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Insects are ectotherms, and as such, rely on a diverse repertoire of thermoreceptors to monitor environmental temperature and control behavioral thermoregulation. Here, we use structural, phylogenetic, genetic, and functional analyses to show that TRPA5 genes, widespread across numerous insect orders, encode a novel class of noxious heat receptors. We show that in the triatomine bugRhodnius prolixus, the primary vector of Chagas disease, Rp-TRPA52differs biophysically and structurally from noxious thermoTRPAs previously described in insects. This includes key changes in the ankyrin repeat domain and the selectivity filter of the channel.In vitro, we find evidence that the homo-tetrameric channel is not activated by voltage, but displays high thermosensitivity with an enthalpy change (ΔH) of 72 kcal/mol associated with the channel activation, with a Q10= 25 and T°half= 58.6°C. Structural analyses reveal parallels in the overall ion channel architecture between fruit fly TRPA1 and Rp-TRPA52; however, functional properties and expression patterns indicate that the role of Rp-TRPA52is more similar to that ofPyrexianoxious heat receptors found in fruit flies.Pyrexiagenes have been lost in true bugs, and our findings suggest that the rapidly evolving insect TRPA gene family has given rise to an independent evolutionary origin of a molecular transducer that is responsive to noxious thermal stimuli.

T-type voltage-gated Ca

Glioblastoma is a lethal brain cancer that commonly recurs after tumor resection and chemotherapy treatment. Depolarized resting membrane potentials and an acidic intertumoral extracellular pH have been associated with a proliferative state and drug resistance, suggesting that forced hyperpolarization and disruption of proton pumps in the plasma membrane could be a successful strategy for targeting glioblastoma overgrowth. We screened 47 compounds and compound combinations, most of which were ion-modulating, at different concentrations in the NG108-15 rodent neuroblastoma/glioma cell line. A subset of these were tested in the U87 human glioblastoma cell line. A FUCCI cell cycle reporter was stably integrated into both cell lines to monitor proliferation and cell cycle response. Immunocytochemistry, electrophysiology, and a panel of physiological dyes reporting voltage, calcium, and pH were used to characterize responses. The most effective treatments on proliferation in U87 cells were combinations of NS1643 and pantoprazole; retigabine and pantoprazole; and pantoprazole or NS1643 with temozolomide. Marker analysis and physiological dye signatures suggest that exposure to bioelectric drugs significantly reduces proliferation, makes the cells senescent, and promotes differentiation. These results, along with the observed low toxicity in human neurons, show the high efficacy of electroceuticals utilizing combinations of repurposed FDA approved drugs.

CACNA1I is implicated in the susceptibility to schizophrenia by large-scale genetic association studies of single nucleotide polymorphisms. However, the channelopathy of CACNA1I in schizophrenia is unknown. CACNA1I encodes CaV3.3, a neuronal voltage-gated calcium channel that underlies a subtype of T-type current that is important for neuronal excitability in the thalamic reticular nucleus and other regions of the brain. Here, we present an extensive functional characterization of 57 naturally occurring rare and common missense variants of CACNA1I derived from a Swedish schizophrenia cohort of more than 10 000 individuals. Our analysis of this allelic series of coding CACNA1I variants revealed that reduced CaV3.3 channel current density was the dominant phenotype associated with rare CACNA1I coding alleles derived from control subjects, whereas rare CACNA1I alleles from schizophrenia patients encoded CaV3.3 channels with altered responses to voltages. CACNA1I variants associated with altered current density primarily impact the ionic channel pore and those associated with altered responses to voltage impact the voltage-sensing domain. CaV3.3 variants associated with altered voltage dependence of the CaV3.3 channel and those associated with peak current density deficits were significantly segregated across affected and unaffected groups (Fisher’s exact test, P = 0.034). Our results, together with recent data from the SCHEMA (Schizophrenia Exome Sequencing Meta-Analysis) cohort, suggest that reduced CaV3.3 function may protect against schizophrenia risk in rare cases. We subsequently modelled the effect of the biophysical properties of CaV3.3 channel variants on thalamic reticular nucleus excitability and found that compared with common variants, ultrarare CaV3.3-coding variants derived from control subjects significantly decreased thalamic reticular nucleus excitability (P = 0.011). When all rare variants were analysed, there was a non-significant trend between variants that reduced thalamic reticular nucleus excitability and variants that either had no effect or increased thalamic reticular nucleus excitability across disease status. Taken together, the results of our functional analysis of an allelic series of >50 CACNA1I variants in a schizophrenia cohort reveal that loss of function of CaV3.3 is a molecular phenotype associated with reduced disease risk burden, and our approach may serve as a template strategy for channelopathies in polygenic disorders.

CACNA1I, a schizophrenia risk gene, encodes a subtype of voltage-gated T-type calcium channel CaV3.3. We previously reported that a patient-derived missense de novo mutation (R1346H) of CACNA1I impaired CaV3.3 channel function. Here, we generated CaV3.3-RH knock-in animals, along with mice lacking CaV3.3, to investigate the biological impact of R1346H (RH) variation. We found that RH mutation altered cellular excitability in the thalamic reticular nucleus (TRN), where CaV3.3 is abundantly expressed. Moreover, RH mutation produced marked deficits in sleep spindle occurrence and morphology throughout non-rapid eye movement (NREM) sleep, while CaV3.3 haploinsufficiency gave rise to largely normal spindles. Therefore, mice harboring the RH mutation provide a patient derived genetic model not only to dissect the spindle biology but also to evaluate the effects of pharmacological reagents in normalizing sleep spindle deficits. Importantly, our analyses highlighted the significance of characterizing individual spindles and strengthen the inferences we can make across species over sleep spindles. In conclusion, this study established a translational link between a genetic allele and spindle deficits during NREM observed in schizophrenia patients, representing a key step toward testing the hypothesis that normalizing spindles may be beneficial for schizophrenia patients.

International audience; ObjectiveVoltage‐gated sodium channels (SCNs) share similar amino acid sequence, structure, and function. Genetic variants in the four human brain‐expressed SCN genes SCN1A/2A/3A/8A have been associated with heterogeneous epilepsy phenotypes and neurodevelopmental disorders. To better understand the biology of seizure susceptibility in SCN‐related epilepsies, our aim was to determine similarities and differences between sodium channel disorders, allowing us to develop a broader perspective on precision treatment than on an individual gene level alone.MethodsWe analyzed genotype‐phenotype correlations in large SCN‐patient cohorts and applied variant constraint analysis to identify severe sodium channel disease. We examined temporal patterns of human SCN expression and correlated functional data from in vitro studies with clinical phenotypes across different sodium channel disorders.ResultsComparing 865 epilepsy patients (504 SCN1A, 140 SCN2A, 171 SCN8A, four SCN3A, 46 copy number variation [CNV] cases) and analysis of 114 functional studies allowed us to identify common patterns of presentation. All four epilepsy‐associated SCN genes demonstrated significant constraint in both protein truncating and missense variation when compared to other SCN genes. We observed that age at seizure onset is related to SCN gene expression over time. Individuals with gain‐of‐function SCN2A/3A/8A missense variants or CNV duplications share similar characteristics, most frequently present with early onset epilepsy (

Overexpression of mouse neurogenin ( Neurog) 2 alone or in combination with mouse Neurog2/1 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can rapidly produce high-yield excitatory neurons. Here, we report a detailed characterization of human neuronal networks induced by the expression of human NEUROG2 together with human NEUROG2/1 in hESCs using molecular, cellular, and electrophysiological measurements over 60 d after induction. Both excitatory synaptic transmission and network firing activity increased over time. Strikingly, inhibitory synaptic transmission and GABAergic cells were identified from NEUROG2/1 induced neurons (iNs). To illustrate the application of such iNs, we demonstrated that the heterozygous knock out of SCN2A, whose loss-of-function mutation is strongly implicated in autism risk, led to a dramatic reduction in network activity in the NEUROG2/1 iNs. Our findings not only extend our understanding of the NEUROG2/1-induced human neuronal network but also substantiate NEUROG2/1 iNs as an in vitro system for modeling neuronal and functional deficits on a human genetic background.-Lu, C., Shi, X., Allen, A., Baez-Nieto, D., Nikish, A., Sanjana, N. E., Pan, J. Q. Overexpression of NEUROG2 and NEUROG1 in human embryonic stem cells produces a network of excitatory and inhibitory neurons.

Ion channels represent nearly a quarter of all targets that currently available medications modulate, and their dysfunction underlies increasing number of human diseases. Functional analysis of ion channels have traditionally been a bottleneck in large-scale analyses. Recent technological breakthroughs in automated planar electrophysiology have democratized the technique to enable high-throughput patch clamping at scale. In this chapter, we describe the methodology to perform a phenotypic screen on voltage-gated calcium channels across many different genetic coding variations and against small-molecule modulators. We first describe the procedures to establish inducible heterologous ion channel expression in HEK293 cells, where each cell incorporates one copy of a target protein cDNA-a step that is critical for producing stable and consistent expression of ion channels. We then describe the experimental and analytical methods for analyzing the function of ion channels using high-throughput planar electrophysiology.

The main role of voltage-gated proton channels (Hv1) is to extrude protons from the intracellular milieu when, mediated by different cellular processes, the H+ concentration increases. Hv1 are exquisitely selective for protons and their structure is homologous to the voltage sensing domain (VSD) of other voltage-gated ion channels like sodium, potassium, and calcium channels. In clear contrast to the classical voltage-dependent channels, Hv1 lacks a pore domain and thus permeation necessarily occurs through the voltage sensing domain. Hv1 channels are activated by depolarizing voltages, and increases in internal proton concentration. It has been proposed that local conformational changes of the transmembrane segment S4, driven by depolarization, trigger the molecular rearrangements that open Hv1. However, it is still unclear how the electromechanical coupling is achieved between the VSD and the potential pore, allowing the proton flux from the intracellular to the extracellular side. Here we provide a revised view of voltage activation in Hv1 channels, offering a comparative scenario with other voltage sensing channels domains.

Ion channels represent nearly a quarter of all targets that currently available medications modulate, and their dysfunction underlies increasing number of human diseases. Functional analysis of ion channels have traditionally been a bottleneck in large-scale analyses. Recent technological breakthroughs in automated planar electrophysiology have democratized the technique to enable high-throughput patch clamping at scale. In this chapter, we describe the methodology to perform a phenotypic screen on voltage-gated calcium channels across many different genetic coding variations and against small-molecule modulators. We first describe the procedures to establish inducible heterologous ion channel expression in HEK293 cells, where each cell incorporates one copy of a target protein cDNA-a step that is critical for producing stable and consistent expression of ion channels. We then describe the experimental and analytical methods for analyzing the function of ion channels using high-throughput planar electrophysiology.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P2 is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P2 binding site located between the TRP box and the S4-S5 linker. At this site, PI(4,5)P2 interacts with the amino acid residues Arg-575 and Arg-579 in the S4-S5 linker and with Lys-694 in the TRP box. We confirmed that PI(4,5)P2 behaves as a channel agonist and found that Arg-575, Arg-579, and Lys-694 mutations to alanine reduce PI(4,5)P2 binding affinity. Additionally, in silico mutations R575A, R579A, and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P2 in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P2 binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which cause an opening of the lower TRPV1 channel gate.

The voltage-gated proton channel (Hv1) is a dimer of subunits containing four transmembrane domains (S1-S4) which play the double role of harboring the voltage-sensing charges and forming the proton conduction pathway. Some biophysical properties of Hv1

The most well-known role of Connexins (Cxs) is to communicate the cytoplasm of two adjacent cells forming a gap junction channel in the apposition zone. On the other hand, Cxs form the so called “hemichannels”, a fully functional voltage-gated non-selective ion channel, which present two gating mechanism called “slow” and “fast” gating. Hemichannels consist of 6 monomers, each one composed of four transmembrane domains. Despite the fact that hemichannels are voltage-activated, they lack the classical voltage-sensing domain described for canonical voltage-gated channels. Furthermore, the molecular determinants associated to the voltage activation in connexins hemichannels are still elusive. In this work we search for the molecular determinants associated to voltage detection in human Cx26 hemichannels. Using two electrode voltage-clamp in Xenopus laevis oocytes, we study how the steady-state conductance-voltage (G/V) relationship and kinetics of gating are affected by the neutralization of different charged residues located within the first transmembrane domain. We have found that Cx26 conductance-voltage curve shows an apparent number of gating charges (zδ) of 1.4 e0 for the slow gating mechanism. On the other hand, we obtained a zδ of 3.9 e0 for the fast gate, which is in agreement with the zδ of Cx26 gap junctions. A simple three-state closed-open1-open2 kinetic model is able to account for the channel voltage dependence. Furthermore, our data shows that neutralizing the K41 residue (K41N) increases the zδ; of the slow gate to 2.5 e0, and decreases the zδ of the fast gate to 2.3 e0.This work is supported by FONDECYT Grants 1110430 (R.L.), 1120802 (C.G.), 1130855 (A.M.); ANILLO Grant ACT1104 (C.G.); Beca para Estudios de Magister en Chile Ano 2014 (B.P.); CINV is a Millennium Institute supported by the Millennium Scientific Initiative of the Ministerio de Economia, Fomento y Turismo.

Mammalian transient receptor potential (TRP) channels consist of at least seven families of related proteins. A subset belong to the TRP channel superfamily, and they are responsible for temperature detection in mammals. These TRP channels (thermoTRPs) are expressed primarily in the dorsal root

Voltage-gated proton (HV) channels are expressed by different cells type including immune cells, microglia, among others. In other voltage-gated cation channels, the pore-forming domains are S5, S6, and the P loop connecting S5 and S6. Since Hv channels do not contain a S5-S6 region, other parts of the channel must form the pore domain. Interestingly, Hv channel present three gating charges in the S4 followed by an asparagine, N264, highly conserved among all Hv channel. Using non-stationary fluctuation analysis we establish the conductance for the dimer and the monomer in 200 and 100 fS, respectively. Mutations at position S191 (S2) and N264 (S4) modified the unitary conductance of Hv channel. Furthermore, mutants S191E/D and N264R removed the H+ current remaining the S4 functioning unaltered, according to voltage-clamp fluorometry experiments. The introduction of an arginine or lysine at position 264 or negative residue at position S191, drastically reduced or abolished the proton currents. Interestingly, mutations of the analogue position S191 of Ciona VSOP, in the voltage sensor domain of a non-conductor Shaker K+ channel produce fully functional voltage gated H+ channels. In the same way, mutation resembling the position N264 of Ciona Hv in the VSD of the voltage-dependent phosphate Ci-VSP spontaneously entail the formation of a voltage-gated H+ channel. Thus, S191 and N264 form the molecular determinants of permeation pathway in Hv channel. Supported by Fondecyt grants ACT 1104 and Fondecyt 1120802 to CG and 1110430 to R.L.

Voltage-gated proton channels are integral membrane proteins with the capacity to permeate elementary particles in a voltage and pH dependent manner. These proteins have been found in several species and are involved in various physiological processes. Although their primary topology is known, lack of details regarding their structures in the open conformation has limited analyses toward a deeper understanding of the molecular determinants of their function and regulation. Consequently, the function-structure relationships have been inferred based on homology models. In the present work, we review the existing proton channel models, their assumptions, predictions and the experimental facts that support them. Modeling proton channels is not a trivial task due to the lack of a close homolog template. Hence, there are important differences between published models. This work attempts to critically review existing proton channel models toward the aim of contributing to a better understanding of the structural features of these proteins.

Potassium channels are particularly important in determining the shape and duration of the action potential, controlling the membrane potential, modulating hormone secretion, epithelial function and, in the case of those K(+) channels activated by Ca(2+), damping excitatory signals. The multiplicity of roles played by K(+) channels is only possible to their mammoth diversity that includes at present 70 K(+) channels encoding genes in mammals. Today, thanks to the use of cloning, mutagenesis, and the more recent structural studies using x-ray crystallography, we are in a unique position to understand the origins of the enormous diversity of this superfamily of ion channels, the roles they play in different cell types, and the relations that exist between structure and function. With the exception of two-pore K(+) channels that are dimers, voltage-dependent K(+) channels are tetrameric assemblies and share an extremely well conserved pore region, in which the ion-selectivity filter resides. In the present overview, we discuss in the function, localization, and the relations between function and structure of the five different subfamilies of K(+) channels: (a) inward rectifiers, Kir; (b) four transmembrane segments-2 pores, K2P; (c) voltage-gated, Kv; (d) the Slo family; and (e) Ca(2+)-activated SK family, SKCa.

In this chapter we discuss the polymodal activation of thermo-TRP channels using as exemplars two of the best characterized members of this class of channels: TRPM8 and TRPV1. Since channel activation by temperature is the hallmark of thermo-TRP channels, we present a detailed discussion on the thermodynamics involved in the gating processes by temperature, voltage, and agonists. We also review recently published data in an effort to put together all the pieces available of the amazing puzzle of thermo-TRP channel activation. Special emphasis is made in the structural components that allow the channel-forming proteins to integrate such diverse stimuli, and in the coupling between the different sensors and the ion conduction pathway. We conclude that the present data is most economically explained by allosteric models in which temperature, voltage, and agonists act separately to modulate channel activity.

Hv1 channels are integral membrane proteins with the capacity to selectively permeate protons in a voltage and pH-dependent manner. As Hv1 lacks a pore domain, permeation must occur through the voltage-sensing domain. Previous reports propose a permeation pathway consisting in a stable water wire which allows proton to permeate by means of a Grotthuss mechanism. Our molecular dynamics simulations do not support the formation of such stable water wire since it shows a dry zone around residue N264 in the wild type and in N264 mutants. Mutations of residues D222 and N264 affects single channel conductance (determined by non-stationary noise analysis) and selectivity, suggesting that both residues are involved in the permeation pathway. Quantum dynamics simulations performed in our model of the open Ci-Hv1 wt and in silico mutants suggest that permeation occur through a proton wire involving residues D160 and D222, a process modulated by N264.Supported by Beca de Doctorado Nacional para Extranjeros de Conicyt (A.P), FONDECYT Grants 1110430 (R.L.), 1120802 (C.G.); ANILLO Grant ACT1104 (C.G.); Postdoctoral Fellowships 3140590 (G.F.C.). CINV is a Millennium Institute.

Every organism needs molecular antennae in order to sense the environmental temperature. In mammals, these antennae are a set of ion channels called thermo Transient Receptor Potential (thermoTRP). TRPV1 is a heat activated channel that belongs to TRP family, with an activation threshold around 42 °C. Experimental evidences show that TRPV1 posses a similar structural architecture to voltage-dependent K+ channels (Kv). Nevertheless, TRPV1 has a weakly voltage dependence (number of apparent gating charges, z = 0.83 e0) and in contrast with the numerous positively charged residues contained in the S4 of Kv channels (∼12 e0 gating charges/channel), it has only one arginine (R557) located in the fourth transmembrane segment (S4). However, the molecular determinant that confers the voltage sensitivity to this channel remains elusive. using site-directed mutagenesis we have neutralized several charged amino acid (positives and negatives) along to the channel. The electrophysiological studies (macro-patch in X. laevis oocytes), surprisingly reveals that the uncharged S4 TRPV1 channels still conserve the same voltage dependence as wt channels. Furthermore, neutralizations in other transmembrane segments or even neutralizations on the pore region do not affect the voltage sensitivity of TRPV1 channels. Despite the weak voltage dependence of TRPV1 channel, the membrane voltage has a huge impact over the temperature activation; changing the activation enthalpy and the ionic current kinetics. At hyperpolarizing potentials the temperature activation shows a delay resembling the Cole-Moore shift. Furthermore, at depolarizing potential the channel kinetics speeds up, revealing an allosteric connection between voltage and thermal stimuli. This work was supported by Fondecyt grant 1110430 to RL, Fondecyt 1120802 to CG and ACT 10224 to CG. CINV is a Scientific Millennium Institute.

TRPM8 channels are a polymodal receptors activated by voltage, low temperatures and agonists. The structural data suggests that the distal C-terminus (1064-1104 amino acid residues) form a coiled-coil motif. Our aim was to investigate the importance of this structure in the thermo-sensitivity of TRPM8. TRPM8 wild type and deletion mutants of the coiled-coil region of this channel were cloned and expressed in Xenopus oocytes. Electrophysiological measurements were performed 48-72 hr after mRNA microinjection. Macroscopic currents were recorded by patch-clamp technique in cell-attached configuration at different temperatures (10- 40°C). An analysis of the conductance-voltage curves determined from tail currents showed that values of the voltage at which the open probability is 0,5 (V1/2) saturate at high and low temperatures. These results and limiting slope analysis of the open probability-voltage curves demonstrated that TRPM8 channel gating is best explained using an allosteric gating model for both voltage and temperature dependence. On the other hand, gradual deletions of the coiled-coil region (from 1064 to 1104 residues) promoted a gradual decrease in the temperature sensitivity of TRPM8 channel, without affecting the voltage dependence. The decrease in the enthalpy (H) promoted by gradual deletions of the distal C-terminus suggests that coiled-coil region is a structural determinant of the thermo sensitivity in TRPM8 channels. Our results also indicate that the voltage and temperature sensors are localized in different structural domains. Supported by FONDECYT grants 1110430 to R.L, CONICYT AT-24100020 to N.R. CINV is a Millennium Scientific Institute.

Thermal TRP channels are directly activated by ambient temperature changes and thus play important functions in thermosensation and pain. TRPM8, in particular, is sensitive to innocuous cold (

Recently, a family of voltage-gated proton channels, called (Hv) was cloned from different organisms. Hv channel shares a common structure with the Voltage Sensor Domain (VSD) of voltage-gated K channels. Despite off the high homology between Hv channel and the VSD, they have distant quaternary structures. Hv channel lacks a pore domain, and is fully functional either as a monomer or dimer. The S4 was proposed as the possible permeation pathway, resembling the omega current present in some mutants of Shaker K+ channel. However the permeation pathway is still elusive. Fluorescence experiments suggests that the voltage activation in Hv is in two steps, where the second step is a cooperative conformational change, involving interactions between subunits that opens both permeation pathways in a concerted way in the dimer. However, the relationship between the H+ pore, and the voltage sensor activation is not fully understood for the monomer. Here we presented the first Hv gating currents recordings from the monomeric Hv form (Hv-ΔNΔC). Interestingly, the voltage sensor activation precedes the H+ conduction, suggesting that voltage sensor and H+ pore, are two different structures allosterically coupled. The Q-V present a zδ of 1.6 e0, in agreement with previous experiments of voltage-clamp fluorometry. The data presented support the hypothesis that the channel opening is associated to a second conformational state of the voltage sensor, associated to an extremely slow component in the gating current. Supported by Fondecyt grants ACT 1104 and Fondecyt 1120802 to CG and 1110430 to R.L.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) has been recognized as an important activator of certain transient receptor potential (TRP) channels. More specifically, TRPV1 is a pain receptor activated by a wide range of stimuli. However, whether or not PI(4,5)P2 is a TRPV1 agonist remains open to debate. Utilizing a combined approach of mutagenesis and molecular modeling, we identified a PI(4,5)P2 binding site located between the TRP box and the S4-S5 linker. At this site PI(4,5)P2 interacts with the amino acid residues R575 and R579 in the S4-S5 linker and with K694 in the TRP box. We confirmed that PI(4,5)P2 behaves as a channel agonist and found that R575, R579 and K694 mutations to alanine reduce PI(4,5)P2 binding affinity. Additionally, in silico mutations R575A, R579A and K694A showed that the reduction in binding affinity results from the delocalization of PI(4,5)P2 in the binding pocket. Molecular dynamics simulations indicate that PI(4,5)P2 binding induces conformational rearrangements of the structure formed by S6 and the TRP domain, which leads to the opening of the lower TRPV1 channel gate.