Unitary Conductance Research Articles

Plumbagin, a novel TRPV2 inhibitor, ameliorates microglia activation and brain injury in a middle cerebral artery occlusion/reperfusion mouse model.

Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable non-selective cation channel. Despite the significant roles of TRPV2 in immunological response, cancer progression and cardiac development, pharmacological probes of TRPV2 remain to be identified. We aimed to discover TRPV2 inhibitors and to elucidate their molecular mechanism of action. Fluorescence-based Ca2+ assay in HEK-293 cells expressing murine TRPV2 was used to identify plumbagin as a novel TRPV2 inhibitor. Patch-clamp, in silico docking and site-directed mutagenesis were applied to investigate the molecular mechanisms critical for plumbagin interaction. ELISA and qPCR were used to assess nitric oxide release and mRNA levels of inflammatory mediators, respectively. si-RNA interference was used to knock down TRPV2 expression, which was validated by western blotting. Neurological and histological analyses were used to examine brain injury of mice following middle cerebral artery occlusion/reperfusion (MCAO/R). Plumbagin is a potent TRPV2 negative allosteric modulator with an IC50 value of 0.85 μM, exhibiting >14-fold selectivity over TRPV1, TRPV3 and TRPV4. Plumbagin suppresses TRPV2 activity by decreasing the channel open probability without affecting the unitary conductance. Moreover, plumbagin binds to an extracellular pocket formed by the pore helix and flexible loop between transmembrane helices S5 and S6 of TRPV2. Plumbagin effectively suppresses LPS-induced inflammation of BV-2 microglia and ameliorates brain injury of MCAO/R mice. Plumbagin is a novel pharmacological probe to study TRPV2 pathophysiology. TRPV2 is a novel molecular target for the treatment of neuroinflammation and ischemic stroke.

Inhibition of Cutaneous TRPV3 Channels by Natural Caffeic Acid for the Alleviation of Skin Inflammation.

Natural caffeic acid (CA) and its analogues have been studied for their potential applications in the treatment of various inflammatory and infectious skin diseases. However, the molecular mechanism underlying the effects of the CA remains largely unknown. Here, we report that CA and its two analogues, caffeic acid phenethyl ester (CAPE) and caffeic acid methyl caffeate (CAMC), inhibit TRPV3 currents in their concentration- and structure-dependent manners with IC50 values ranging from 102 to 410 μM. At the single-channel level, CA reduces the channel open probability and open frequency without alteration of unitary conductance. CA selectively inhibits TRPV3 relative to other subtypes of thermo-TRPs, such as TRPA1, TRPV1, TRPV4, and TRPM8. Molecular docking combined with site-specific mutagenesis reveals that a residue T636 in the Pore-loop is critical for CA binding to TRPV3. Further in vivo evaluation shows that CA significantly reverses TRPV3-mediated skin inflammation induced by skin sensitizer carvacrol. Altogether, our findings demonstrate that CA exerts its anti-inflammatory effects by selectively inhibiting TRPV3 through binding to the pocket formed by the Pore-loop and the S6. CA may serve as a lead for further modification and identification of specific TRPV3 channel inhibitors.

Functional characterization of native Piezo1 as calcium and magnesium influx pathway in human myeloid leukemia cells.

Piezo1 is a Ca2+-permeable mechanically activated ion channel that is involved in various physiological processes and cellular responses to mechanical stimuli. The study of biophysical characteristics of Piezo1 is important for understanding the mechanisms of its function and regulation. Stretch activation, a routine approach that is applied to stimulate Piezo1 activity in the plasma membrane, has a number of significant limitations that complicate precise single-channel analysis. Here, we aimed to determine pore properties of native Piezo1, specifically to examine permeation for physiologically relevant signaling divalent ions (calcium and magnesium) in human myeloid leukemia K562 cells using Piezo1-specific chemical agonist, Yoda1. Using a combination of low-noise single-current patch-clamp recordings of Piezo1 activity in response to Yoda1, we have determined single-channel characteristics of native Piezo1 under various ionic conditions. Whole-cell assay allowed us to directly measure Piezo1 single currents carried by Ca2+ or Mg2+ ions in the absence of other permeable cations in the extracellular solutions; unitary conductance values estimated at various concentrations of Mg2+ revealed strong saturation effect. Patch clamp data complemented with fluorescent imaging clearly evidenced Ca2+ and Mg2+ entry via native Piezo1 channel in human leukemia K562 cells. Mg2+ influx via Piezo1 was detected under quasi-physiological conditions, thus showing that Piezo1 channels could potentially provide the physiological relevant pathway for Mg2+ ion transport and contribute to the regulation of Mg2+-dependent intracellular signaling.

Impact of Unitary Synaptic Inhibition on Spike Timing in Ventral Tegmental Area Dopamine Neurons.

Midbrain dopamine neurons receive convergent synaptic input from multiple brain areas, which perturbs rhythmic pacemaking to produce the complex firing patterns observed in vivo. This study investigated the impact of single and multiple inhibitory inputs on ventral tegmental area (VTA) dopamine neuron firing in mice of both sexes using novel experimental measurements and modeling. We first measured unitary inhibitory postsynaptic currents produced by single axons using both minimal electrical stimulation and minimal optical stimulation of rostromedial tegmental nucleus and ventral pallidum afferents. We next determined the phase resetting curve, the reversal potential for GABAA receptor-mediated inhibitory postsynaptic currents (IPSCs), and the average interspike membrane potential trajectory during pacemaking. We combined these data in a phase oscillator model of a VTA dopamine neuron, simulating the effects of unitary inhibitory postsynaptic conductances (uIPSGs) on spike timing and rate. The effect of a uIPSG on spike timing was predicted to vary according to its timing within the interspike interval or phase. Simulations were performed to predict the pause duration resulting from the synchronous arrival of multiple uIPSGs and the changes in firing rate and regularity produced by asynchronous uIPSGs. The model data suggest that asynchronous inhibition is more effective than synchronous inhibition, because it tends to hold the neuron at membrane potentials well positive to the IPSC reversal potential. Our results indicate that small fluctuations in the inhibitory synaptic input arriving from the many afferents to each dopamine neuron are sufficient to produce highly variable firing patterns, including pauses that have been implicated in reinforcement.

One-shot design elevates functional expression levels of a voltage-gated potassium channel.

Membrane proteins play critical physiological roles as receptors, channels, pumps, and transporters. Despite their importance, however, low expression levels often hamper the experimental characterization of membrane proteins. We present an automated and web-accessible design algorithm called mPROSS (https://mPROSS.weizmann.ac.il), which uses phylogenetic analysis and an atomistic potential, including an empirical lipophilicity scale, to improve native-state energy. As a stringent test, we apply mPROSS to the Kv1.2-Kv2.1 paddle chimera voltage-gated potassium channel. Four designs, encoding 9-26 mutations relative to the parental channel, were functional and maintained potassium-selective permeation and voltage dependence in Xenopus oocytes with up to 14-fold increase in whole-cell current densities. Additionally, single-channel recordings reveal no significant change in the channel-opening probability nor in unitary conductance, indicating that functional expression levels increase without impacting the activity profile of individual channels. Our results suggest that the expression levels of other dynamic channels and receptors may be enhanced through one-shot design calculations.

Direct Inhibition of BK Channels by Cannabidiol, One of the Principal Therapeutic Cannabinoids Derived from Cannabis sativa.

Cannabidiol (CBD), one of the main Cannabis sativa bioactive compounds, is utilized in the treatment of major epileptic syndromes. Its efficacy can be attributed to a multimodal mechanism of action that includes, as potential targets, several types of ion channels. In the brain, CBD reduces the firing frequency in rat hippocampal neurons, partly prolonging the duration of action potentials, suggesting a potential blockade of voltage-operated K+ channels. We postulate that this effect might involve the inhibition of the large-conductance voltage- and Ca2+-operated K+ channel (BK channel), which plays a role in the neuronal action potential's repolarization. Thus, we assessed the impact of CBD on the BK channel activity, heterologously expressed in HEK293 cells. Our findings, using the patch-clamp technique, revealed that CBD inhibits BK channel currents in a concentration-dependent manner with an IC50 of 280 nM. The inhibition is through a direct interaction, reducing both the unitary conductance and voltage-dependent activation of the channel. Additionally, the cannabinoid significantly delays channel activation kinetics, indicating stabilization of the closed state. These effects could explain the changes induced by CBD in action potential shape and duration, and they may contribute to the observed anticonvulsant activity of this cannabinoid.

Estimating the Ca2+ Block of NMDA Receptors with Single-Channel Electrophysiology.

In vertebrate central neurons, NMDA receptors are glutamate- and glycine-gated ion channels that allow the passage of Na+ and Ca2+ ions into the cell when these neurotransmitters are simultaneously present. The passage of Ca2+ is critical for initiating the cellular processes underlying various forms of synaptic plasticity. These Ca2+ ions can autoregulate the NMDA receptor signal through multiple distinct mechanisms to reduce the total flux of cations. One such mechanism is the ability of Ca2+ ions to exclude the passage of Na+ ions resulting in a reduced unitary current conductance. In contrast to the well-characterized Mg2+ block, this "channel block" mechanism is voltage-independent. In this chapter, we discuss theoretical and experimental considerations for the study of channel block by Ca2+ using single-channel patch-clamp electrophysiology. We focus on two classic methodologies to quantify the dependence of unitary channel conductance on external concentrations of Ca2+ as the basis for quantifying Ca2+ block.

Selective inhibition of overactive warmth-sensitive Ca2+-permeable TRPV3 channels by antispasmodic agent flopropione for alleviation of skin inflammation

The temperature-sensitive Ca2+-permeable TRPV3 ion channel is robustly expressed in the skin keratinocytes, and its gain-of-function mutations are involved in the pathology of skin lesions. Here, we report the identification of an antispasmodic agent flopropione that alleviates skin inflammation by selective inhibition of TRPV3. In whole-cell patch clamp recordings, flopropione selectively inhibits macroscopic TRPV3 currents in a concentration-dependent manner with an IC50 value of 17.8± 3.5μM. At the single-channel level, flopropione inhibits TRPV3 channel open probability without alteration of its unitary conductance. In an invivo mouse model of skin inflammation induced by the skin sensitizer DNFB, flopropione also alleviates dorsal skin lesions and ear skin swelling. Further molecular docking combined with site-directed mutagenesis reveals that two residues E501 and I505 in the channel S2-helix are critical for flopropione-mediated inhibition of TRPV3. Taken together, our findings demonstrate that the spasmolytic drug flopropione as a selective inhibitor of TRPV3 channel not only provides a valuable tool molecule for understanding of TRPV3 channel pharmacology but also holds repurposing potential for therapy of skin disorders, such as dermatitis and pruritus.

Capsazepine activates amiloride-insensitive ENaC-like channels in human leukemia cells

Sodium influx carried out by ion channels is one of the main regulators of water-salt and volume balance in cells of blood origin. Previously, we described amiloride-insensitive ENaC-like channels in human myeloid leukemia K562 cells; the intracellular regulatory mechanisms of the channels are associated with actin cytoskeleton dynamics. Recently, an extracellular mechanism of ENaC-like channels activation in K562 cells by the action of serine protease trypsin has been revealed. The other extracellular pathways that modulate ENaC (epithelial Na+ channel) activity and sodium permeability in transformed blood cells are not yet fully investigated. Here, we study the action of capsazepine (CPZ), as δ-ENaC activator, on single channel activity in K562 cells in whole-cell patch clamp experiments. Addition of CPZ (2 μM) to the extracellular solution caused an activation of sodium channels with typical features; unitary conductance was 15.1 ± 0.8 pS. Amiloride derivative benzamil (50 μM) did not inhibit their activity. Unitary currents and conductance of CPZ-activated channels were higher in Na+-containing extracellular solution than in Li+, that is one of the main fingerprints of δ-ENaC. The results of RT-PCR analysis and immunofluorescence staining also confirmed the expression of δ-hENaC (as well as α-, β-, γ-ENaC) at the mRNA and protein level. These findings allow us to speculate that CPZ activates amiloride-insensitive ENaC-like channels that contain δ-ENaC in К562 cells. Our data reveal a novel extracellular mechanism for ENaC-like activation in human leukemia cells.

A mechano- and heat-gated two-pore domain K+ channel controls excitability in adult zebrafish skeletal muscle.

TRAAK channels are mechano-gated two-pore-domain K+ channels. Up to now, activity of these channels has been reported in neurons but not in skeletal muscle, yet an archetype of tissue challenged by mechanical stress. Using patch clamp methods on isolated skeletal muscle fibers from adult zebrafish, we show here that single channels sharing properties of TRAAK channels, i.e., selective to K+ ions, of 56 pS unitary conductance in the presence of 5 mM external K+, activated by membrane stretch, heat, arachidonic acid, and internal alkaline pH, are present in enzymatically isolated fast skeletal muscle fibers from adult zebrafish. The kcnk4b transcript encoding for TRAAK channels was cloned and found, concomitantly with activity of mechano-gated K+ channels, to be absent in zebrafish fast skeletal muscles at the larval stage but arising around 1 mo of age. The transfer of the kcnk4b gene in HEK cells and in the adult mouse muscle, that do not express functional TRAAK channels, led to expression and activity of mechano-gated K+ channels displaying properties comparable to native zebrafish TRAAK channels. In whole-cell voltage-clamp and current-clamp conditions, membrane stretch and heat led to activation of macroscopic K+ currents and to acceleration of the repolarization phase of action potentials respectively, suggesting that heat production and membrane deformation associated with skeletal muscle activity can control muscle excitability through TRAAK channel activation. TRAAK channels may represent a teleost-specific evolutionary product contributing to improve swimming performance for escaping predators and capturing prey at a critical stage of development.

Determination of single-molecule transport activity of OATP2B1 by measuring the number of transporter molecules using electrophysiological approach

Transporter-mediated clearance is determined by two factors, its single-molecule clearance, and expression level. However, no reliable method has been developed to evaluate them separately. This study aimed to develop a reliable method for evaluating the single-molecule activity of membrane transporters, such as organic anion transporting polypeptide (OATP) 2B1.HEK293 cells that co-expressed large conductance calcium-activated potassium (BK) channel and OATP2B1 were established and used for the following experiments. i) BK channel-mediated whole-cell conductance was measured using patch-clamp technique and divided by its unitary conductance to estimate the number of channels on plasma membrane (QI). ii) Using plasma membrane fraction, quantitative targeted absolute proteomics determined the stoichiometric ratio (ρ) of OATP2B1 to BK channel. iii) The uptake of estrone 3-sulfate was evaluated to calculate the Michaelis constant and uptake clearance (CL) per cell. Single-molecule clearance (CLint) was calculated by dividing CL by QI·ρ.QI and ρ values were estimated to be 916 and 2.16, respectively, yielding CLint of 5.23 fL/min/molecule. We successfully developed a novel method to reliably measure the single-molecule activity of a transporter, which could be used to evaluate the influences of factors such as genetic variations and post-translational modifications on the intrinsic activity of transporters.

Cx43 Hemichannel and Panx1 Channel Modulation by Gap19 and 10Panx1 Peptides.

Cx43 hemichannels (HCs) and Panx1 channels are two genetically distant protein families. Despite the lack of sequence homology, Cx43 and Panx1 channels have been the subject of debate due to their overlapping expression and the fact that both channels present similarities in terms of their membrane topology and electrical properties. Using the mimetic peptides Gap19 and 10Panx1, this study aimed to investigate the cross-effects of these peptides on Cx43 HCs and Panx1 channels. The single-channel current activity from stably expressing HeLa-Cx43 and C6-Panx1 cells was recorded using patch-clamp experiments in whole-cell voltage-clamp mode, demonstrating 214 pS and 68 pS average unitary conductances for the respective channels. Gap19 was applied intracellularly while 10Panx1 was applied extracellularly at different concentrations (100, 200 and 500 μM) and the average nominal open probability (NPo) was determined for each testing condition. A concentration of 100 µM Gap19 more than halved the NPo of Cx43 HCs, while 200 µM 10Panx1 was necessary to obtain a half-maximal NPo reduction in the Panx1 channels. Gap19 started to significantly inhibit the Panx1 channels at 500 µM, reducing the NPo by 26% while reducing the NPo of the Cx43 HCs by 84%. In contrast 10Panx1 significantly reduced the NPo of the Cx43 HCs by 37% at 100 µM and by 83% at 200 µM, a concentration that caused the half-maximal inhibition of the Panx1 channels. These results demonstrate that 10Panx1 inhibits Cx43 HCs over the 100-500 µM concentration range while 500 µM intracellular Gap19 is necessary to observe some inhibition of Panx1 channels.

Functional characterization of the transient receptor potential melastatin 2 (TRPM2) cation channel from Nematostella vectensis reconstituted into lipid bilayer

Transient receptor potential melastatin 2 (TRPM2) cation channel activity is required for insulin secretion, immune cell activation and body heat control. Channel activation upon oxidative stress is involved in the pathology of stroke and neurodegenerative disorders. Cytosolic Ca2+, ADP-ribose (ADPR) and phosphatidylinositol-4,5-bisphosphate (PIP2) are the obligate activators of the channel. Several TRPM2 cryo-EM structures have been resolved to date, yet functionality of the purified protein has not been tested. Here we reconstituted overexpressed and purified TRPM2 from Nematostella vectensis (nvTRPM2) into lipid bilayers and found that the protein is fully functional. Consistent with the observations in native membranes, nvTRPM2 in lipid bilayers is co-activated by cytosolic Ca2+ and either ADPR or ADPR-2′-phosphate (ADPRP). The physiological metabolite ADPRP has a higher apparent affinity than ADPR. In lipid bilayers nvTRPM2 displays a large linear unitary conductance, its open probability (Po) shows little voltage dependence and is stable over several minutes. Po is high without addition of exogenous PIP2, but is largely blunted by treatment with poly-l-Lysine, a polycation that masks PIP2 headgroups. These results indicate that PIP2 or some other activating phosphoinositol lipid co-purifies with nvTRPM2, suggesting a high PIP2 binding affinity of nvTRPM2 under physiological conditions.

Interactions between selectivity filter and pore helix control filter gating in the MthK channel.

K+ channel activity can be limited by C-type inactivation, which is likely initiated in part by dissociation of K+ ions from the selectivity filter and modulated by the side chains that surround it. While crystallographic and computational studies have linked inactivation to a "collapsed" selectivity filter conformation in the KcsA channel, the structural basis for selectivity filter gating in other K+ channels is less clear. Here, we combined electrophysiological recordings with molecular dynamics simulations, to study selectivity filter gating in the model potassium channel MthK and its V55E mutant (analogous to KcsA E71) in the pore-helix. We found that MthK V55E has a lower open probability than the WT channel, due to decreased stability of the open state, as well as a lower unitary conductance. Simulations account for both of these variables on the atomistic scale, showing that ion permeation in V55E is altered by two distinct orientations of the E55 side chain. In the "vertical" orientation, in which E55 forms a hydrogen bond with D64 (as in KcsA WT channels), the filter displays reduced conductance compared to MthK WT. In contrast, in the "horizontal" orientation, K+ conductance is closer to that of MthK WT; although selectivity filter stability is lowered, resulting in more frequent inactivation. Surprisingly, inactivation in MthK WT and V55E is associated with a widening of the selectivity filter, unlike what is observed for KcsA and reminisces recent structures of inactivated channels, suggesting a conserved inactivation pathway across the potassium channel family.

Mechanism of external K+ sensitivity of KCNQ1 channels.

KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.

Characterization of human AQP5 mutations found in patients suffering from palmoplantar keratosis (Bothnian type).

Aquaporins are structurally well conserved within all kingdoms of life and can be divided into two main groups: (i) orthodox aquaporins conducting only water molecules, and (ii) aquaglyceroporins channeling water and other small uncharged solutes. Water transport through these integral membrane channels takes place in a single-file fashion through the pores of each of the protomers that form tetrameric units in cell membranes.[PP1] Here we focus on the functional consequences of mutations in the orthodox aquaporin-5 (Aqp5), commonly expressed in salivary glands, digestive and respiratory systems, kidneys, the CNS, and sweat glands. These mutations cause hereditary palmoplantar keratoma (Bothnia type; PPKB), marked by hyperkeratotic lesions on the surface of palms and soles. They differ in the positions of the substituted amino acids as well as backbone interactions. We overexpressed some of the mutations in S. cerevisiae as YFP-fusion proteins, purified and reconstituted the water channels into lipid vesicles, and monitored osmotic vesicle deflation under stopped-flow conditions. Additionally assessing the number of reconstituted channels by fluorescence correlation spectroscopy allowed calculation of unitary water channel conductances. The significance of the alterations in water channeling capabilities is discussed with respect to disease genesis.

Function modulation of MDSC mHv1 channel by N terminal length.

Voltage-gated proton channel (Hv1) regulate the immunosuppressive function of myeloid-derived suppressor cells (MDSC) in mice. We asked if there were Hv1 isoforms present in MDSC. A prediction made by the NCBI showed that there could be longer isoforms of mouse Hv1, of 42 and four extra amino acid residues, called mHv1.2 and mHv1.3, respectively. These additional residues are predicted to express at the very beginning of the N terminal domain. We validated the prediction in vitro by RT-PCR, founding two isoforms of mHv1 and cloned them. We expressed these isoforms into Xenopus laevis oocytes for proton current recording studies by macropatch voltage clamp. All these isoforms studied satisfy the typical proton channel properties regarding the selectivity and channel activation kinetics. However, these isoforms exhibited clear differences both in their voltage-dependent gating and kinetics: the longer isoform, mHv1.2 exhibited the most right-displaced V0.5, followed by mHv1.3 and mHv1.1, while the most accelerated opening kinetics belonged to mHv1.1, followed by mHv1.3 and finally mHv1.2. We studied the unitary conductance of these isoforms by non-stationary noise analysis, evidencing values related to the fS order. All the cloned isoforms are expressed in the membrane as dimers, as shown by the estimation of the charge coupled to the opening by limit slope technique. These results strongly suggest that all these isoforms were typical Hv1 channels that possess unique biophysical properties. Since the different isoforms exhibited exclusively differences in their N terminal, the longer the N terminal length, the slower the opening kinetics and the shifted to the right the GV curve. We propose that the differences in the voltage-dependent gating exhibited by the isoforms are due by the extension of their N terminal.

The unique structural characteristics of the Kir 7.1 inward rectifier potassium channel: a novel player in energy homeostasis control.

The inward rectifier potassium channel Kir7.1, encoded by the KCNJ13 gene, is a tetramer composed of two-transmembrane domain-spanning monomers, closer in homology to Kir channels associated with potassium transport such as Kir1.1, 1.2, and 1.3. Compared with other channels, Kir7.1 exhibits small unitary conductance and low dependence on external potassium. Kir7.1 channels also show a phosphatidylinositol 4,5-bisphosphate (PIP2) dependence for opening. Accordingly, retinopathy-associated Kir7.1 mutations mapped at the binding site for PIP2 resulted in channel gating defects leading to channelopathies such as snowflake vitreoretinal degeneration and Leber congenital amaurosis in blind patients. Lately, this channel's role in energy homeostasis was reported due to the direct interaction with the melanocortin type 4 receptor (MC4R) in the hypothalamus. As this channel seems to play a multipronged role in potassium homeostasis and neuronal excitability, we will discuss what is predicted from a structural viewpoint and its possible implications for hunger control.

Alkaline-sensitive two-pore domain potassium channels form functional heteromers in pancreatic β-cells

Two-pore domain K+ channels (K2P channels), active as dimers, produce inhibitory currents regulated by a variety of stimuli. Among them, TWIK1-related alkalinization-activated K+ channel 1 (TALK1), TWIK1-related alkalinization-activated K+ channel 2 (TALK2), and TWIK1-related acid-sensitive K+ channel 2 (TASK2) form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding these proteins are clustered at chromosomal region 6p21 and coexpressed in multiple tissues, including the pancreas. The question whether these channels form functional heteromers remained open. By analyzing single-cell transcriptomic data, we show that these channels are coexpressed in insulin-secreting pancreatic β-cells. Using in situ proximity ligation assay and electrophysiology, we show that they form functional heterodimers both upon heterologous expression and under native conditions in human pancreatic β-cells. We demonstrate that heteromerization of TALK2 with TALK1 or with TASK2 endows TALK2 with sensitivity to extracellular alkalosis in the physiological range. We further show that the association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of the potential physiological and pathophysiological roles of TALK1/TALK2/TASK2 channels, not only in insulin-secreting cells but also in the many other tissues in which they are coexpressed.

Unitary conductance of the voltage-sensitive proton channel HV1

Unitary conductance of the voltage-sensitive proton channel HV1