cAMP binding to closed pacemaker ion channels is cooperative.

The cooperative action of the subunits in oligomeric receptors enables fine-tuning of receptor activation, as demonstrated for the regulation of voltage-activated HCN pacemaker ion channels by relating cAMP binding to channel activation in ensemble signals. HCN channels generate electric rhythmicity in specialized brain neurons and cardiomyocytes. There is conflicting evidence on whether binding cooperativity does exist independent of channel activation or not, as recently reported for detergent-solubilized receptors positioned in zero-mode waveguides. Here, we show positive cooperativity in ligand binding to closed HCN2 channels in native cell membranes by following the binding of individual fluorescence-labeled cAMP molecules. Kinetic modeling reveals that the affinity of the still empty binding sites rises with increased degree of occupation and that the transition of the channel to a flip state is promoted accordingly. We conclude that ligand binding to the subunits in closed HCN2 channels not pre-activated by voltage is already cooperative. Hence, cooperativity is not causally linked to channel activation by voltage. Our analysis also shows that single-molecule binding measurements at equilibrium can quantify cooperativity in ligand binding to receptors in native membranes.

Fast functional mapping of ligand-gated ion channels.

Ligand-gated ion channels are formed by three to five subunits that control the opening of the pore in a cooperative fashion. We developed a microfluidic chip-based technique for studying ion currents and fluorescence signals in either excised membrane patches or whole cells to measure activation and deactivation kinetics of the channels as well as ligand binding and unbinding when using confocal patch-clamp fluorometry. We show how this approach produces in a few seconds either unidirectional concentration-activation relationships at or near equilibrium and, moreover, respective time courses of activation and deactivation for a large number of freely designed steps of the ligand concentration. The short measuring period strongly minimizes the contribution of disturbing superimposing effects such as run-down phenomena and desensitization effects. To validate gating mechanisms, complex kinetic schemes are quantified without the requirement to have data at equilibrium. The new method has potential for functionally analyzing any ligand-gated ion channel and, beyond, also for other receptors.

Kinetic fingerprinting of metabotropic glutamate receptors.

Dimeric metabotropic glutamate receptors (mGluRs) are abundantly expressed in neurons. In mammals, eight subunit isoforms, mGluR1-8, have been identified, forming the groups I, II, and III. We investigated receptor dimerization and kinetics of these mGluR isoforms in excised membrane patches by FRET and confocal patch-clamp fluorometry. We show that 5 out of 8 homodimeric receptors develop characteristic glutamate-induced on- and off-kinetics, as do 11 out of 28 heterodimers. Glutamate-responsive heterodimers were identified within each group, between groups I and II as well as between groups II and III, but not between groups I and III. The glutamate-responsive heterodimers showed heterogeneous activation and deactivation kinetics. Interestingly, mGluR7, not generating a kinetic response in homodimers, showed fast on-kinetics in mGluR2/7 and mGluR3/7 while off-kinetics retained the speed of mGluR2 or mGluR3 respectively. In conclusion, glutamate-induced conformational changes in heterodimers appear within each group and between groups if one group II subunit is present.

Subunit promotion energies for channel opening in heterotetrameric olfactory CNG channels.

Cyclic nucleotide-gated (CNG) ion channels of olfactory sensory neurons contain three types of homologue subunits, two CNGA2 subunits, one CNGA4 subunit and one CNGB1b subunit. Each subunit carries an intracellular cyclic nucleotide binding domain (CNBD) whose occupation by up to four cyclic nucleotides evokes channel activation. Thereby, the subunits interact in a cooperative fashion. Here we studied 16 concatamers with systematically disabled, but still functional, binding sites and quantified channel activation by systems of intimately coupled state models transferred to 4D hypercubes, thereby exploiting a weak voltage dependence of the channels. We provide the complete landscape of free energies for the complex activation process of heterotetrameric channels, including 32 binding steps, in both the closed and open channel, as well as 16 closed-open isomerizations. The binding steps are specific for the subunits and show pronounced positive cooperativity for the binding of the second and the third ligand. The energetics of the closed-open isomerizations were disassembled to elementary subunit promotion energies for channel opening, [Formula: see text], adding to the free energy of the closed-open isomerization of the empty channel, E0. The [Formula: see text] values are specific for the four subunits and presumably invariant for the specific patterns of liganding. In conclusion, subunit cooperativity is confined to the CNBD whereas the subunit promotion energies for channel opening are independent.

Probing the leak pathway: Live-cell imaging of macromolecule passage through epithelia.

Epithelia compartmentalize multicellular organisms and provide interfacing between the inside and outside. Apart from regulating the exchange of solutes, uptake of nutrients, and excretion of waste products, their major function is to prevent uncontrolled access of foreign material to immune-competent compartments. Progress in understanding this barrier function toward larger solutes and its possible defects, as can be seen in a variety of diseases, is largely hampered by a lack of methods to spatiotemporally resolve transepithelial passage of macromolecules. Using different cell culture epithelia, we applied biotinylated dextran tracers carrying an acceptor fluorophore. These bind to cell-adherent avidin carrying donor fluorophore at the basolateral membranes of single-layered epithelial sheets. Confocal fluorescence microscopy was applied to living epithelia in order to image apical-to-basolateral tracer passage as a Förster resonance energy transfer signal of the fluorescent dextran-avidin pair over time. Stimulated macromolecule passage using barrier-perturbing agents proved its effectiveness for the leak imaging method presented herein. Over hours of imaging, spontaneous leaks were rare, occurring transiently on the scale of minutes and for the most part associated with rearranging cell junctions. The discussed approach to leak imaging is expected to promote the understanding of epithelial barriers, particularly, the nature and dynamics of the epithelial cell leak pathway.

Bayesian inference of kinetic schemes for ion channels by Kalman filtering.

Inferring adequate kinetic schemes for ion channel gating from ensemble currents is a daunting task due to limited information in the data. We address this problem by using a parallelized Bayesian filter to specify hidden Markov models for current and fluorescence data. We demonstrate the flexibility of this algorithm by including different noise distributions. Our generalized Kalman filter outperforms both a classical Kalman filter and a rate equation approach when applied to patch-clamp data exhibiting realistic open-channel noise. The derived generalization also enables inclusion of orthogonal fluorescence data, making unidentifiable parameters identifiable and increasing the accuracy of the parameter estimates by an order of magnitude. By using Bayesian highest credibility volumes, we found that our approach, in contrast to the rate equation approach, yields a realistic uncertainty quantification. Furthermore, the Bayesian filter delivers negligibly biased estimates for a wider range of data quality. For some data sets, it identifies more parameters than the rate equation approach. These results also demonstrate the power of assessing the validity of algorithms by Bayesian credibility volumes in general. Finally, we show that our Bayesian filter is more robust against errors induced by either analog filtering before analog-to-digital conversion or by limited time resolution of fluorescence data than a rate equation approach.

Functional and structural characterization of interactions between opposite subunits in HCN pacemaker channels.

Hyperpolarization-activated and cyclic nucleotide (HCN) modulated channels are tetrameric cation channels. In each of the four subunits, the intracellular cyclic nucleotide-binding domain (CNBD) is coupled to the transmembrane domain via a helical structure, the C-linker. High-resolution channel structures suggest that the C-linker enables functionally relevant interactions with the opposite subunit, which might be critical for coupling the conformational changes in the CNBD to the channel pore. We combined mutagenesis, patch-clamp technique, confocal patch-clamp fluorometry, and molecular dynamics (MD) simulations to show that residue K464 of the C-linker is relevant for stabilizing the closed state of the mHCN2 channel by forming interactions with the opposite subunit. MD simulations revealed that in the K464E channel, a rotation of the intracellular domain relative to the channel pore is induced, which is similar to the cAMP-induced rotation, weakening the autoinhibitory effect of the unoccupied CL-CNBD region. We suggest that this CL-CNBD rotation is considerably involved in activation-induced affinity increase but only indirectly involved in gate modulation. The adopted poses shown herein are in excellent agreement with previous structural results.

A strategy for determining the equilibrium constants for heteromeric ion channels in a complex model.

Ligand-gated ion channels are oligomers containing several binding sites for the ligands. However, the signal transmission from the ligand binding site to the pore has not yet been fully elucidated for any of these channels. In heteromeric channels, the situation is even more complex than in homomeric channels. Using published data for concatamers of heteromeric cyclic nucleotide-gated channels, we show that, on theoretical grounds, multiple functional parameters of the individual subunits can be determined with high precision. The main components of our strategy are (1) the generation of a defined subunit composition by concatenating multiple subunits, (2) the construction of 16 concatameric channels, which differ in systematically permutated binding sites, (3) the determination of respectively differing concentration-activation relationships, and (4) a complex global fit analysis with corresponding intimately coupled Markovian state models. The amount of constraints in this approach is exceedingly high. Furthermore, we propose a stochastic fit analysis with a scaled unitary start vector of identical elements to avoid any bias arising from a specific start vector. Our approach enabled us to determine 23 free parameters, including 4 equilibrium constants for the closed-open isomerizations, 4 disabling factors for the mutations of the different subunits, and 15 virtual equilibrium-association constants in the context of a 4-D hypercube. From the virtual equilibrium-association constants, we could determine 32 equilibrium-association constants of the subunits at different degrees of ligand binding. Our strategy can be generalized and is therefore adaptable to other ion channels.

Thermodynamic profile of mutual subunit control in a heteromeric receptor.

Cyclic nucleotide-gated (CNG) ion channels of olfactory neurons are tetrameric membrane receptors that are composed of two A2 subunits, one A4 subunit, and one B1b subunit. Each subunit carries a cyclic nucleotide-binding domain in the carboxyl terminus, and the channels are activated by the binding of cyclic nucleotides. The mechanism of cooperative channel activation is still elusive. Using a complete set of engineered concatenated olfactory CNG channels, with all combinations of disabled binding sites and fit analyses with systems of allosteric models, the thermodynamics of microscopic cooperativity for ligand binding was subunit- and state-specifically quantified. We show, for the closed channel, that preoccupation of each of the single subunits increases the affinity of each other subunit with a Gibbs free energy (ΔΔG) of ∼-3.5 to ∼-5.5 kJ ⋅ mol-1, depending on the subunit type, with the only exception that a preoccupied opposite A2 subunit has no effect on the other A2 subunit. Preoccupation of two neighbor subunits of a given subunit causes the maximum affinity increase with ΔΔG of ∼-9.6 to ∼-9.9 kJ ⋅ mol-1 Surprisingly, triple preoccupation leads to fewer negative ΔΔG values for a given subunit as compared to double preoccupation. Channel opening increases the affinity of all subunits. The equilibrium constants of closed-open isomerizations systematically increase with progressive liganding. This work demonstrates, on the example of the heterotetrameric olfactory CNG channel, a strategy to derive detailed insights into the specific mutual control of the individual subunits in a multisubunit membrane receptor.

Unravelling the intricate cooperativity of subunit gating in P2X2 ion channels.

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive. Here we kinetically unraveled and quantified subunit activation in P2X2 receptors by an extensive global fit approach with four complex and intimately coupled kinetic schemes to currents obtained from wild type and mutated receptors using ATP and its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP). We show that the steep concentration-activation relationship in wild type channels is caused by a subunit flip reaction with strong positive cooperativity, overbalancing a pronounced negative cooperativity for the three ATP binding steps, that the net probability fluxes in the model generate a marked hysteresis in the activation-deactivation cycle, and that the predicted fATP binding matches the binding measured by fluorescence. Our results shed light into the intricate activation process of P2X channels.

Relating ligand binding to activation gating in P2X2 receptors using a novel fluorescent ATP derivative.

Ionotropic purinergic receptors (P2X receptors) are non-specific cation channels that are activated by the binding of ATP at their extracellular side. P2X receptors contribute to multiple functions, including the generation of pain, inflammation, or synaptic transmission. The channels are trimers and structural information on several of their isoforms is available. In contrast, the cooperation of the subunits in the activation process is poorly understood. We synthesized a novel fluorescent ATP derivative, 2-[DY-547P1]-AET-ATP (fATP) to unravel the complex activation process in P2X2 and mutated P2X2 H319K channels with enhanced apparent affinity by characterizing the relation between ligand binding and activation gating. fATP is a full agonist with respect to ATP that reports the degree of binding by bright fluorescence. For quantifying the binding, a fast automated algorithm was employed on human embryonic kidney cell culture images. The concentrations of half maximum occupancy and activation as well as the respective Hill coefficients were determined. All Hill coefficients exceeded unity, even at an occupancy <10%, suggesting cooperativity of the binding even for the first and second binding step. fATP shows promise for continuative functional studies on other purinergic receptors and, beyond, any other ATP-binding proteins.

Occludin knockdown is not sufficient to induce transepithelial macromolecule passage.

Occludin, a tight junction protein, has been reported to regulate barrier function - particularly the leak pathway for larger solutes - in epithelia. Therefore, we aimed to precisely define its role in macromolecule passage at single cell-cell junctions. A combination of varying occludin expression by transient and stable knockdown including systematic seeding strategies was employed to achieve a broad and defined pattern of variance in occludin expression over epithelia. This variance model enabled us to examine occludin function in the leak pathway using global and local analysis, i.e. to analyze macromolecule flux across epithelia and macromolecule passage at single-cell level. Macromolecular flux was found not to correlate with occludin expression in intestinal epithelial cells. In fact, by spatially resolving macromolecular permeation sites using a recently developed method we uncovered leaky cell junctions at the edge of Transwells resulting in increased passage. This demonstrates that rare leaks can determine net flux of macromolecules across epithelia while the vast majority of cellular junctions do not contribute significantly. Hence, concomitant local analysis of macromolecule passage across epithelial barriers is indispensable for interpretation of global flux data. By combining this new approach with cell culture models of the leak pathway, we can present evidence that lack of occludin is not sufficient to stimulate the epithelial leak pathway.

Novel Fluorescent Cyclic Nucleotide Derivatives to Study CNG and HCN Channel Function.

A highly specific molecular interaction of diffusible ligands with their receptors belongs to the key processes in cellular signaling. Because an appropriate method to monitor the unitary binding events is still missing, most of our present knowledge is based on ensemble signals recorded from a big number of receptors, such as ion currents or fluorescence changes of suitably labeled receptors, and reasoning from these data to the ligand binding. To study the binding process itself, appropriately tagged ligands are required that fully activate the receptors and report the binding at the same time. Herein, we tailored a series of 18 novel fluorescent cyclic nucleotide derivatives by attaching 6 different dyes via different alkyl linkers to the 8-position of the purine ring of cGMP or cAMP. The biological activity was determined in inside-out macropatches containing either homotetrameric (CNGA2), heterotetrameric (CNGA2:CNGA4:CNGB1b), or hyperpolarization-activated cyclic nucleotide-modulated (HCN2) channels. All these novel fluorescent ligands are efficient to activate the channels, and the potency of most of them significantly exceeded that of the natural cyclic nucleotides cGMP or cAMP. Moreover, some of them showed an enhanced brightness when bound to the channels. The best of our derivatives bear great potential to systematically analyze the activation mechanism in CNG and HCN channels, at both the level of ensemble and single-molecule analyses.

Stepwise activation of a class C GPCR begins with millisecond dimer rearrangement.

G protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the "light receptor" rhodopsin has been shown to activate with a rearrangement of the transmembrane (TM) helix bundle within ∼1 ms, while all other receptors are thought to become activated within ∼50 ms to seconds at saturating concentrations. Here, we investigate synchronous stimulation of a dimeric GPCR, the metabotropic glutamate receptor type 1 (mGluR1), by two entirely different methods: (i) UV light-triggered uncaging of glutamate in intact cells or (ii) piezo-driven solution exchange in outside-out patches. Submillisecond FRET recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations, the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1 ∼ 1-2 ms and requires the occupancy of both binding sites in the mGluR1 dimer. These rapid changes were followed by significantly slower conformational changes in the TM domain (τ2 ∼ 20 ms). Receptor deactivation occurred with time constants of ∼40 and ∼900 ms for the inter- and intrasubunit conformational changes, respectively. Together, these data show that, at high glutamate concentrations, the initial intersubunit activation of mGluR1 proceeds with millisecond speed, that there is loose coupling between this initial step and activation of the TM domain, and that activation and deactivation follow a cyclic pathway, including-in addition to the inactive and active states-at least two metastable intermediate states.

Corrigendum: Quantifying the cooperative subunit action in a multimeric membrane receptor.

This corrects the article DOI: 10.1038/srep20974.

Activation gating in HCN2 channels.

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels control electrical rhythmicity in specialized brain and heart cells. We quantitatively analysed voltage-dependent activation of homotetrameric HCN2 channels and its modulation by the second messenger cAMP using global fits of hidden Markovian models to complex experimental data. We show that voltage-dependent activation is essentially governed by two separable voltage-dependent steps followed by voltage-independent opening of the pore. According to this model analysis, the binding of cAMP to the channels exerts multiple effects on the voltage-dependent gating: It stabilizes the open pore, reduces the total gating charge from ~8 to ~5, makes an additional closed state outside the activation pathway accessible and strongly accelerates the ON-gating but not the OFF-gating. Furthermore, the open channel has a much slower computed OFF-gating current than the closed channel, in both the absence and presence of cAMP. Together, these results provide detailed new insight into the voltage- and cAMP-induced activation gating of HCN channels.

Publisher Correction: Hysteresis of ligand binding in CNGA2 ion channels.

This corrects the article DOI: 10.1038/ncomms3866.

A novel method for imaging sites of paracellular passage of macromolecules in epithelial sheets.

Understanding the dynamics of intestinal barrier function is key to elucidating oral delivery routes of therapeutics as well as to understanding various diseases that involve the mucosal immune system. Passage of macromolecules across barrier-forming epithelia is classically analyzed by means of various tracer flux measurements. This approach averages over contributions from many cells and lacks labeling of passage-sites. Thus, abundance and nature of involved cells have remained unidentified. We present a novel method that allowed for optical analysis of passage of various macromolecules on large-scale and single-cell level. To achieve tracking of passage loci in epithelia at submicrometer resolution we used biotinylated and fluorescent macromolecules that bind to basolateral membranes pre-labeled with cell-adherent avidin. We applied this method to epithelial cell lines and isolated mucosae in order to 3-dimensionally determine barrier leak properties over time. Tracer passage was found in all epithelia examined. However, it was infrequent, strikingly inhomogeneous, depended on culture duration and tightness of the monolayer. Stimulating passage with barrier-perturbing agents increased the number of leaks exposition time-dependently in cell lines and explanted mucosae. After stepwise opening of the paracellular passage pathway, integrated tracer-signal measured by our assay strictly correlated to simultaneously performed standard fluxes. Thus, our assay allows for the study of transepithelial macromolecule passage in various physiological and pathological conditions.

Quantifying the cooperative subunit action in a multimeric membrane receptor.

In multimeric membrane receptors the cooperative action of the subunits prevents exact knowledge about the operation and the interaction of the individual subunits. We propose a method that permits quantification of ligand binding to and activation effects of the individual binding sites in a multimeric membrane receptor. The power of this method is demonstrated by gaining detailed insight into the subunit action in olfactory cyclic nucleotide-gated CNGA2 ion channels.