Nitrate is a primary nutrient for plant growth, but its levels in soil can fluctuate by several orders of magnitude. Previous studies have identified Arabidopsis NRT1.1 as a dual-affinity nitrate transporter, which can take up nitrate over a wide range of concentrations. The mode of action of NRT1.1 is controlled by phosphorylation of a key residue, Thr101. Yet how this posttranslational modification switches the transporter between two affinity states remains unclear. Here we report the crystal structure of unphosphorylated NRT1.1, which reveals an unexpected homodimer in the inward-facing conformation. In this low-affinity state, the Thr101 phosphorylation site is embedded in a pocket immediately adjacent to the dimer interface, linking the phosphorylation status of the transporter to its oligomeric state. Using a cell-based fluorescence resonance energy transfer assay, we show that functional NRT1.1 indeed dimerizes in the cell membrane and the phosphomimetic mutation of Thr101 converts the protein into a monophasic high affinity transporter by structurally decoupling the dimer. Together with analyses of the substrate transport tunnel, our results establish a phosphorylation-controlled dimerization switch that allows NRT1.1 to uptake nitrate with two distinct affinity modes.

A long-sought, and thus far elusive, goal has been to develop drugs to manage diseases of excitability. One such disease that affects millions each year is cardiac arrhythmia, which occurs when electrical impulses in the heart become disordered, sometimes causing sudden death. Pharmacological management of cardiac arrhythmia has failed because it is not possible to predict how drugs that target cardiac ion channels, and have intrinsically complex dynamic interactions with ion channels, will alter the emergent electrical behavior generated in the heart. Here, we applied a computational model, which was informed and validated by experimental data, that defined key measurable parameters necessary to simulate the interaction kinetics of the anti-arrhythmic drugs flecainide and lidocaine with cardiac sodium channels. We then used the model to predict the effects of these drugs on normal human ventricular cellular and tissue electrical activity in the setting of a common arrhythmia trigger, spontaneous ventricular ectopy. The model forecasts the clinically relevant concentrations at which flecainide and lidocaine exacerbate, rather than ameliorate, arrhythmia. Experiments in rabbit hearts and simulations in human ventricles based on magnetic resonance images validated the model predictions. This computational framework initiates the first steps toward development of a virtual drug-screening system that models drug-channel interactions and predicts the effects of drugs on emergent electrical activity in the heart.

The cardiac-delayed rectifier K ؉ current (IKS) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensor domains and the pore, and auxiliary KCNE1 (E1) subunits, required for the characteristic IKS voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1-TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and E1-TM to Cys, coexpressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide-bond formation. Cys-flanking E1-TM readily formed disulfides with Cysflanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 on different subunits of Q1. The salient functional effects of selected cross-links were as follows. A disulfide from E1 K41C to S1 I145C strongly slowed deactivation, and one from E1 L42C to S6 V324C eliminated deactivation. Given that E1-TM is between S1 and S6 and that K41C and L42C are likely to point approximately oppositely, these two cross-links are likely to favor similar axial rotations of E1-TM. In the opposite orientation, a disulfide from E1 K41C to S6 V324C slightly slowed activation, and one from E1 L42C to S1 I145C slightly speeded deactivation. Thus, the first E1 orientation strongly favors the open state, while the approximately opposite orientation favors the closed state.arrhythmias ͉ cardiac repolarization ͉ electrophysiology ͉ atrial fibrillation ͉ S1T he slow, outwardly rectifying K ϩ current (I KS ) is one of two delayed rectifier K ϩ currents critical for repolarization of the heart, particularly during sympathetic nervous system stimulation (1, 2). The I KS channel is composed of four pore-forming KCNQ1 (Q1) subunits and two auxiliary KCNE1 (E1) subunits (3-5). Several human mutations in Q1 and E1 cause variants of long QT syndrome (6), short QT syndrome (7), or atrial fibrillation (8, 9).Although a tetramer of Q1 subunits alone forms a voltagegated channel, only Q1 and E1 together form a channel with the slow activation and deactivation kinetics and the minimal inactivation characteristics of I KS (10, 11). Furthermore, E1 is necessary for sympathetic modulation of I KS (12). How E1 exerts its effect on Q1 is not yet fully understood.There have been a number of conclusions about Q1-E1 interactions in the I KS channel, not all of which are compatible. There is evidence for (13) and against (14, 15) the contribution of E1 to the pore wall and its accessibility from the pore. There is also evidence that E1 interacts with the pore domain, although not necessarily exposed in the pore (16,17), that the E1 TM helix (E1-TM) interacts directly with Q1 S4 helix (18), that E1 modulates Q1 through its C terminus (19-21), and that E1 interacts with the cytoplasmic Q1 S4-S5 linker (22).More recently, a site of possible Q1-E1 interaction was suggested by the association of mutations in Q1 S...

Rationale The antianginal ranolazine blocks the hERG-based current IKr at therapeutic concentrations and causes QT interval prolongation. Thus, ranolazine is contraindicated for patients with preexisting long QT and those with repolarization abnormalities. However, with its preferential targeting of late INa (INaL), patients with disease resulting from increased INaL from inherited defects (e.g. Long QT syndrome type-3, LQT3), or disease induced electrical remodeling (e.g. ischemic heart failure), might be exactly the ones to most benefit from the presumed antiarrhythmic properties of ranolazine. Objective We developed a computational model to predict if therapeutic effects of pharmacological targeting of late INa by ranolazine prevailed over the off-target block of hERG in the setting of inherited LQT3 and heart failure. Methods and Results We developed computational models describing the kinetics the interaction of ranolazine with cardiac Na+ channels in the setting of normal physiology, LQT3 linked ΔKPQ mutation, and heart failure. We then simulated clinically relevant concentrations of ranolazine and predicted the combined effects of Na+ channel and hERG blockade by both the parent compound ranolazine and its active metabolites, which have shown potent blocking effects in the therapeutically relevant range. Our simulations suggest that ranolazine is effective at normalizing arrhythmia triggers in bradycardia-dependent arrhythmias in LQT3 as well tachyarrhythmogenic triggers arising from heart failure-induced remodeling. Conclusions Our model predictions suggest that acute targeting of late INa with ranolazine may be an effective therapeutic strategy in diverse arrhythmia provoking situations that arise from a common pathway of increased pathologic late INa.

Ion channels operate in intact tissues as part of large macromolecular complexes that can include cytoskeletal proteins, scaffolding proteins, signaling molecules, and a litany of other molecules. The proteins that make up these complexes can influence the trafficking, localization, and biophysical properties of the channel. TRIP8b (tetratricopetide repeat-containing Rab8b-interacting protein) is a recently discovered accessory subunit of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that contributes to the substantial dendritic localization of HCN channels in many types of neurons. TRIP8b interacts with the carboxyl-terminal region of HCN channels and regulates their cell-surface expression level and cyclic nucleotide dependence. Here we examine the molecular determinants of TRIP8b binding to HCN2 channels. Using a singlemolecule fluorescence bleaching method, we found that TRIP8b and HCN2 form an obligate 4:4 complex in intact channels. Fluorescence-detection size-exclusion chromatography and fluorescence anisotropy allowed us to confirm that two different domains in the carboxyl-terminal portion of TRIP8b-the tetratricopepide repeat region and the TRIP8b conserved region-interact with two different regions of the HCN carboxyl-terminal region: the carboxyl-terminal three amino acids (SNL) and the cyclic nucleotidebinding domain, respectively. And finally, using X-ray crystallography, we determined the atomic structure of the tetratricopepide region of TRIP8b in complex with a peptide of the carboxy-terminus of HCN2. Together, these experiments begin to uncover the mechanism for TRIP8b binding and regulation of HCN channels.ver the past two decades, it has become apparent that in living tissues ion channels do not function in isolation, but instead exist as macromolecular complexes that include auxiliary subunits, signaling molecules, scaffolding proteins, and cytoskeletal proteins (1-3). These accessory proteins associate with channels and alter their gating, expression levels, localization, and response to signals such as phosphorylation or internal calcium. Until recently, little was known about which proteins may associate with and modulate hyperpolarization-activated cyclic nucleotide-gated (HCN) channels under physiological conditions. HCN channels are members of the superfamily of voltage-gated channels that contain four subunits around a centrally located pore. Unlike most of the members of the family, however, HCN channels open in response to hyperpolarizing voltages (4, 5). In addition to their unique voltage dependence, activation of HCN channels is facilitated by direct binding of cAMP to a cyclic nucleotide-binding domain (CNBD) in the carboxyl-terminal region of each subunit. The CNBD is connected to the pore region via a gating ring formed from the C-linker of each subunit. The binding of cAMP to the CNBD causes a conformational change in the C-linker and potentiation of the pore opening, which leads to a speeding of channel activation and a depolarizing shift in the voltage ...

Summary Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels underlie the cationic Ih current present in many neurons. The direct binding of cAMP to HCN channels increases the rate and extent of channel opening and results in a depolarizing shift in the voltage dependence of activation. TRIP8b is an accessory protein that regulates the cell surface expression and dendritic localization of HCN channels and reduces the cyclic nucleotide dependence of these channels. Here we use electron paramagnetic resonance (EPR) to show that TRIP8b binds to the apo state of the cyclic nucleotide-binding domain (CNBD) of HCN2 channels without changing the overall domain structure. With EPR and nuclear magnetic resonance (NMR), we locate TRIP8b relative to the HCN channel and identify the binding interface on the CNBD. These data provide a structural framework for understanding how TRIP8b regulates the cyclic nucleotide dependence of HCN channels.

The ability to measure the properties of proteins at the single-molecule level offers an unparalleled glimpse into biological systems at the molecular scale. The interpretation of single-molecule time series has often been rooted in statistical mechanics and the theory of Markov processes. While existing analysis methods have been useful, they are not without significant limitations including problems of model selection and parameter nonidentifiability. To address these challenges, we introduce the use of nonparametric Bayesian inference for the analysis of single-molecule time series. These methods provide a flexible way to extract structure from data instead of assuming models beforehand. We demonstrate these methods with applications to several diverse settings in single-molecule biophysics. This approach provides a well-constrained and rigorously grounded method for determining the number of biophysical states underlying single-molecule data.

Background SCN5A encodes the α-subunit (Nav1.5) of the principle Na+ channel in the human heart. Genetic lesions in SCN5A can cause congenital long QT syndrome (LQTS) variant 3 (LQT-3) in adults by disrupting inactivation of the Nav1.5 channel. Pharmacological targeting of mutation-altered Na+ channels has proven promising in developing a gene-specific therapeutic strategy to manage specifically this LQTS variant. SCN5A mutations that cause similar channel dysfunction may also contribute to sudden infant death syndrome (SIDS) and other arrhythmias in newborns, but the prevalence, impact, and therapeutic management of SCN5A mutations may be distinct in infants compared with adults.Methods and ResultsHere, in a multidisciplinary approach, we report a de novo SCN5A mutation (F1473C) discovered in a newborn presenting with extreme QT prolongation and differential responses to the Na+ channel blockers flecainide and mexiletine. Our goal was to determine the Na+ channel phenotype caused by this severe mutation and to determine whether distinct effects of different Na+ channel blockers on mutant channel activity provide a mechanistic understanding of the distinct therapeutic responsiveness of the mutation carrier. Sequence analysis of the proband revealed the novel missense SCN5A mutation (F1473C) and a common variant in KCNH2 (K897T). Patch clamp analysis of HEK 293 cells transiently transfected with wild-type or mutant Na+ channels revealed significant changes in channel biophysics, all contributing to the proband's phenotype as predicted by in silico modeling. Furthermore, subtle differences in drug action were detected in correcting mutant channel activity that, together with both the known genetic background and age of the patient, contribute to the distinct therapeutic responses observed clinically.SignificanceThe results of our study provide further evidence of the grave vulnerability of newborns to Na+ channel defects and suggest that both genetic background and age are particularly important in developing a mutation-specific therapeutic personalized approach to manage disorders in the young.