Inhibition of the Aquaporin-1 Cation Conductance by Selected Furan Compounds Reduces Red Blood Cell Sickling

In sickle cell disease (SCD), the pathological shift of red blood cells (RBCs) into distorted morphologies under hypoxic conditions follows activation of a cationic leak current (Psickle) and cell dehydration. Prior work showed sickling was reduced by 5-hydroxylmethyl-2-furfural (5-HMF), which stabilized mutant hemoglobin and also blocked the Psickle current in RBCs, though the molecular basis of this 5-HMF-sensitive cation current remained a mystery. Work here is the first to test the hypothesis that Aquaporin-1 (AQP1) cation channels contribute to the monovalent component of Psickle. Human AQP1 channels expressed in Xenopus oocytes were evaluated for sensitivity to 5-HMF and four derivatives known to have differential efficacies in preventing RBC sickling. Ion conductances were measured by two-electrode voltage clamp, and osmotic water permeability by optical swelling assays. Compounds tested were: 5-HMF; 5-PMFC (5-(phenoxymethyl)furan-2-carbaldehyde); 5-CMFC (5-(4-chlorophenoxymethyl)furan-2-carbaldehyde); 5-NMFC (5-(2-nitrophenoxymethyl)-furan-2-carbaldehyde); and VZHE006 (tert-butyl (5-formylfuran-2-yl)methyl carbonate). The most effective anti-sickling agent, 5-PMFC, was the most potent inhibitor of the AQP1 ion conductance (98% block at 100 µM). The order of sensitivity of the AQP1 conductance to inhibition was 5-PMFC > VZHE006 > 5-CMFC ≥ 5-NMFC, which corresponded with effectiveness in protecting RBCs from sickling. None of the compounds altered AQP1 water channel activity. Combined application of a selective AQP1 ion channel blocker AqB011 (80 µM) with a selective hemoglobin modifying agent 5-NMFC (2.5 mM) increased anti-sickling effectiveness in red blood cells from human SCD patients. Another non-selective cation channel known to be expressed in RBCs, Piezo1, was unaffected by 2 mM 5-HMF. Results suggest that inhibition of AQP1 ion channels and capacity to modify hemoglobin are combined features of the most effective anti-sickling agents. Future therapeutics aimed at both targets could hold promise for improved treatments for SCD.

The Chloride Conductance Inhibitor NS3623 Enhances the Activity of a Non-selective Cation Channel in Hyperpolarizing Conditions

Handbooks of physiology state that the strategy adopted by red blood cells (RBCs) to preserve cell volume is to maintain membrane permeability for cations at its minimum. However, enhanced cation permeability can be measured and observed in specific physiological and pathophysiological situations such as in vivo senescence, storage at low temperature, sickle cell anemia and many other genetic defects affecting transporters, membrane or cytoskeletal proteins. Among cation pathways, cation channels are able to dissipate rapidly the gradients that are built and maintained by the sodium and calcium pumps. These situations are very well-documented but a mechanistic understanding of complex electrophysiological events underlying ion transports is still lacking. In addition, non-selective cation (NSC) channels present in the RBC membrane have proven difficult to molecular identification and functional characterization. For instance, NSC channel activity can be elicited by Low Ionic Strength conditions (LIS): the associated change in membrane potential triggers its opening in a voltage dependent manner. But, whereas this depolarizing media produces a spectacular activation of NSC channel, Gárdos channel-evoked hyperpolarization's have been shown to induce sodium entry through a pathway thought to be conductive and termed Pcat. Using the CCCP method, which allows to follow fast changes in membrane potential, we show here (i) that hyperpolarization elicited by Gárdos channel activation triggers sodium entry through a conductive pathway, (ii) that chloride conductance inhibition unveils such conductive cationic conductance, (iii) that the use of the specific chloride conductance inhibitor NS3623 (a derivative of Neurosearch compound NS1652), at concentrations above what is needed for full anion channel block, potentiates the non-selective cation conductance. These results indicate that a non-selective cation channel is likely activated by the changes in the driving force for cations rather than a voltage dependence mechanism per se.

In vivo optogenetics reveals control of cochlear sensitivity by supporting cells

Cochlear sensitivity, essential for communication and exploiting the acoustic environment, is due to the sensory-motor outer hair cells (OHCs) that operate in the structural scaffold of supporting cells and extracellular spaces in the cochlear organ of Corti (OoC). It is unclear whether supporting cells (e.g., Deiters cells [DCs] and outer pillar cells [OPCs]) control cochlear sensitivity in vivo. Here we employed optogenetics to measure in vivo sound-induced cochlear mechanical and electrical responses, and ex vivo light-induced DC electrical responses in the OoC of mice that conditionally expressed channelrhodopsins (ChR2) specifically in DCs and OPCs. Illumination activated a nonselective ChR2 cation conductance and depolarized the DCs. This transient action reversibly blocked continuous, normally occurring, minor adjustments of tone-evoked basilar membrane displacements, and OHC voltage responses to tones at and close to their characteristic frequency, and speeded recovery from temporary acoustic desensitization. This is the first direct evidence for the interdependency of the structural, mechanical, and electrochemical arrangement of OHCs and OoC supporting cells which together fine control cochlear sensitivity.

Thrombospondin-1/CD47 signaling modulates transmembrane cation conductance, survival, and deformability of human red blood cells

Background Thrombospondin-1 (TSP-1), a Ca2+-binding trimeric glycoprotein secreted by multiple cell types, has been implicated in the pathophysiology of several clinical conditions. Signaling involving TSP-1, through its cognate receptor CD47, orchestrates a wide array of cellular functions including cytoskeletal organization, migration, cell-cell interaction, cell proliferation, autophagy, and apoptosis. In the present study, we investigated the impact of TSP-1/CD47 signaling on Ca2+ dynamics, survival, and deformability of human red blood cells (RBCs). Methods Whole-cell patch-clamp was employed to examine transmembrane cation conductance. RBC intracellular Ca2+ levels and multiple indices of RBC cell death were determined using cytofluorometry analysis. RBC morphology and microvesiculation were examined using imaging flow cytometry. RBC deformability was measured using laser-assisted optical rotational cell analyzer. Results Exposure of RBCs to recombinant human TSP-1 significantly increased RBC intracellular Ca2+ levels. As judged by electrophysiology experiments, TSP-1 treatment elicited an amiloride-sensitive inward current alluding to a possible Ca2+ influx via non-selective cation channels. Exogenous TSP-1 promoted microparticle shedding as well as enhancing Ca2+- and nitric oxide-mediated RBC cell death. Monoclonal (mouse IgG1) antibody-mediated CD47 ligation using 1F7 recapitulated the cell death-inducing effects of TSP-1. Furthermore, TSP-1 treatment altered RBC cell shape and stiffness (maximum elongation index). Conclusions Taken together, our data unravel a new role for TSP-1/CD47 signaling in mediating Ca2+ influx into RBCs, a mechanism potentially contributing to their dysfunction in a variety of systemic diseases.

Conductance Mechanisms of Rapidly Desensitizing Cation Channelrhodopsins from Cryptophyte Algae

ABSTRACT Channelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. “Bacteriorhodopsin-like” cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, the sequences of BCCR match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here, we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption, Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function. IMPORTANCE Cation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-visible (UV-vis) spectroscopy and photoelectrophysiology reveal that the desensitization is caused by rapid accumulation of a nonconductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

Conductance mechanisms of rapidly desensitizing cation channelrhodopsins from cryptophyte algae

ABSTRACTChannelrhodopsins guide algal phototaxis and are widely used as optogenetic probes for control of membrane potential with light. “Bacteriorhodopsin-like” cation channelrhodopsins (BCCRs) from cryptophytes differ in primary structure from other CCRs, lacking usual residues important for their cation conductance. Instead, BCCR sequences match more closely those of rhodopsin proton pumps, containing residues responsible for critical proton transfer reactions. We report 19 new BCCRs, which, together with the earlier 6 known members of this family, form three branches (subfamilies) of a phylogenetic tree. Here we show that the conductance mechanisms in two subfamilies differ with respect to involvement of the homolog of the proton donor in rhodopsin pumps. Two BCCRs from the genus Rhodomonas generate photocurrents that rapidly desensitize under continuous illumination. Using a combination of patch clamp electrophysiology, absorption and Raman spectroscopy, and flash photolysis, we found that the desensitization is due to rapid accumulation of a long-lived nonconducting intermediate of the photocycle with unusually blue-shifted absorption with a maximum at 330 nm. These observations reveal diversity within the BCCR family and contribute to deeper understanding of their independently evolved cation channel function.IMPORTANCECation channelrhodopsins, light-gated channels from flagellate green algae, are extensively used as optogenetic photoactivators of neurons in research and recently have progressed to clinical trials for vision restoration. However, the molecular mechanisms of their photoactivation remain poorly understood. We recently identified cryptophyte cation channelrhodopsins, structurally different from those of green algae, which have separately evolved to converge on light-gated cation conductance. This study reveals diversity within this new protein family and describes a subclade with unusually rapid desensitization that results in short transient photocurrents in continuous light. Such transient currents have not been observed in the green algae channelrhodopsins and are potentially useful in optogenetic protocols. Kinetic UV-vis spectroscopy and photoelectrophysiology reveal the desensitization is caused by rapid accumulation of a non-conductive photointermediate in the photochemical reaction cycle. The absorption maximum of the intermediate is 330 nm, the shortest wavelength reported in any rhodopsin, indicating a novel chromophore structure.

Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate

ABSTRACTChannelrhodopsins (ChR) are cation channels that can be expressed heterologously in various living tissues, including cardiac and neuronal cells. To tune spatial and temporal control of potentials across ChR-enriched cell membranes, it is essential to understand how pore hydration impacts the ChR photocycle kinetics. Here, we measure channel opening and closing rates of channelrhodopsin chimera and selected variants (C1C2 wild type, C1C2-N297D, C1C2-N297V, and C1C2-V125L) and correlate them with changes in chemical interactions among functionally important residues in both closed and open states. Kinetic results substantiate that replacement of helices I and II in ChR2 with corresponding residues from ChR1, to make the chimera C1C2, affects the kinetics of channelrhodopsin pore gating significantly, making C1C2 a unique channel. As a prerequisite for studies of ion transport, detailed understanding of the water pathway within a ChR channel is important. Our atomistic simulations confirm that opening of the channel and initial hydration of the previously dry gating regions between helices I, II, III, and VII of the channel occurs with 1) the presence of 13-cis retinal; 2) deprotonation of a glutamic acid gating residue, E129; and 3) subsequent weakening of the central gate hydrogen bond between the same glutamic acid E129 and asparagine N282 in the central region of the pore. Also, an aspartate (D292) is the unambiguous primary proton acceptor for the retinal Schiff base in the hydrated channel.SIGNIFICANCEChannelrhodopsins (ChR) are light-sensitive ion channels used in optogenetics, a technique that applies light to selectively and non-invasively control cells (e.g., neurons) that have been modified genetically to express those channels. Using electrophysiology, we measured the opening and closing rates of a ChR chimera, and several variants, and correlated those rates with changes in chemical interactions determined from atomistic simulations. Significant new insights include correlation of single-point-mutations with four factors associated with pore hydration and cation conductance. Additionally, our work unambiguously identifies the primary proton acceptor for the retinal chromophore in the channel open state. These new insights add to mechanistic understanding of light-gated membrane transport and should facilitate future efforts to control membrane potentials spatially and temporally in optogenetics.

Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

Although Channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light-adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well-understood from a molecular perspective. Herein, we address these open questions using single turn-over electrophysiology, time-resolved step-scan FTIR and Raman spectroscopy of fully dark adapted ChR2. This yields a unifying parallel photocycle model explaining all data: in dark-adapted ChR2, the protonated Schiff base retinal chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long lived P480-state, which has been previously assigned to a late intermediate in a single photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn-cycle and we show that deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light-adaptation. Parallel anti- and syn-photocycles explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.Significance statementUnderstanding the mechanisms of photoactivated biological processes facilitates the development of new molecular tools, engineered for specific optogenetic applications, allowing the control of neuronal activity with light. Here, we use a variety of experimental and theoretical techniques to examine the precise nature of the light-activated ion channel in one of the most important molecular species used in optogenetics, channelrhodopsin-2. Existing models for the photochemical and photophysical pathway after light absorption by the molecule fail to explain many aspects of its observed behavior including the inactivation of the photocurrent under continuous illumination. We resolve this by proposing a new branched photocycle explaining electrical and photochemical channel properties and establishing the structure of intermediates during channel turnover.