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High K+ supplementation is correlated with a lower risk of the composite of death, major cardiovascular events, and ameliorated blood pressure, but the exact mechanisms have not been established. Inwardly rectifying K+ (Kir) channels expressed in the basolateral membrane of the distal nephron play an essential role in maintaining electrolyte homeostasis. Mutations in this channel family have been shown to result in strong disturbances in electrolyte homeostasis, among other symptoms. Kir7.1 is a member of the ATP-regulated subfamily of Kir channels. However, its role in renal ion transport and its effect on blood pressure have yet to be established. Our results indicate the localization of Kir7.1 to the basolateral membrane of aldosterone-sensitive distal nephron cells. To examine the physiological implications of Kir7.1, we generated a knockout of Kir7.1 (Kcnj13) in Dahl salt-sensitive (SS) rats and deployed chronic infusion of a specific Kir7.1 inhibitor, ML418, in the wild-type Dahl SS strain. Knockout of Kcnj13 (Kcnj13-/-) resulted in embryonic lethality. Heterozygous Kcnj13þ/- rats revealed an increase in K+ excretion on a normal-salt diet but did not exhibit a difference in blood pressure development or plasma electrolytes after 3 wk of a high-salt diet. Wild-type Dahl SS rats exhibited increased renal Kir7.1 expression when dietary K+ was increased. K+ supplementation also demonstrated that Kcnj13þ/- rats excreted more K+ on normal salt. The development of hypertension was not different when rats were challenged with high salt for 3 wk, although Kcnj13þ/- rats excrete less Na+. Interestingly, chronic infusion of ML418 significantly increased Na+ and Cl- excretion after 14 days of high salt but did not alter salt-induced hypertension development. Here, we found that reduction of Kir7.1 function, either through genetic ablation or pharmacological inhibition, can influence renal electrolyte excretion but not to a sufficient degree to impact the development of SS hypertension. [ABSTRACT FROM AUTHOR]

We created a single-compartment computer model of a C[O.sub.2] chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in C[O.sub.2] chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current ([I.sub.KA]), a delayed rectifier current ([I.sub.KDR]), a calcium-activated potassium current ([I.sub.KCa]), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation, pH-dependent inhibition of [I.sub.KA] initiated the response to C[O.sub.2], but hypercapnic inhibition of [I.sub.KDR] and [I.sub.KCa] affected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH ([pH.sub.i]) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to C[O.sub.2] chemosensitivity, but the primary sensor is probably [I.sub.KA]. [pH.sub.i] may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either [pH.sub.i] or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of [pH.sub.i] regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of [pH.sub.i] regulation during hypercapnia probably depends on intracellular activation of phi regulation but extracellular inhibition of [pH.sub.i] regulation. These general principles are applicable to all C[O.sub.2] chemosensory cells in vertebrate and invertebrate neurons. hypercapnia; potassium channels; computer modeling; central chemoreceptors

Elevated levels of carbon dioxide increase lung ventilation in Helix aspersa. The hypercapnic response originates from a discrete respiratory chemosensory region in the dorsal subesophageal ganglia that contains C[O.sub.2]-sensitive neurons. We tested the hypothesis that pH-dependent inhibition of potassium channels in neurons in this region mediated the chemosensory response to C[O.sub.2]. Cells isolated from the dorsal subesophageal ganglia retained C[O.sub.2] chemosensitivity and exhibited membrane depolarization and/or an increase in input resistance during an acid challenge. Isolated somata expressed two voltage-dependent potassium channels, an A-type and a delayed-rectifier-type channel ([I.sub.KA] and [I.sub.KDR]). Both conductances were inhibited during hypercapnia. The pattern of voltage dependence indicated that [I.sub.KA] was affected by extracellular or intracellular pH, but the activity of [I.sub.KDR] was modulated by extracellular pH only. Application of inhibitors of either channel mimicked many of the effects of acidification in isolated cells and neurons in situ. We also detected evidence of a pH-sensitive calcium-activated potassium channel ([I.sub.KCa]) in neurons in situ. The results of these studies support the hypothesis that [I.sub.KA] initiates the chemosensory response, and [I.sub.KDR] and [I.sub.KCa] prolong the period of activation of C[O.sub.2]-sensitive neurons. Thus multiple potassium channels are inhibited by acidosis, and the combined effect of pH-dependent inhibition of these channels enhances neuronal excitability and mediates C[O.sub.2] chemosensory responses in H. aspersa. We did not find a single 'chemosensory channel,' and the chemosensitive channels that we did find were not unique in any way that we could detect. The protein 'machinery' of C[O.sub.2] chemosensitivity is probably widespread among neurons, and the selection process whereby a neuron acts or does not act as a respiratory C[O.sub.2] chemosensor probably depends on the resting membrane potential and synaptic connectivity. carbon dioxide

Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryoelectron microscopy (cryo-EM) structures of nonnative LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c-/-HCT116 cells are poorly activated by low intracellular ionic strength (m) and insensitive to cell swelling with normal m. Combining low m with swelling modestly activates LRRC8A, allowing characterization of pore properties. VRACs are strongly inhibited by 10 mM 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, an LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ~90% by 10 mM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly m dependent. At normal intracellular m, LRRC8A þ LRRC8C heteromers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular m. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage dependence under low m conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heteromeric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homomeric channels. [ABSTRACT FROM AUTHOR]

Diuretics acting on specific nephron segments to inhibit Na+ reabsorption have been used clinically for decades; however, drug interactions, tolerance, and derangements in serum K+ complicate their use to achieve target blood pressure. ROMK is an attractive diuretic target, in part, because its inhibition is postulated to indirectly inhibit the bumetanide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the amiloride- and benzamil-sensitive epithelial Na+ channel (ENaC). The development of small-molecule ROMK inhibitors has created opportunities for exploring the physiological responses to ROMK inhibition. The present study evaluated how inhibition of ROMK alone or in combination with NKCC2, ENaC, or the hydrochlorothiazide (HCTZ) target NCC alter fluid and electrolyte transport in the nephron. The ROMK inhibitor VU591 failed to induce diuresis when administered orally to rats. However, another ROMK inhibitor, termed compound A, induced a robust natriuretic diuresis without kaliuresis. Compound A produced additive effects on urine output and Na+ excretion when combined with HCTZ, amiloride, or benzamil, but not when coadministered with bumetanide, suggesting that the major diuretic target site is the thick ascending limb (TAL). Interestingly, compound A inhibited the kaliuretic response induced by bumetanide and HCTZ, an effect we attribute to inhibition of ROMK-mediated K+ secretion in the TAL and CD. Compound A had no effect on heterologously expressed flow-sensitive large-conductance Ca2+-activated K+ channels (Slo1/β1). In conclusion, compound A represents an important new pharmacological tool for investigating the renal consequences of ROMK inhibition and therapeutic potential of ROMK as a diuretic target. [ABSTRACT FROM AUTHOR]

Changes in intracellular pH (pH(sub i)) but not changes in extracellular pH regulate alpha,beta,gamma-epithelial sodium channel currents. Channel activity was reduced by acidification and increased by alkalization via a voltage-independent mechanism. Single-channel open probability and single-channel open time were reduced and single-channel closed time were increased by a reduction of pH(sub i), without any change in single-channel conductance.

As the molecular revolution continues to inform a deeper understanding of disease mechanisms and pathways, there exist unprecedented opportunities for translating discoveries at the bench into novel therapies for improving human health. Despite the availability of several different classes of antihypertensive medications, only about half of the 67 million Americans with hypertension manage their blood pressure appropriately. A broader selection of structurally diverse antihypertensive drugs acting through different mechanisms would provide clinicians with greater flexibility in developing effective treatment regimens for an increasingly diverse and aging patient population. An emerging body of physiological, genetic, and pharmacological evidence has implicated several renal ion-transport proteins, or regulators thereof, as novel, yet clinically unexploited, diuretic targets. These include the renal outer medullary potassium channel, ROMK (Kir1.1), Kir4.1/5.1 potassium channels, ClC-Ka/b chloride channels, UTA/B urea transporters, the chloride/bicarbonate exchanger pendrin, and the STE20/SPS1-related proline/alanine- rich kinase (SPAK). The molecular pharmacology of these putative targets is poorly developed or lacking altogether; however, recent efforts by a few academic and pharmaceutical laboratories have begun to lessen this critical barrier. Here, we review the evidence in support of the aforementioned proteins as novel diuretic targets and highlight examples where progress toward developing small-molecule pharmacology has been made. [ABSTRACT FROM AUTHOR]

Elevated levels of carbon dioxide increase lung ventilation in Helix aspersa. The hypercapnic response originates from a discrete respiratory chemosensory region in the dorsal subesophageal ganglia that contains CO2-sensitive neurons. We tested the hypothesis that pH-dependent inhibition of potassium channels in neurons in this region mediated the chemosensory response to CO2. Cells isolated from the dorsal subesophageal ganglia retained CO2 chemosensitivity and exhibited membrane depolarization and/or an increase in input resistance during an acid challenge. Isolated somata expressed two voltage-dependent potassium channels, an A-type and a delayed-rectifier-type channel (IRA and IKDR). Both conductances were inhibited during hypercapnia. The pattern of voltage dependence indicated that IKA was affected by extracellular or intracellular pH, but the activity of IKDR was modulated by extracellular pH only. Application of inhibitors of either channel mimicked many of the effects of acidification in isolated cells and neurons in situ. We also detected evidence of a pH-sensitive calcium-activated potassium channel (IKCa) in neurons in situ. The results of these studies support the hypothesis that IKA initiates the chemosensory response, and IKDR and IKCa prolong the period of activation of CO2-sensitive neurons. Thus multiple potassium channels are inhibited by acidosis, and the combined effect of pH-dependent inhibition of these channels enhances neuronal excitability and mediates CO2 chemosensory responses in H. aspersa. We did not find a single ‘chemosensory channel,’ and the chemosensitive channels that we did find were not unique in any way that we could detect. The protein ‘machinery’ of CO2 chemosensitivity is probably widespread among neurons, and the selection process whereby a neuron acts or does not act as a respiratory CO2 chemosensor probably depends on the resting membrane potential and synaptic connectivity. [ABSTRACT FROM AUTHOR]

We created a single-compartment computer model of a CO2 chemosensory neuron using differential equations adapted from the Hodgkin-Huxley model and measurements of currents in CO2 chemosensory neurons from Helix aspersa. We incorporated into the model two inward currents, a sodium current and a calcium current, three outward potassium currents, an A-type current (IKA), a delayed rectifier current (IKDR), a calcium-activated potassium current (IKCa), and a proton conductance found in invertebrate cells. All of the potassium channels were inhibited by reduced pH. We also included the pH regulatory process to mimic the effect of the sodium-hydrogen exchanger (NHE) described in these cells during hypercapnic stimulation. The model displayed chemosensory behavior (increased spike frequency during acid stimulation), and all three potassium channels participated in the chemosensory response and shaped the temporal characteristics of the response to acid stimulation, pH-dependent inhibition of IKA initiated the response to CO2, but hypercapnic inhibition of IKDR and IKCa affected the duration of the excitatory response to hypercapnia. The presence or absence of NHE activity altered the chemosensory response over time and demonstrated the inadvisability of effective intracellular pH (pHi) regulation in cells designed to act as chemostats for acid-base regulation. The results of the model indicate that multiple channels contribute to CO2 chemosensitivity, but the primary sensor is probably IKA. pHi may be a sufficient chemosensory stimulus, but it may not be a necessary stimulus: either pHi or extracellular pH can be an effective stimuli if chemosensory neurons express appropriate pH-sensitive channels. The lack of pHi regulation is a key feature determining the neuronal activity of chemosensory cells over time, and the balanced lack of pHi regulation during hypercapnia probably depends on intracellular activation of pHi regulation but extracellular inhibition of pHi regulation. These general principles are applicable to all CO2 chemosensory cells in vertebrate and invertebrate neurons. [ABSTRACT FROM AUTHOR]

States that alpha, beta, gamma-epithelial sodium channel (ENaC) currents were regulated by changes in intracellular pH. Characterization of the ENaC currents; How acidification inhibits alpha, beta, gamma-ENaC currents; Effect of intracellular pH on single-channel ENaC currents.

High K + supplementation is correlated with a lower risk of the composite of death, major cardiovascular events, and ameliorated blood pressure, but the exact mechanisms have not been established. Inwardly rectifying K + (K ir ) channels expressed in the basolateral membrane of the distal nephron play an essential role in maintaining electrolyte homeostasis. Mutations in this channel family have been shown to result in strong disturbances in electrolyte homeostasis, among other symptoms. K ir 7.1 is a member of the ATP-regulated subfamily of K ir channels. However, its role in renal ion transport and its effect on blood pressure have yet to be established. Our results indicate the localization of K ir 7.1 to the basolateral membrane of aldosterone-sensitive distal nephron cells. To examine the physiological implications of K ir 7.1, we generated a knockout of K ir 7.1 ( Kcnj13 ) in Dahl salt-sensitive (SS) rats and deployed chronic infusion of a specific K ir 7.1 inhibitor, ML418, in the wild-type Dahl SS strain. Knockout of Kcnj13 ( Kcnj13 -/- ) resulted in embryonic lethality. Heterozygous Kcnj13 +/- rats revealed an increase in K + excretion on a normal-salt diet but did not exhibit a difference in blood pressure development or plasma electrolytes after 3 wk of a high-salt diet. Wild-type Dahl SS rats exhibited increased renal K ir 7.1 expression when dietary K + was increased. K + supplementation also demonstrated that Kcnj13 +/- rats excreted more K + on normal salt. The development of hypertension was not different when rats were challenged with high salt for 3 wk, although Kcnj13 +/- rats excrete less Na + . Interestingly, chronic infusion of ML418 significantly increased Na + and Cl - excretion after 14 days of high salt but did not alter salt-induced hypertension development. Here, we found that reduction of K ir 7.1 function, either through genetic ablation or pharmacological inhibition, can influence renal electrolyte excretion but not to a sufficient degree to impact the development of SS hypertension. NEW & NOTEWORTHY To investigate the role of the K ir 7.1 channel in salt-sensitive hypertension, its function was examined using complementary genetic and pharmacological approaches. The results revealed that although reducing K ir 7.1 expression had some impact on maintaining K + and Na + balance, it did not lead to a significant change in the development or magnitude of salt-induced hypertension. Hence, it is probable that K ir 7.1 works in conjunction with other basolateral K + channels to fine-tune membrane potential.

Inward rectifying potassium (Kir) channels play important roles in both excitable and nonexcitable cells of various organ systems and could represent valuable new drug targets for cardiovascular, metabolic, immune, and neurological diseases. In nonexcitable epithelial cells of the kidney tubule, for example, Kir1.1 ( KCNJ1 ) and Kir4.1 ( KCNJ10 ) are linked to sodium reabsorption in the thick ascending limb of Henle's loop and distal convoluted tubule, respectively, and have been explored as novel-mechanism diuretic targets for managing hypertension and edema. G protein-coupled Kir channels (Kir3) channels expressed in the central nervous system are critical effectors of numerous signal transduction pathways underlying analgesia, addiction, and respiratory-depressive effects of opioids. The historical dearth of pharmacological tool compounds for exploring the therapeutic potential of Kir channels has led to a molecular target-based approach using high-throughput screen (HTS) of small-molecule libraries and medicinal chemistry to develop "next-generation" Kir channel modulators that are both potent and specific for their targets. In this article, we review recent efforts focused specifically on discovery and improvement of target-selective molecular probes. The reader is introduced to fluorescence-based thallium flux assays that have enabled much of this work and then provided with an overview of progress made toward developing modulators of Kir1.1 (VU590, VU591), Kir2.x (ML133), Kir3.X (ML297, GAT1508, GiGA1, VU059331), Kir4.1 (VU0134992), and Kir7.1 (ML418). We discuss what is known about the small molecules' molecular mechanisms of action, in vitro and in vivo pharmacology, and then close with our view of what critical work remains to be done.

Leucine-rich repeat-containing 8 (LRRC8) volume-regulated anion channels (VRACs) play important physiological roles in diverse cell types and may represent therapeutic targets for various diseases. To date, however, the pharmacological tools for evaluating the druggability of VRACs have been limited to inhibitors, as no activators of the channel have been reported. We therefore performed a fluorescence-based high-throughput screening (HTS) of 1,184 Food and Drug Administration-approved drugs for compounds that increase VRAC activity. The most potent VRAC potentiator identified was zinc pyrithione (ZPT), which is used commercially as an antifouling agent and for treating dandruff and other skin disorders. In intracellular Yellow Fluorescent Protein YFP(F46L/H148Q/I152L)-quenching assays, ZPT potentiates the rate and extent of swelling-induced iodide influx dose dependently with a half-maximal effective concentration (EC 50 ) of 5.7 µM. Whole cell voltage-clamp experiments revealed that coapplication of hypotonic solution and 30 µM ZPT to human embryonic kidney 293 or human colorectal carcinoma 116 cells increases the rate of swelling-induced VRAC activation by approximately 10-fold. ZPT potentiates swelling-induced VRAC currents after currents have reached a steady state and activates currents in the absence of cell swelling. Neither ZnCl 2 nor free pyrithione activated VRAC; however, treating cells with a mixture of ZnCl 2 and pyrithione led to robust channel activation. Finally, the effects of ZPT on VRAC were inhibited by reactive oxygen species (ROS) scavenger N -acetylcysteine (NAC) and NAD(P)H oxidase inhibitor diphenyleneiodonium chloride, suggesting the mechanism of action involves ROS generation. The discovery of ZPT as a potentiator/activator of VRAC demonstrates the utility of HTS for identifying small-molecule modulators of VRAC and adds to a growing repertoire of pharmacological tool compounds for probing the molecular physiology and regulation of this important channel.

Swelling-activated volume-regulated anion channels (VRACs) are heteromeric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryoelectron microscopy (cryo-EM) structures of nonnative LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c -/- HCT116 cells are poorly activated by low intracellular ionic strength (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A, allowing characterization of pore properties. VRACs are strongly inhibited by 10 µM 4-[(2-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1 H -inden-5-yl)oxy]butanoic acid (DCPIB) in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, an LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ∼90% by 10 µM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ dependent. At normal intracellular µ, LRRC8A + LRRC8C heteromers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heteromeric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homomeric channels.

Volume-regulated anion channels (VRACs) encoded by the leucine-rich repeat containing 8 ( LRRC8 ) gene family play critical roles in myriad cellular processes and might represent druggable targets. The dearth of pharmacological compounds available for studying VRAC physiology led us to perform a high-throughput screen of 1,184 of US Food and Drug Administration-approved drugs for novel VRAC modulators. We discovered the cysteinyl leukotriene receptor 1 (CysLT1R) antagonist, pranlukast, as a novel inhibitor of endogenous VRAC expressed in human embryonic kidney 293 (HEK293) cells. Pranlukast inhibits VRAC voltage-independently, reversibly, and dose-dependently with a maximal efficacy of only ~50%. The CysLT1R pathway has been implicated in activation of VRAC in other cell types, prompting us to test whether pranlukast requires the CysLT1R for inhibition of VRAC. Quantitative PCR analysis demonstrated that CYSLTR1 mRNA is virtually undetectable in HEK293 cells. Furthermore, the CysLT1R agonist leukotriene D4 had no effect on VRAC activity and failed to stimulate G q -coupled receptor signaling. Heterologous expression of the CysLT1R reconstituted LTD4-CysLT1R- G q -calcium signaling in HEK293 cells but had no effect on VRAC inhibition by pranlukast. Finally, we show the CysLT1R antagonist zafirlukast inhibits VRAC with an IC 50 of ~17 µM and does so with full efficacy. Our data suggest that both pranlukast and zafirlukast are likely direct channel inhibitors that work independently of the CysLT1R. This study provides clarifying insights into the putative role of leukotriene signaling in modulation of VRAC and identifies two new chemical scaffolds that can be used for development of more potent and specific VRAC inhibitors.