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Background Serum free light chain (sFLC) assays are interpreted using a sFLC-ratio-based reference interval (manufacturer’s interval) that was defined using a cohort of healthy patients. However, renal impairment elevates the sFLC-ratio, leading to a high false positive rate when using the manufacturer’s interval. Prior studies have developed renal-specific reference intervals; however, this approach has not been widely adopted due to practical limitations. Thus, there remains a critical need for a renally robust sFLC interpretation method. Methods Retrospective data mining was used to define patient cohorts that reflect the spectrum of renal function seen in clinical practice. Two new reference intervals, one based on the sFLC-ratio and one based on a novel principal component analysis (PCA)-based metric, were developed for the FREELITE assay (Binding Site) on the Roche Cobas c501 instrument (Roche). Results Compared to the manufacturer’s reference interval, both new methods exhibited significantly lower false positive rates and greater robustness to renal function while maintaining equivalent sensitivity for monoclonal gammopathy (MG) diagnosis. While not significantly different, the point estimate for sensitivity was highest for the PCA-based approach. Conclusion Renally robust sFLC interpretation using a single reference interval is possible given a reference cohort that reflects the variation in renal function observed in practice. Further studies are needed to achieve sufficient power and determine if the novel PCA-based metric offers superior sensitivity for MG diagnosis. These new methods offer the practical advantages of not requiring an estimated glomerular filtration rate result or multiple reference intervals, thereby lowering practical barriers to implementation.

Background Methods of machine learning provide opportunities to use real-world data to solve complex problems. Applications of these methods in laboratory medicine promise to increase diagnostic accuracy and streamline laboratory operations leading to improvement in the quality and efficiency of healthcare delivery. However, machine learning models are vulnerable to learning from undesirable patterns in the data that reflect societal biases. As a result, irresponsible application of machine learning may lead to the perpetuation, or even amplification, of existing disparities in healthcare outcomes. Content In this work, we review what it means for a model to be unfair, discuss the various ways that machine learning models become unfair, and present engineering principles emerging from the field of algorithmic fairness. These materials are presented with a focus on the development of machine learning models in laboratory medicine. Summary We hope that this work will serve to increase awareness, and stimulate further discussion, of this important issue among laboratorians as the field moves forward with the incorporation of machine learning models into laboratory practice.

The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD+ ) and activate immunity. In vitro and in vivo assays were conducted to determine if HopAM1 hydrolyzes NAD+ and if the activity is essential for HopAM1's suppression of plant immunity and contribution to virulence. HPLC and LC-MS were utilized to analyze metabolites produced from NAD+ by HopAM1 in vitro and in both yeast and plants. Agrobacterium-mediated transient expression and in planta inoculation assays were performed to determine HopAM1's intrinsic enzymatic activity and virulence contribution. HopAM1 is catalytically active and hydrolyzes NAD+ to produce nicotinamide and a novel cADPR variant (v2-cADPR). Expression of HopAM1 triggers cell death in yeast and plants dependent on the putative catalytic residue glutamic acid 191 (E191) within the TIR domain. Furthermore, HopAM1's E191 residue is required to suppress both pattern-triggered immunity and effector-triggered immunity and promote P. syringae virulence. HopAM1 manipulates endogenous NAD+ to produce v2-cADPR and promote pathogenesis. This work suggests that HopAM1's TIR domain possesses different catalytic specificity than other TIR domain-containing NAD+ hydrolases and that pathogens exploit this activity to sabotage NAD+ metabolism for immune suppression and virulence.

Cyclic adenosine diphosphate (ADP)–ribose (cADPR) isomers are signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via nicotinamide adenine dinucleotide (oxidized form) (NAD + ) hydrolysis. We show that v-cADPR (2′cADPR) and v2-cADPR (3′cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of 2′cADPR-producing TIR domains reveal conformational changes that lead to an active assembly that resembles those of Toll-like receptor adaptor TIR domains. Mutagenesis reveals a conserved tryptophan that is essential for cyclization. We show that 3′cADPR is an activator of ThsA effector proteins from the bacterial antiphage defense system termed Thoeris and a suppressor of plant immunity when produced by the effector HopAM1. Collectively, our results reveal the molecular basis of cADPR isomer production and establish 3′cADPR in bacteria as an antiviral and plant immunity–suppressing signaling molecule.

Cyclic ADP ribose (cADPR) isomers are important signaling molecules produced by bacterial and plant Toll/interleukin-1 receptor (TIR) domains via NAD+ hydrolysis, yet their chemical structures are unknown. We show that v-cADPR (2’cADPR) and v2-cADPR (3’cADPR) isomers are cyclized by O-glycosidic bond formation between the ribose moieties in ADPR. Structures of v-cADPR (2’cADPR)-producing TIR domains reveal that conformational changes are required for the formation of the active assembly that resembles those of Toll-like receptor adaptor TIR domains, and mutagenesis data demonstrate that a conserved tryptophan is essential for cyclization. We show that v2-cADPR (3’cADPR) is a potent activator of ThsA effector proteins from Thoeris anti-phage defence systems and is responsible for suppression of plant immunity by the effector HopAM1. Collectively, our results define new enzymatic activities of TIR domains, reveal the molecular basis of cADPR isomer production, and establish v2-cADPR (3’cADPR) as an antiviral signaling molecule and an effector-mediated signaling molecule for plant immunity suppression.One-Sentence SummaryThe chemical structures of two O-glycosidic bond-containing cyclic ADP ribose isomers, the molecular basis of their production, and their function in antiviral and plant immunity suppression by bacteria are reported.

Protein domain-based approaches to analyzing sequence data are valuable tools for examining and exploring genomic architecture across genomes of different organisms. Here, we present a complete dataset of domains from the publicly available sequence data of 9,051 reference viral genomes. The data provided contain information such as sequence position and neighboring domains from 30,947 pHMM-identified domains from each reference viral genome. Domains were identified from viral whole-genome sequence using automated profile Hidden Markov Models (pHMM). This study also describes the framework for constructing “domain neighborhoods”, as well as the dataset representing it. These data can be used to examine shared and differing domain architectures across viral genomes, to elucidate potential functional properties of genes, and potentially to classify viruses., Measurement(s)Protein Domain • RNA viral genome • DNA viral genome • protein domain neighborhoods • protein domain clusterTechnology Type(s)digital curation • bioinformatics method • Cluster AnalysisFactor Type(s)Viral GenomeSample Characteristic - OrganismViruses Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12319631

Cellular phenotypes emerge from a hierarchy of molecular interactions: proteins interact to form complexes, pathways, and phenotypes. We show that hierarchical networks of protein interactions can be extracted from the statistical pattern of proteome variation as measured across thousands of bacteria and that these hierarchies reflect the emergence of complex bacterial phenotypes. We describe the mathematics underlying our statistical approach and validate our results through gene-set enrichment analysis and comparison to existing experimentally-derived hierarchical databases. We demonstrate the biological utility of our unbiased hierarchical models by creating a model of motility in Pseudomonas aeruginosa and using it to discover a previously unappreciated genetic effector of twitch-based motility. Overall, our approach, SCALES (Spectral Correlation Analysis of Layered Evolutionary Signals), predicts hierarchies of protein interaction networks describing emergent biological function using only the statistical pattern of bacterial proteome variation.

Receptor proteins sense stimuli and generate downstream signals via sensor and effector domains. Presently, the structural constraints on sensor-effector organization across receptor protein superfamilies are not clear. Here, we perform statistical coupling analysis (SCA) on the transient receptor potential (TRP) and voltage-gated potassium (Kv) ion channel superfamilies to characterize the networks of coevolving residues, or protein sectors, that mediate their receptor functions. Comparisons to structural and functional studies reveal a conserved “core” sector that extends from the pore and mediates effector functions, including pore gating and sensor-pore coupling, while sensors correspond to family-specific “accessory” sectors and localize according to three principles: Sensors (1) may emerge in any region with access to the core, (2) must maintain contact with the core, and (3) must preserve the integrity of the core. This sensor-core architecture may represent a conserved and generalizable paradigm for the structure-function relationships underlying the evolution of receptor proteins.

The Pseudomonas syringae DC3000 type III effector HopAM1 suppresses plant immunity and contains a Toll/interleukin-1 receptor (TIR) domain homologous to immunity-related TIR domains of plant nucleotide-binding leucine-rich repeat receptors that hydrolyze nicotinamide adenine dinucleotide (NAD

Objective Clinicians order laboratory tests in an effort to reduce diagnostic or therapeutic uncertainty. Information theory provides the opportunity to quantify the degree to which a test result is expected to reduce diagnostic uncertainty. We sought to apply information theory toward the evaluation and optimization of a diagnostic test threshold and to determine if the results would differ from those of conventional methodologies. We used a heparin/PF4 immunoassay (PF4 ELISA) as a case study. Materials and Methods The laboratory database was queried for PF4 ELISA and serotonin release assay (SRA) results during the study period, with the latter serving as the gold standard for the disease heparin-induced thrombocytopenia (HIT). The optimized diagnostic threshold of the PF4 ELISA test was compared using conventional versus information theoretic approaches under idealized (pretest probability = 50%) and realistic (pretest probability = 2.4%) testing conditions. Results Under ideal testing conditions, both analyses yielded a similar optimized optical density (OD) threshold of OD > 0.79. Under realistic testing conditions, information theory suggested a higher threshold, OD > 1.5 versus OD > 0.6. Increasing the diagnostic threshold improved the global information value, the value of a positive test and the noise content with only a minute change in the negative test value. Discussion Our information theoretic approach suggested that the current FDA approved cutoff (OD > 0.4) is overly permissive leading to loss of test value and injection of noise into an already complex diagnostic dilemma. Because our approach is purely statistical and takes as input data that are readily accessible in the clinical laboratory it offers a scalable and data-driven strategy for optimizing test value that may be widely applicable in the domain of laboratory medicine. Conclusion Information theory provides more meaningful measures of test value than the widely used accuracy-based metrics.

In voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes sequential activation from the resting state to the intermediate state and activated state to trigger pore opening via electro–mechanical (E–M) coupling. However, the spatial and temporal details underlying E–M coupling remain elusive. Here, utilizing KV7.1’s unique two open states, we report a two-stage E–M coupling mechanism in voltage-dependent gating of KV7.1 as triggered by VSD activations to the intermediate and then activated state. When the S4 segment transitions to the intermediate state, the hand-like C-terminus of the VSD-pore linker (S4-S5L) interacts with the pore in the same subunit. When S4 then proceeds to the fully-activated state, the elbow-like hinge between S4 and S4-S5L engages with the pore of the neighboring subunit to activate conductance. This two-stage hand-and-elbow gating mechanism elucidates distinct tissue-specific modulations, pharmacology, and disease pathogenesis of KV7.1, and likely applies to numerous domain-swapped KV channels., In voltage-gated potassium (KV) channels, the voltage-sensing domain (VSD) undergoes activation states to trigger pore opening via electro–mechanical (E–M) coupling. Here authors show that KV7.1 undergoes a two-stage E–M coupling mechanism during voltage-dependent activation.

Red meat allergy is a tick-associated hypersensitivity reaction to galactose-α-1,3-galactose (α-gal) and is characterized by anaphylaxis, angioedema, urticaria and/or gastrointestinal symptoms occurring 3–6 hours after ingesting red meat such as beef, pork, or lamb. Diagnosis of red meat allergy is challenging due to the unusually long delay in symptom onset and poor sensitivity of skin prick tests with commercial meat extracts. The primary diagnostic tools available for this disease are quantification of α-gal-, beef-, pork-, and/or lamb-specific IgE in serum, however the diagnostic performance of these tests has not been reported. Using patient data for n=135 patients with red meat allergy and n=37 controls, we found that measurement of α-gal-specific IgE using the bovine thyroglobulin (bTG) ImmunoCAP method had the best overall sensitivity (100%) and specificity (92.3%) for diagnosis of red meat allergy. Measuring α-gal-specific IgE using the streptavidin (SA)-CAP technique or beef- or pork-specific IgE using ImmunoCAP were also effective tests with high sensitivities (89–92%) and variable specificities (65–82%). Lamb-specific IgE and total IgE had essentially no diagnostic value for red meat allergy. Positive and negative predictive values mirrored these trends. Taken together, these findings indicate that the α-gal-specific IgE test by bTG ImmunoCAP is the most useful for establishing a diagnosis of red meat allergy, although α-gal-specific IgE by SA-CAP and beef- and pork-specific IgE by ImmunoCAP are also effective tests.

Mechanisms Mark A. Zaydman,†,‡,§ Jonathan R. Silva,†,‡,§ and Jianmin Cui*,†,‡,§ †Department of Biomedical Engineering, Washington University, Saint Louis, Missouri 63130, United States ‡Center for the Investigation of Membrane Excitability Disorders, Washington University, Saint Louis, Missouri 63130, United States Cardiac Bioelectricity and Arrhythmia Center, Washington University, Saint Louis, Missouri 63130, United States

The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1–S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1–R4) can restore current, demonstrating that R1–R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)− could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)+ could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES− but repels MTSET+. We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET+ modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1–R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.

Background— Dysregulation of voltage-gated cardiac Na + channels (Na V 1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the Na V 1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na + current ( I Na ) have not been specified. Our aim was to correlate I Na kinetics with conformational changes within the 4 (DI–DIV) VSDs to define molecular mechanisms of Na V 1.5 modulation. Method and Results— Four Na V 1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry. Each VSD displayed unique kinetics, consistent with distinct roles in determining I Na . In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late I Na . We then used the DII-VSD construct to probe the molecular pathology of 2 Brugada syndrome mutations (A735V and G752R). A735V shifted DII-VSD voltage dependence to depolarized potentials, whereas G752R significantly slowed DII-VSD kinetics. Both mutations slowed I Na activation, although DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate of I Na activation and myocyte excitability. Conclusions— Our results reveal novel mechanisms whereby the Na V 1.5 VSDs regulate channel activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal Brugada syndrome mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications, and antiarrhythmic drugs alter Na V 1.5 at the molecular level.

The IKs current controls action potential duration in the heart, and abnormal function of this current causes cardiac arrhythmias. The IKs current is carried by the voltage activated KCNQ1 potassium channel associated with KCNE1 β-subunits. 18 years of study have shown that KCNE1 drastically modulates every characteristic of KCNQ1, such that it would appear as if KCNQ1 and KCNQ1+ KCNE1 were completely unrelated channels. However, no coherent mechanism has been provided that can explain all these drastic changes, which are essential for the physiological role of IKs. Here we show that KCNE1 alters the state-dependent interactions (coupling) between the voltage-sensing and pore-gate domains of KCNQ1 and that this sole mechanism is sufficient to explain all these changes. Contrary to conventional belief that the voltage-sensing domain must reach the fully-activated state before promoting pore-opening, we found that the KCNQ1 channels can open when the voltage-sensing domain is at intermediate and fully-activated states. Importantly, the intermediate-open and activated-open channels differ in voltage-dependence, ion-permeation, pharmacology and dependence on PIP2, a cofactor for coupling between the voltage-sensing and pore-gate domains. By changing the coupling, KCNE1 prevents the intermediate-open state and changes the properties of the activated-open state, thereby bringing about the characteristics of the IKs current. These results indicate that, during voltage-dependent ion channel gating, every-state of the voltage-sensing domain along its activation pathway is coupled to the conformation of the pore domain through a unique set of protein-protein and protein-lipid interactions. These interactions determine both the open-probability and the open-pore properties.

Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (β) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 β-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore. DOI: http://dx.doi.org/10.7554/eLife.03606.001, eLife digest Cells are surrounded by a membrane that prevents charged molecules from flowing directly into or out of the cell. Instead ions move through channel proteins within the cell membrane. Most ion channel proteins are selective and only allow one or a few types of ion to cross. Ion channels can also be ‘gated’, and have a central pore that can open or close to allow or stop the flow of selected ions. This gating can be affected by the channel sensing changes in conditions, such as changes in the voltage across the cell membrane. Research conducted more than half a century ago—before the discovery of channel proteins—led to a mathematical model of the flow of potassium ions across a membrane in response to changes in voltage. This model made a number of assumptions, many of which are still widely accepted. However, Zaydman et al. have now called into question some of the assumptions of this model. Based on the original model, it has been long assumed that the voltage-sensing domains that open or close the central pore in response to changes in voltage must be fully activated to allow the channel to open. It had also been assumed that the voltage-sensing domains do not affect the flow of ions once the channel is open. Zaydman et al. have now shown that these assumptions are not valid for a specific voltage-gated potassium channel called KCNQ1. Instead, this ion channel opens when its voltage-sensing domains are either partially or fully activated. Zaydman found that the intermediate-open and activated-open states had different preferences for passing various types of ion; therefore, the gating of the channel and the flow of ions through the open channel are both dependent on the state of the voltage-sensing domains. This is in direct contrast to what had previously been assumed. The original model cannot reproduce the gating of KCNQ1, nor can any other established model. Therefore, Zaydman et al. devised a new model to understand how the interactions between different states of the voltage-sensing domains and the pore lead to gating. Zaydman et al. then used their model to address how another protein called KCNE1 is able to alter properties of the KCNQ1 channel. KCNE1 is a protein that is expressed in the heart muscle cell and mutations affecting KCNQ1 or KCNE1 have been associated with potentially fatal heart conditions. Based on the assumptions of the original model, it had been difficult to understand how KCNE1 was able to affect different properties of the KCNQ1 channel. Thus, for nearly 20 years it has been debated whether KCNE1 primarily affects the activation of the voltage-sensing domains or the opening of the pore. Zaydman et al. found instead that KCNE1 alters the interactions between the voltage-sensing domains and the pore, which prevented the intermediate-open state and modified the properties of the activated-open state. This mechanism provides one of the most complete explanations for the action of the KCNE1 protein. DOI: http://dx.doi.org/10.7554/eLife.03606.002

Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein–protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP 2 . This result is due to loss of coupling because PIP 2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP 2 -dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K + channels, suggesting that lipids play an important role in coupling in many ion channels.

Functional eukaryotic Na+ channels are composed of a single monomer with four homologous domains (DI-DIV), each with six transmembrane segments (S1-S6). In each domain, the fourth segment, S4, contains positive charges that transduce changes in voltage into channel gating. Previously, voltage clamp fluorometry (VCF) was applied to track the S4 voltage sensors in the rat muscle sodium channel, rNaV1.4 (Cha et al, Neuron, 1999 22(1):73-87). Our aim was to apply a similar methodology to the human cardiac sodium channel, hNaV1.5, which initiates the cardiac action potential, is the target of numerous anti-arrhythmic drugs and carries >100 mutations that are linked to inherited cardiac diseases. VCF reports on protein motion via an extracellular cysteine that is conjugated with a small fluorescent molecule, in our case tetramethylrhodamine maleimide. Within the extracellular S4 of each NaV1.5 domain, we have identified positions for cysteine substitutions that, when fluorescently tagged, display a voltage-sensitive change in fluorescence. Consistent with motions previously identified in rNaV1.4, and reflecting the role of each domain in gating, initial results show ultra-rapid outward movement of the S4 segments in DI, DII and DIII correlating with activation, but much slower translocation of the DIV S4 that is closer to the time scale of inactivation.

Voltage-activated K(+) (K(V)) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. K(V) channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many K(V) channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the K(V) β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca(2+)- and voltage-activated K(+) (BK) channels. These TM β subunits affect the voltage-dependent activation of K(V) α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD-PGD coupling. These results reveal some common characteristics and mechanistic insights into K(V) channel modulation by TM β subunits.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is necessary for the function of various ion channels. The potassium channel, I Ks , is important for cardiac repolarization and requires PIP 2 to activate. Here we show that the auxiliary subunit of I Ks , KCNE1, increases PIP 2 sensitivity 100-fold over channels formed by the pore-forming KCNQ1 subunits alone, which effectively amplifies current because native PIP 2 levels in the membrane are insufficient to activate all KCNQ1 channels. A juxtamembranous site in the KCNE1 C terminus is a key structural determinant of PIP 2 sensitivity. Long QT syndrome associated mutations of this site lower PIP 2 affinity, resulting in reduced current. Application of exogenous PIP 2 to these mutants restores wild-type channel activity. These results reveal a vital role of PIP 2 for KCNE1 modulation of I Ks channels that may represent a common mechanism of auxiliary subunit modulation of many ion channels.

The channel-forming α subunit of voltage-gated Na+ (NaV) channels contains four domains (DI-DIV) each with six membrane-spanning segments (S1-S6). Voltage-clamp fluorometry (VCF) allows the tracking of the fourth charged segment (S4) in each domain with a site-directed fluorophore, which reports voltage-dependent changes in local protein conformation. We have created four novel channel constructs that enable us to study the voltage sensor movements within each of the domains in the human cardiac voltage-gated Na+ channel, hNaV1.5, in conjunction with the cut-open oocyte technique. Our results with hNaV1.5 show significant differences compared to previous VCF results from the rat skeletal muscle isoform (rNaV1.4). Previously, the rNaV1.4 DIII-S4 was shown to immobilize with kinetics that correlated with fast inactivation. In contrast, we show that hNaV1.5 DIII-S4 rapidly returns to the down-state even when fast inactivation has not yet recovered. Based on this result, we hypothesize that the local anesthetic lidocaine, which was shown to alter the DIII-S4 voltage dependence in rNaV1.4, also has a distinct interaction with hNaV1.5. Supporting this hypothesis, we show no significant change in DIII-S4 voltage-dependence with the application of lidocaine to hNaV1.5. Thus, previous lidocaine-hNaV1.5 results that conflict with rNaV1.4 VCF observations are now reconciled by our observation that the mechanism of interaction with the DIII-S4 differs between the two isoforms. In conclusion, despite high homology, significant functional differences exist between hNav1.5 and rNav1.4, in particular in the links between the DIII-S4, inactivation gating and lidocaine interaction.

Rate adaptation is the physiological shortening of the cardiac action potential duration as heart rate increases. Rate adaptation protects the diastolic interval and maintains electrical stability of the cardiac myocyte. Inherited mutations that decrease the cardiac IKs current predispose patients to arrythmias that manifest during stress or exercise, suggesting that IKs plays a prominent role in limiting action potential duration under conditions where heart rate is fast and beta-adrenergic tone is elevated. Using computational models of IKs, Silva et al. were able to reproduce rate adaptive behavior. In their model, IKs channel can occupy readily recruitable- (shallow) or functionally silent- (deep) closed states at rest. However, the molecular basis of the deep-closed states was unknown, and it was empirically modeled as an additional, slow voltage-sensor transition. In a recent study of Kv7.1, the principal subunit of the IKs channel, we found that binding of the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) is required to couple voltage-sensor activation to pore-opening. Here we study the properties of PIP2 interaction with hIKs channels and describe three phenomena: a large reserve of PIP2 unbound channels that exists in biological membranes, the kinetics of PIP2 binding and unbinding are slow, and the activated-open state has a much greater apparent affinity for PIP2 compared to other gating states. Based on these experimentally observed properties we propose that PIP2 binding is the molecular basis for the mode switching behavior in the model by Silva et al., and it underlies spontaneous adaptation of IKs current to changes in cycle length. We test these hypotheses using kinetic and cellular computational models and experimental protocols simulating fast heart rates.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important regulator of ion channel activity. While PIP2 modulation of voltage-independent Kir channels has been extensively studied, the mechanism of how PIP2 potentiates the activity of voltage-dependent ion channels remains uknown. In this work, we study PIP2 activation of a voltage-dependent potassium channel (Kv7.1). Kv7.1 channels have a canonical Kv structure with a central pore-gate domain (PGD) surrounded by four peripheral voltage-sensing domains (VSDs). Kv channel activation is thought to involve two general steps. First, membrane depolarization is sensed by gating charges in the VSDs resulting in conformational changes within the VSDs from the resting state to the activated state. After all four VSDs have been activated, the second general step in Kv activation involves a concerted motion during which the PGD opens to allow ion permeation. In this study we ask if PIP2 regulates activation of Kv7.1 by potentiating the early VSD conformational changes, the concerted opening of the PGD, or both. Using the voltage-clamp fluorometry (VCF) technique to assay local conformation changes in the VSDs and the PGD simultaneously, we are able to show that although depletion of PIP2 eliminates the ionic current, the early conformational changes in the VSD do not require PIP2. This result indicates that the later conformational changes that couple VSD activation to the opening of the PGD are eliminated by PIP2 depletion. We continue this work by dissecting the molecular details of why PIP2 is required for opening of the PGD in response to VSD activation. These results may provide insights on the common principle of how Kv channels are modulated by PIP2.

The fourth transmembrane segment of Kv channels, S4, contains a series of positively charged residues that imparts voltage sensitivity to the channel. Because the insertion of a highly charged peptide into a hydrophobic lipid environment is energetically unfavorable, electrostatic interactions with countercharges in the protein and phospholipids are required to lower this energy barrier. However, once the protein has been inserted into the membrane, what further role do these interactions play? In functional channels, electrostatic interactions are assumed to stabilize voltage sensor movement from a resting to an activated conformation. Although this assumption is at the crux of many models of voltage dependent gating, experimental evidence specifically examining these interactions in functional channels is incomplete. Here, we demonstrate in Kv7.1 channels that the first glutamate in S2, E160 (E1), form state dependent electrostatic interactions with arginines in S4. We used charged MTS reagents to directly probe the environment around E1 after mutating E1 to cysteine. We found that MTSES- but not MTSET+ modifies E1C, suggesting a positively charged environment around E1. Mutations neutralizing or reversing the charge of the first or fourth arginine in S4 (R1 or R4) change the polarity of the environment around E1C such that MTSET+ modifies E1C in the presence of these secondary mutations. Therefore, R1 and R4 both contribute to the positive electrostatic environment around E1. Moreover, MTSET+ modification of E1C with R1E could only occur at hyperpolarizing voltages but not at depolarizing voltages, suggesting that R1 is proximal to E1 only at the resting state but moves distally at the activated state. Overall, our data is consistent with a mechanism where arginines interact sequentially with E1 as S4 moves from a resting to an activated conformation.

Conserved positively charged arginines in the fourth transmembrane segment (S4) of Kv channels are responsible for imparting voltage sensitivity to the channel. There are several forces that may influence these arginines including the membrane potential and electrostatic interactions with countercharges. In Shaker channels, the first four arginines are the primary gating charges that sense the membrane potential. Kv7.1 has fewer positively charged S4 residues than Shaker, notably with the third arginine in Shaker replaced by a glutamine (Q3). Further loss of charge induced by charge reversal at R1 (R1E) in Kv7.1 results in constitutively activated channels, perhaps due to insufficient charge in S4. Consistent with this idea, introduction of a positive charge at Q3 (Q3R) can restore voltage dependent activation to R1E, suggesting that Q3R may substitute for the loss of gating charge at R1E. In a related study, we have demonstrated in Kv7.1 channels that residues corresponding to the first four arginines in Shaker channels (R1-R4) interact sequentially with the first conserved glutamate in S2 (E1) during gating. Here we show via intragenic suppression that S4 arginines also interact electrostatically with the second conserved glutamate in S2 (E2), and these electrostatic interactions play an important role in voltage sensing of S4. Therefore, a network of electrostatic interactions and the membrane potential act on S4 arginines, and the balance of these forces stabilize the conformation of the voltage sensor at different states. The combination of these interactions acts uniquely on each arginine such that each arginine plays a different role in voltage dependent gating. In Kv7.1, the first two arginines (R1, R2) stabilize the resting state while the last three charged residues (R4, H5, R6) stabilize the activated state.

Coupling between the voltage-sensing domain (VSD) and pore-gate domain (PGD) is required for the voltage-dependent gating of ion channels, but the molecular mechanisms of coupling are unclear. Previous studies have identified protein-protein interactions that are important for coupling, while structural and recent functional data demonstrate that membrane lipids also play a role. In a recent study of Kv7.1, we found that phosphatidylinositol 4,5-bisphosphate (PIP2) binding at the VSD-PD interface is required to couple the activated-state of the VSD to the open-state of the PD. We devised a method to directly measure PIP2 mediated coupling and an allosteric framework for describing such coupling. These advances provide new tools to investigate the mechanisms of VSD-PGD coupling. We also identified a putative PIP2 binding site and found that mutations of residues within this site reduce PIP2-mediated coupling. Paradoxically, a set of mutations near the PIP2 binding site increased the macroscopic current. Using a combined computational and experimental approach to study these gain of function mutations, we now identify a PIP2-interaction that is preferred by the resting-closed channel. Using the KCNE1 accessory subunit as an experimental tool, we are able to resolve the functional effects of this resting-closed state interaction. These results allow us to propose a novel mechanism for voltage-dependent gating in which repositioning of cofactor lipids at the VSD-PD represents a critical step in the transitions between resting-closed and activated-open states. This model can be used to explain the phenomena of cooperativity and concerted motion in voltage-gated channels.

Voltage-gated ion channels are vital for the generation of action potentials that orchestrate various physiological processes. Phosphatidylinositol 4,5-bisphophate (PIP2) is a membrane lipid that modulates several voltage-gated channels (TRP, HCN, CaV, Kv, KCNQ). KCNQ channels are unique in that they cannot be activated by voltage in the absence of PIP2 and the loss of KCNQ channel activity due to PIP2 depletion is known to be physiologically relevant. Although residues governing PIP2 sensitivity have been identified for several KCNQ channels, it is unclear where PIP2 binds and what it does to channel gating. Voltage-dependent gating involves coupled conformational changes in the voltage-sensing domain (VSD) and the pore domain (PD) providing three possible mechanisms of PIP2 action: VSD activation, PD opening, or coupling between VSD activation and PD opening. We combine voltage-clamp fluorometry wih expression of a voltage-sensitive lipid-phosphatase in order to measure VSD activation and PD opening simultaneously while manipulating PIP2 abundance. We find that PIP2 is not necessary for VSD activation; however, PIP2 is required for VSD-PD coupling. Deriving a simple mathematical model, we find that PIP2 regulation of coupling is sufficient to recapitulate the experimental results without any direct effects on PD opening. These findings suggest PIP2 may bind at the VSD-PD interface that, in KCNQ1, is densely populated with basic residues near the intracellular face of the membrane. By mutating these residues and measuring the effects on channel function, PIP2 sensitivity and VSD-PD coupling, we propose a PIP2 binding site. In our resulting model, the negatively charged PIP2 headgroup resolves the electrostatic repulsion between basic residues on the VSD and the PD, thus holding the two domains together and allowing the transfer of conformational energy between them.