious heat and functional coupling between the two channels has also been shown. We used live confocal imaging of Cl- channel activity, proximity ligation and super-resolution microscopy to investigate physical and functional relationships between TMEM16A and TRPV1 channels, and IP3 receptors in rat dorsal root ganglion (DRG) neurons. (i) Simultaneous monitoring of Cl- channel activity and Ca2þ dynamics in DRG neurons transfected with a halide-sensitive EYFP mutant (H148Q/I152L) and loaded with fura-2 revealed that the TRPV1 ligand, capsaicin, can robustly activate CaCC. However, ER store depletion significantly reduced (but not abolished) the capsaicininduced Ca2þ rises and CaCC activation, suggesting that Ca2þ release from the ER contributes significantly to TRPV1-mediated CaCC activation. (ii) Proximity ligation assays established that in DRG neurons, TMEM16A and TRPV1 is often in close (less than 40 nm) proximity; similarly, close proximity between TMEM16A and IP3R1 was also observed. (iii) Stochastic optical reconstruction (STORM) microscopy confirmed close association of TMEM16A and TRPV1 channels in DRG neurons, representing 21% of the total population of labeled channels. We also observed close proximity of TMEM16A and IP3R1, and between TRPV1 and IP3R1, within 100 nm, although these molecules could be somewhat further apart, compared to membrane-localized TMEM16A/TRPV1 complexes, consistent with the ER membrane localization of IP3R1. Taken together, our data reveal the composition of TMEM16A-containing signaling complexes in DRG neurons and suggest that coupling between TRPV1 and TMEM16A requires ER Ca2þ release, which may be necessary to boost activation of TMEM16A, as this channel has a relatively low Ca2þ affinity. 1244-Pos Board B312 Endothelial Caveolae-TRPV4-SK Channel Interactions in Shear Stress-Mediated Coronary Arteriole Dilation Hon-Chi Lee, Tong Lu, Qiang Chai, Xiao-Li Wang. Internal Medicine, Mayo Clinic, Rochester, MN, USA. We have previously reported that shear stress-mediated dilation (SSD) of coronary arterioles is caveolae-dependent. We found that both TRPV4 and SK channels are targeted to the low-buoyant density caveolin-rich membrane frac-
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tion in freshly isolated bovine coronary endothelial cells (BCEC) and TRPV4 and SK3 channels are co-immunoprecipitated by anti-caveolin-1 antibody. Isoproterenol (1 mM) enhanced the whole-cell Kþ currents in BCEC by 5-fold that are inhibited by apamin (200 nM), an SK channel-specific inhibitor, but not by iberiotoxin (100 nM) or Tram34 (200 nM). Exposure of the BCEC to the TRPV4 activator, GSK1016790A (150 nM) resulted in a 3.5-fold activation of SK currents. Acute exposure of BCEC seeded in a capillary tube to 10 dynes/ cm2 of shear stress (SS) resulted in a 4-fold increase in TRPV4 currents that are inhibited by HC067047 (200 nM), a TRPV4-specific inhibitor, and a 3.5-fold increase in SK currents that are inhibited by apamin (200 nM). However, after incubation with HC067047, a TRPV4 specific inhibitor, SK currents could no longer be activated by SS, suggesting SK activation by SS was mediated through TRPV4. In addition, pre-incubation of isolated coronary arterioles with apamin (200 nM) resulted in a significant diminution of SSD while preincubation with HC067047 (150 nM) results in vasoconstriction by SS. Exposure of BECE to SS (10 dynes/cm2 12 h) enhanced the production of endothelium-derived vasodilators, NO and PGI2, and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated [Ca2þ]i increase in BECE. These novel results indicate that there is a dynamic interaction in the vascular endothelium between caveolae, TRPV4 and SK channels in the modulation of SSD. 1245-Pos Board B313 Statin Therapy in Long QT Syndrome Type II Elsa Ronzier, Xiaorong Xu Parks, Coeli Lopes. University of Rochester, Rochester, NY, USA. Statins are among the most commonly prescribed drug classes for prevention and treatment of coronary artery disease, and their use is expected to increase due to recent changes in therapy guidelines. Although statins have an overall very favorable safety profile in the general population, the effect of this drug class on cardiac electrophysiology, in particular for non-heart-failure patients, has been incompletely studied. Indeed, some reports show statins decreasing repolarizing potassium channel currents in the heart, an effect expected to prolong QTc interval and increase the risk for cardiac arrhythmias. Our data suggest statins may be detrimental to patients with mutations in the KCNH2 gene, increasing their risk of cardiac events. Thus, to understand the molecular mechanism of statins, we tested the effect of these drugs in wild-type and mutant KCNH2 channels both alone and in combination to KCNH2 targeting drugs using Western Blots and Patch Clamp techniques. We found that statins inhibited current, membrane expression and trafficking of KCNH2 channels at pharmacological concentrations. Importantly, KCNH2 is a common drug target, with drugs that inhibit the channel current also showing a protective effect by stabilizing channel protein folding. Our data shows that statin inhibits the increase in membrane expression caused by channel inhibitors, thus may increase their inhibitory effects. Our data suggest that statin may have pro-arrhythmic effects for patients with mutations in the KCNH2 gene and in combination with QT prolonging drugs that target the KNCH2 channel by inhibiting KCNH2 trafficking and function. 1246-Pos Board B314 Regulation of the Human Ether-A-Go-Go-Related Gene (hERG) Potassium Channel by the Ubiquitin Ligase Rififylin (RFFL) Karim Roder, Karni S. Moshal, An Xie, Tae Yun Kim, Kevin R. Murphy, Nil€ufer Nermin, Turan Dural, Yichun Lu, Bum-Rak Choi, Gideon Koren. Medicine, Rhode Island Hospital, Providence, RI, USA. A recent study by Newton-Cheh and colleagues identified an association, with genome-wide significance, between a single nucleotide polymorphism approximately 12 kb downstream of the gene encoding the RING finger ubiquitin ligase rifiylin (RFFL) and the QT interval. We sought to determine the role of RFFL in cardiac electrophysiology. Digital imaging of transduced neonatal rabbit cardiomyocytes demonstrated that RFFL overexpression significantly prolonged action potential duration (APD) by approximately 20% (control: 300 5 5 ms; RFFL: 360 5 5 ms), whereas the RING deletion of RFFL shortened APD significantly (RFFL-DRING: 220 5 6 ms). Additionally, RFFL overepxression resulted in an approximately 50% decrease of total hERG. Using transfections of HEK293 cells, western blot, immunoprecipitation and immunocytochemistry, we demonstrate that RFFL and the hERG potassium channel interact and co-localize. Furthermore, RFFL overexpression lead to increased polyubiquitination and proteasomal degradation of total and membrane hERG protein and a decrease of IKr density by approximately 70%, which was dependent on the intact RING but not FYVE-like domain of RFFL. Taken together, there is strong evidence that RFFL is an important regulator of action potential duration in larger animals likely via effects on hERG channels.
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tion in freshly isolated bovine coronary endothelial cells (BCEC) and TRPV4 and SK3 channels are co-immunoprecipitated by anti-caveolin-1 antibody. Isoproterenol (1 mM) enhanced the whole-cell Kþ currents in BCEC by 5-fold that are inhibited by apamin (200 nM), an SK channel-specific inhibitor, but not by iberiotoxin (100 nM) or Tram34 (200 nM). Exposure of the BCEC to the TRPV4 activator, GSK1016790A (150 nM) resulted in a 3.5-fold activation of SK currents. Acute exposure of BCEC seeded in a capillary tube to 10 dynes/ cm2 of shear stress (SS) resulted in a 4-fold increase in TRPV4 currents that are inhibited by HC067047 (200 nM), a TRPV4-specific inhibitor, and a 3.5-fold increase in SK currents that are inhibited by apamin (200 nM). However, after incubation with HC067047, a TRPV4 specific inhibitor, SK currents could no longer be activated by SS, suggesting SK activation by SS was mediated through TRPV4. In addition, pre-incubation of isolated coronary arterioles with apamin (200 nM) resulted in a significant diminution of SSD while preincubation with HC067047 (150 nM) results in vasoconstriction by SS. Exposure of BECE to SS (10 dynes/cm2 12 h) enhanced the production of endothelium-derived vasodilators, NO and PGI2, and facilitated the translocation of TRPV4 to the caveolae. Inhibition of TRPV4 abolished the SS-mediated [Ca2þ]i increase in BECE. These novel results indicate that there is a dynamic interaction in the vascular endothelium between caveolae, TRPV4 and SK channels in the modulation of SSD. 1245-Pos Board B313 Statin Therapy in Long QT Syndrome Type II Elsa Ronzier, Xiaorong Xu Parks, Coeli Lopes. University of Rochester, Rochester, NY, USA. Statins are among the most commonly prescribed drug classes for prevention and treatment of coronary artery disease, and their use is expected to increase due to recent changes in therapy guidelines. Although statins have an overall very favorable safety profile in the general population, the effect of this drug class on cardiac electrophysiology, in particular for non-heart-failure patients, has been incompletely studied. Indeed, some reports show statins decreasing repolarizing potassium channel currents in the heart, an effect expected to prolong QTc interval and increase the risk for cardiac arrhythmias. Our data suggest statins may be detrimental to patients with mutations in the KCNH2 gene, increasing their risk of cardiac events. Thus, to understand the molecular mechanism of statins, we tested the effect of these drugs in wild-type and mutant KCNH2 channels both alone and in combination to KCNH2 targeting drugs using Western Blots and Patch Clamp techniques. We found that statins inhibited current, membrane expression and trafficking of KCNH2 channels at pharmacological concentrations. Importantly, KCNH2 is a common drug target, with drugs that inhibit the channel current also showing a protective effect by stabilizing channel protein folding. Our data shows that statin inhibits the increase in membrane expression caused by channel inhibitors, thus may increase their inhibitory effects. Our data suggest that statin may have pro-arrhythmic effects for patients with mutations in the KCNH2 gene and in combination with QT prolonging drugs that target the KNCH2 channel by inhibiting KCNH2 trafficking and function. 1246-Pos Board B314 Regulation of the Human Ether-A-Go-Go-Related Gene (hERG) Potassium Channel by the Ubiquitin Ligase Rififylin (RFFL) Karim Roder, Karni S. Moshal, An Xie, Tae Yun Kim, Kevin R. Murphy, Nil€ufer Nermin, Turan Dural, Yichun Lu, Bum-Rak Choi, Gideon Koren. Medicine, Rhode Island Hospital, Providence, RI, USA. A recent study by Newton-Cheh and colleagues identified an association, with genome-wide significance, between a single nucleotide polymorphism approximately 12 kb downstream of the gene encoding the RING finger ubiquitin ligase rifiylin (RFFL) and the QT interval. We sought to determine the role of RFFL in cardiac electrophysiology. Digital imaging of transduced neonatal rabbit cardiomyocytes demonstrated that RFFL overexpression significantly prolonged action potential duration (APD) by approximately 20% (control: 300 5 5 ms; RFFL: 360 5 5 ms), whereas the RING deletion of RFFL shortened APD significantly (RFFL-DRING: 220 5 6 ms). Additionally, RFFL overepxression resulted in an approximately 50% decrease of total hERG. Using transfections of HEK293 cells, western blot, immunoprecipitation and immunocytochemistry, we demonstrate that RFFL and the hERG potassium channel interact and co-localize. Furthermore, RFFL overexpression lead to increased polyubiquitination and proteasomal degradation of total and membrane hERG protein and a decrease of IKr density by approximately 70%, which was dependent on the intact RING but not FYVE-like domain of RFFL. Taken together, there is strong evidence that RFFL is an important regulator of action potential duration in larger animals likely via effects on hERG channels.