- Email: [email protected]
T Cell Receptor Clustering - A Mechanism of Signal Transduction
Sunday, February 12, 2017 (palmitoylation-capable and -incapable) with mCherry, (2) Express KChIP2 isoforms in COS-7 cells alone or with GFP-Kv4.3, (3) Track KChIP2a/c distribution and relationship with Kv4.3 with confocal microscopy, (4) Quantify Itof channel function and subcellular distribution with patch clamp and subcellular fractionation/immunoblots, (5) Quantify native KChIP2 and Kv4 proteins and Itof channel function in ventricular myocytes from young and old spontaneously hypertensive rats (SY and SO, 4-6 and 22-24 months respectively; the latter suffers from severe hypertrophy/heart failure). Results: (1) When expressed alone, KChIP2a is strong in vesicles, Golgi and plasma membrane (PM), while KChIP2c is strong in nuclei and weak in PM. Pharmacological inhibition of palmitoylation directs KChIP2a from PM to nuclei, but does not alter nuclei localization of KChIP2c. (2) Kv4.3 coexpression leads to Kv4.3/KChIP2a overlap in Golgi, post-Golgi vesicles and PM. KChIP2c and Kv4.3 overlap on PM. While both KChIP2a and KChIP2c accelerate Kv4.3 recovery from inactivation, KChIP2a but not KChIP2c increases the current amplitude. (3) Relative to SY, Itof density is markedly reduced in epicardial myocytes (Epi VMs) of SO hearts. Kv4.2 and Kv4.3 proteins are modestly or not lowered in Epi VMs. While KChIP2a protein is markedly reduced, KChIP2c protein is significantly increased. Conclusions: Reversible palmitoylation confers dynamic KChIP2a localization between PM and nuclei, while KChIP2c has a more static distribution. Chronic hypertension/hypertrophy differentially impacts the expression of KChIP2a and 2c. 184-Plat Effect of Drugs on Repolarization of IPSCD-Cardiomyocytes Mark Nowak1, Aidan Coon2, Sanjot Singh2, Shimin Wang2, Randall Rasmusson3, G. Bett4. 1 Cytocybernetics, Buffalo, NY, USA, 2SUNY, University at Buffalo, Buffalo, NY, USA, 3Physiology and Biophysics, SUNY, University at Buffalo, Buffalo, NY, USA, 4Obstetrics and Gynecology, SUNY, University at Buffalo, Buffalo, NY, USA. The FDA has proposed a Comprehensive in Vitro Pro-Arrhythmia Assay (CiPA Initiative) to address the clinical potential for cardiotoxicity during pre-clinical drug development in part, this involves the use of human-induced pluripotent adult stem cell-derived cardiac myocytes (hiPSC-CM) as a model system to measure the effects of drugs on the cardiac action potential (AP). hiPSCCMs, however, have little or no IK1, resulting in unstable APs and anomalous behavior in the presence of some drugs. We stabilized APs from hiPSC-CMs through the electronic addition of IK1 via dynamic clamp. hiPSC-CMs (iCell, Inc., WI) were cultured according to manufacturer’s instructions, and studied using the Amphotericin B perforated patch technique. Cardiac APs (1.52.0 nA stimulus pulse for 1.5 ms, 0.5 Hz) were measured in the absence and presence of increasing drug concentrations. As expected, quinidine, dofetilide, cisapride and sotalol (known IKR blockers) caused dose-dependent increases in APD. In 10 mM quinidine APD90 increased 144512%; 1420 nM dofetilide increased APD90 12853%; 103 nM cisapride increased, APD90 17056%; 324 mM sotalol increased APD90 139514%). Further, qualitative differences in potentiation were observed in that cisapride and sotalol, but not dofetilide, caused a pronounced flattening in Phase 2 of the AP. Quinidine (10 mM) also decreased dV/dtmax by 68518%, which is consistent with Naþ channel block. Nifedipine (300nM), a Ca2þ channel blocker, significantly decreased APD90 by 3551%. The ability to measure stable cardiac APs by the electronic addition of IK1 allows for more accurate comprehensive evaluation of drug effects on the ion channel components comprising the cardiac AP, particularly in the case of sodium channel block, and may aid in the identification of pro-arrhythmic changes in AP morphology.
Symposium: Single-Molecule Membrane Protein Dynamics 185-Symp Submillisecond Dynamics of the NMDA Receptor Hugo Sanabria. Clemson University, Central, SC, USA. Observation of single molecules has impacted the way we look at biomolecular machines. Particularly, because it gave us a tool to understand how biomachines move as they carry out specific functions. Single molecule fluorescence experiments, done in surface immobilized conditions or freely diffusing, take advantage of the fact that fluorescence occurs in the nanosecond timescale to map a wide range of biologically relevant dynamics, covering over 10 orders of magnitude in time without gaps. Single molecule Fo¨rster Resonance Energy Transfer (smFRET) experiments have evolved to the point where it is possible to work with complex systems including membrane proteins. However, many challenges have limited the impact on the field, mostly because there is the common conception that the obtained
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structural information is very limited. In addition, multiple experimental artifacts can complicate the interpretation of data. However, recent advances in smFRET experiments, particularly using Multiparameter Fluorescence Detection (MFD), have improved its accuracy and precision, to the point that it is possible to overcome this mindset. As an example, we use MFD to identify low populated conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptors, quantify molecular dynamics in the sub millisecond regime and identify how conformational dynamics sheds light into the mechanism of partial agonism in the GluN1 subunit of the NMDA receptor. 186-Symp Measuring the Free Energy of ClC-ec1 Dimerization in Membranes using Single Molecule Photobleaching Analysis Janice L. Robertson. Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, USA. Why do greasy membrane proteins interact with their greasy protein partners instead of the similarly greasy lipid bilayer? This is a question at the root of membrane protein oligomerization and folding in membranes. Recently, we turned to the homodimeric ClC-ec1 Cl-/Hþ antiporter, in order to develop a new model system that can be used to investigate this question. The ClC-ec1 ˚ 2) and lined by ~20 non-polar residues, dimerization interface is large (1200 A forming an interaction surface exhibiting high shape complementarity. Previous studies showed that ClC-ec1 is folded and functional in both monomeric and dimeric states. Using a single-molecule microscopy approach, we measured the photobleaching distributions across a wide range of subunit/lipid dilutions, with the lowest representing a sub-biological condition of 1 subunit per 40 E. coli membranes. The data shows that wild-type ClC-ec1 follows a monomer to dimer reaction that is reversible and fits to an equilibrium isotherm, allowing for determination of the dimerization free energy in 2:1 POPE/POPG lipid bilayers (Chadda et al., eLife 2016). Next, two approaches were used to investigate how residues at the interaction interface influence the free energy of dimerization. First, bulky TRP residues were introduced resulting in a destabilization of the dimer by 2 kcal/mole per TRP. Second, subtractive substitutions to ALA were constructed introducing cavities at the interface. While most of the ALA substitutions appear to have no effect, L194A destabilizes the dimer by 2 kcal/mole in lipid bilayers while maintaining both fold and function. These studies present ClC-ec1 as a robust model system for probing the physical forces driving protein association in membranes. 187-Symp T Cell Receptor Clustering - A Mechanism of Signal Transduction Katharina Gaus. Centre for Vascular Research, University of New South Wales, Sydney, Australia. Antigen recognition by the T cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. We use single-molecule localization microscopy and novel analysis to quantify the organization of TCRCD3 complexes into nanoscale clusters and to distinguish between triggered and non-triggered TCR-CD3 complexes. For example, we found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. In summary, we propose a model in whcih TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex and the formation of dense signaling-competent clusters is a process of antigen discrimination.
Symposium: Mechanotransduction to Physiology 188-Symp The Mechanism of Activation of Piezo Ion Channels Jorg Grandl. Duke University, Durham, NC, USA. Piezo ion channels mediate the conversion of mechanical touch into electrical signals and are critical for the organism’s responsiveness to mechanical forces. How Piezos detect mechanical stimuli is unknown. The apparent mechanical sensitivity of Piezo1 varies substantially across cellular environments, stimulating methods and protocols, raising the fundamental questions of what precise physical stimulus activates the channel and
Symposium: Single-Molecule Membrane Protein Dynamics 185-Symp Submillisecond Dynamics of the NMDA Receptor Hugo Sanabria. Clemson University, Central, SC, USA. Observation of single molecules has impacted the way we look at biomolecular machines. Particularly, because it gave us a tool to understand how biomachines move as they carry out specific functions. Single molecule fluorescence experiments, done in surface immobilized conditions or freely diffusing, take advantage of the fact that fluorescence occurs in the nanosecond timescale to map a wide range of biologically relevant dynamics, covering over 10 orders of magnitude in time without gaps. Single molecule Fo¨rster Resonance Energy Transfer (smFRET) experiments have evolved to the point where it is possible to work with complex systems including membrane proteins. However, many challenges have limited the impact on the field, mostly because there is the common conception that the obtained
37a
structural information is very limited. In addition, multiple experimental artifacts can complicate the interpretation of data. However, recent advances in smFRET experiments, particularly using Multiparameter Fluorescence Detection (MFD), have improved its accuracy and precision, to the point that it is possible to overcome this mindset. As an example, we use MFD to identify low populated conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptors, quantify molecular dynamics in the sub millisecond regime and identify how conformational dynamics sheds light into the mechanism of partial agonism in the GluN1 subunit of the NMDA receptor. 186-Symp Measuring the Free Energy of ClC-ec1 Dimerization in Membranes using Single Molecule Photobleaching Analysis Janice L. Robertson. Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, USA. Why do greasy membrane proteins interact with their greasy protein partners instead of the similarly greasy lipid bilayer? This is a question at the root of membrane protein oligomerization and folding in membranes. Recently, we turned to the homodimeric ClC-ec1 Cl-/Hþ antiporter, in order to develop a new model system that can be used to investigate this question. The ClC-ec1 ˚ 2) and lined by ~20 non-polar residues, dimerization interface is large (1200 A forming an interaction surface exhibiting high shape complementarity. Previous studies showed that ClC-ec1 is folded and functional in both monomeric and dimeric states. Using a single-molecule microscopy approach, we measured the photobleaching distributions across a wide range of subunit/lipid dilutions, with the lowest representing a sub-biological condition of 1 subunit per 40 E. coli membranes. The data shows that wild-type ClC-ec1 follows a monomer to dimer reaction that is reversible and fits to an equilibrium isotherm, allowing for determination of the dimerization free energy in 2:1 POPE/POPG lipid bilayers (Chadda et al., eLife 2016). Next, two approaches were used to investigate how residues at the interaction interface influence the free energy of dimerization. First, bulky TRP residues were introduced resulting in a destabilization of the dimer by 2 kcal/mole per TRP. Second, subtractive substitutions to ALA were constructed introducing cavities at the interface. While most of the ALA substitutions appear to have no effect, L194A destabilizes the dimer by 2 kcal/mole in lipid bilayers while maintaining both fold and function. These studies present ClC-ec1 as a robust model system for probing the physical forces driving protein association in membranes. 187-Symp T Cell Receptor Clustering - A Mechanism of Signal Transduction Katharina Gaus. Centre for Vascular Research, University of New South Wales, Sydney, Australia. Antigen recognition by the T cell receptor (TCR) is a hallmark of the adaptive immune system. When the TCR engages a peptide bound to the restricting major histocompatibility complex molecule (pMHC), it transmits a signal via the associated CD3 complex. How the extracellular antigen recognition event leads to intracellular phosphorylation remains unclear. We use single-molecule localization microscopy and novel analysis to quantify the organization of TCRCD3 complexes into nanoscale clusters and to distinguish between triggered and non-triggered TCR-CD3 complexes. For example, we found that only TCR-CD3 complexes in dense clusters were phosphorylated and associated with downstream signaling proteins, demonstrating that the molecular density within clusters dictates signal initiation. Both pMHC dose and TCR-pMHC affinity determined the density of TCR-CD3 clusters, which scaled with overall phosphorylation levels. In summary, we propose a model in whcih TCR-CD3 clustering translates antigen recognition by the TCR into signal initiation by the CD3 complex and the formation of dense signaling-competent clusters is a process of antigen discrimination.
Symposium: Mechanotransduction to Physiology 188-Symp The Mechanism of Activation of Piezo Ion Channels Jorg Grandl. Duke University, Durham, NC, USA. Piezo ion channels mediate the conversion of mechanical touch into electrical signals and are critical for the organism’s responsiveness to mechanical forces. How Piezos detect mechanical stimuli is unknown. The apparent mechanical sensitivity of Piezo1 varies substantially across cellular environments, stimulating methods and protocols, raising the fundamental questions of what precise physical stimulus activates the channel and