Extracellular hemin is a reverse use-dependent gating modifier of cardiac voltage-gated Na+ channels.

Heme (Fe2+-protoporphyrin IX) is a well-known protein prosthetic group; however, heme and hemin (Fe3+-protoporphyrin IX) are also increasingly viewed as signaling molecules. Among the signaling targets are numerous ion channels, with intracellular-facing heme-binding sites modulated by heme and hemin in the sub-µM range. Much less is known about extracellular hemin, which is expected to be more abundant, in particular after hemolytic insults. Here we show that the human cardiac voltage-gated sodium channel hNaV1.5 is potently inhibited by extracellular hemin (IC 50 ≈ 80 nM), while heme, dimethylhemin, and protoporphyrin IX are ineffective. Hemin is selective for hNaV1.5 channels: hNaV1.2, hNaV1.4, hNaV1.7, and hNaV1.8 are insensitive to 1 µM hemin. Using domain chimeras of hNaV1.5 and rat rNaV1.2, domain II was identified as the critical determinant. Mutation N803G in the domain II S3/S4 linker largely diminished the impact of hemin on the cardiac channel. This profile is reminiscent of the interaction of some peptide voltage-sensor toxins with NaV channels. In line with a mechanism of select gating modifiers, the impact of hemin on NaV1.5 channels is reversely use dependent, compatible with an interaction of hemin and the voltage sensor of domain II. Extracellular hemin thus has potential to modulate the cardiac function.

Large-conductance Ca2+- and voltage-gated K+ channels form and break interactions with membrane lipids during each gating cycle.

Membrane depolarization and intracellular Ca2+ promote activation of the large-conductance Ca2+- and voltage-gated (Slo1) big potassium (BK) channel. We examined the physical interactions that stabilize the closed and open conformations of the ion conduction gate of the human Slo1 channel using electrophysiological and computational approaches. The results show that the closed conformation is stabilized by intersubunit ion-ion interactions involving negative residues (E321 and E324) and positive residues (329RKK331) at the cytoplasmic ends of the transmembrane S6 segments ("RKK ring"). When the channel gate is open, the RKK ring is broken and the positive residues instead make electrostatic interactions with nearby membrane lipid oxygen atoms. E321 and E324 are stabilized by water. When the 329RKK331 residues are mutated to hydrophobic amino acids, these residues form even stronger hydrophobic interactions with the lipid tails to promote the open conformation, shifting the voltage dependence of activation to the negative direction by up to 400 mV and stabilizing the selectivity filter region. Thus, the RKK segment forms electrostatic interactions with oxygen atoms from two sources, other amino acid residues (E321/E324), and membrane lipids, depending on the gate status. Each time the channel opens and closes, the aforementioned interactions are formed and broken. This lipid-dependent Slo1 gating may explain how amphipathic signaling molecules and pharmacologically active agents influence the channel activity, and a similar mechanism may be operative in other ion channels.

Impact of intracellular hemin on N-type inactivation of voltage-gated K+ channels.

N-type inactivation of voltage-gated K+ channels is conferred by the N-terminal "ball" domains of select pore-forming α subunits or of auxiliary ß subunits, and influences electrical cellular excitability. Here, we show that hemin impairs inactivation of K+ channels formed by Kv3.4 α subunits as well as that induced by the subunits Kvß1.1, Kvß1.2, and Kvß3.1 when coexpressed with α subunits of the Kv1 subfamily. In Kvß1.1, hemin interacts with cysteine and histidine residues in the N terminus (C7 and H10) with high affinity (EC50 100 nM). Similarly, rapid inactivation of Kv4.2 channels induced by the dipeptidyl peptidase-like protein DPP6a is also sensitive to hemin, and the DPP6a mutation C13S eliminates this dependence. The results suggest a common mechanism for a dynamic regulation of Kv channel inactivation by heme/hemin in N-terminal ball domains of Kv α and auxiliary ß subunits. Free intracellular heme therefore has the potential to regulate cellular excitability via modulation of Kv channel inactivation.

Modulation of K+ channel N-type inactivation by sulfhydration through hydrogen sulfide and polysulfides.

Fast N-type inactivation of voltage-gated K+ (Kv) channels is important in fine-tuning of cellular excitability. To serve diverse cellular needs, N-type inactivation is regulated by numerous mechanisms. Here, we address how reactive sulfur species-the gaseous messenger H2S and polysulfides-affect N-type inactivation of the mammalian Kv channels Kv1.4 and Kv3.4. In both channels, the H2S donor NaHS slowed down inactivation with varying potency depending on the "aging" of NaHS solution. Polysulfides were > 1000 times more effective than NaHS with the potency increasing with the number of sulfur atoms (Na2S2 < Na2S3 < Na2S4). In Kv1.4, C13 in the N-terminal ball domain mediates the slowing of inactivation. In recombinant protein exposed to NaHS or Na2S4, a sulfur atom is incorporated at C13 in the protein. In Kv3.4, the N terminus harbors two cysteine residues (C6, C24), and C6 is of primary importance for channel regulation by H2S and polysulfides, with a minor contribution from C24. To fully eliminate the dependence of N-type inactivation on sulfhydration, both cysteine residues must be removed (C6S:C24S). Sulfhydration of a single cysteine residue in the ball-and-chain domain modulates the speed of inactivation but does not remove it entirely. In both Kv1.4 and Kv3.4, polysulfides affected the N-terminal cysteine residues when assayed in the whole-cell configuration; on-cell recordings confirmed that polysulfides also modulate K+ channel inactivation with undisturbed cytosol. These findings have collectively identified reactive sulfur species as potent modulators of N-type inactivation in mammalian Kv channels.

Atomic determinants of BK channel activation by polyunsaturated fatty acids.

Docosahexaenoic acid (DHA), a polyunsaturated ω-3 fatty acid enriched in oily fish, contributes to better health by affecting multiple targets. Large-conductance Ca2+- and voltage-gated Slo1 BK channels are directly activated by nanomolar levels of DHA. We investigated DHA-channel interaction by manipulating both the fatty acid structure and the channel composition through the site-directed incorporation of unnatural amino acids. Electrophysiological measurements show that the para-group of a Tyr residue near the ion conduction pathway has a critical role. To robustly activate the channel, ionization must occur readily by a fatty acid for a good efficacy, and a long nonpolar acyl tail with a Z double bond present at the halfway position for a high affinity. The results suggest that DHA and the channel form an ion-dipole bond to promote opening and demonstrate the channel druggability. DHA, a marine-derived nutraceutical, represents a promising lead compound for rational drug design and discovery.

Reactive species modify NaV1.8 channels and affect action potentials in murine dorsal root ganglion neurons.

Dorsal root ganglion (DRG) neurons are important relay stations between the periphery and the central nervous system and are essential for somatosensory signaling. Reactive species are produced in a variety of physiological and pathophysiological conditions and are known to alter electric signaling. Here we studied the influence of reactive species on the electrical properties of DRG neurons from mice with the whole-cell patch-clamp method. Even mild stress induced by either low concentrations of chloramine-T (10 µM) or low-intensity blue light irradiation profoundly diminished action potential frequency but prolonged single action potentials in wild-type neurons. The impact on evoked action potentials was much smaller in neurons deficient of the tetrodotoxin (TTX)-resistant voltage-gated sodium channel NaV1.8 (NaV1.8(-/-)), the channel most important for the action potential upstroke in DRG neurons. Low concentrations of chloramine-T caused a significant reduction of NaV1.8 peak current and, at higher concentrations, progressively slowed down inactivation. Blue light had a smaller effect on amplitude but slowed down NaV1.8 channel inactivation. The observed effects were less apparent for TTX-sensitive NaV channels. NaV1.8 is an important reactive-species-sensitive component in the electrical signaling of DRG neurons, potentially giving rise to loss-of-function and gain-of-function phenomena depending on the type of reactive species and their effective concentration and time of exposure.

A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder.

We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G>A, p.Arg394His). KCNH7 c.1181G>A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G>A had the highest enrichment among individuals with bipolar spectrum disorder (χ(2) = 7.3) and the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031) and any major affective disorder (P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.

Mechanism of the modulation of BK potassium channel complexes with different auxiliary subunit compositions by the omega-3 fatty acid DHA.

Large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels are well known for their functional versatility, which is bestowed in part by their rich modulatory repertoire. We recently showed that long-chain omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA) found in oily fish lower blood pressure by activating vascular BK channels made of Slo1+ß1 subunits. Here we examined the action of DHA on BK channels with different auxiliary subunit compositions. Neuronal Slo1+ß4 channels were just as well activated by DHA as vascular Slo1+ß1 channels. In contrast, the stimulatory effect of DHA was much smaller in Slo1+ß2, Slo1+LRRC26 (γ1), and Slo1 channels without auxiliary subunits. Mutagenesis of ß1, ß2, and ß4 showed that the large effect of DHA in Slo1+ß1 and Slo1+ß4 is conferred by the presence of two residues, one in the N terminus and the other in the first transmembrane segment of the ß1 and ß4 subunits. Transfer of this amino acid pair from ß1 or ß4 to ß2 introduces a large response to DHA in Slo1+ß2. The presence of a pair of oppositely charged residues at the aforementioned positions in ß subunits is associated with a large response to DHA. The Slo1 auxiliary subunits are expressed in a highly tissue-dependent fashion. Thus, the subunit composition-dependent stimulation by DHA demonstrates that BK channels are effectors of omega-3 fatty acids with marked tissue specificity.

Omega-3 fatty acids lower blood pressure by directly activating large-conductance Ca²âº-dependent K⁺ channels.

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA), found abundantly in oily fish, may have diverse health-promoting effects, potentially protecting the immune, nervous, and cardiovascular systems. However, the mechanisms underlying the purported health-promoting effects of DHA remain largely unclear, in part because molecular signaling pathways and effectors of DHA are only beginning to be revealed. In vascular smooth muscle cells, large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels provide a critical vasodilatory influence. We report here that DHA with an EC50 of ∼500 nM rapidly and reversibly activates BK channels composed of the pore-forming Slo1 subunit and the auxiliary subunit ß1, increasing currents by up to ∼20-fold. The DHA action is observed in cell-free patches and does not require voltage-sensor activation or Ca(2+) binding but involves destabilization of the closed conformation of the ion conduction gate. DHA lowers blood pressure in anesthetized wild-type but not in Slo1 knockout mice. DHA ethyl ester, contained in dietary supplements, fails to activate BK channels and antagonizes the stimulatory effect of DHA. Slo1 BK channels are thus receptors for long-chain omega-3 fatty acids, and these fatty acids--unlike their ethyl ester derivatives--activate the channels and lower blood pressure. This finding has practical implications for the use of omega-3 fatty acids as nutraceuticals for the general public and also for the critically ill receiving omega-3-enriched formulas.

Heme impairs the ball-and-chain inactivation of potassium channels.

Fine-tuned regulation of K(+) channel inactivation enables excitable cells to adjust action potential firing. Fast inactivation present in some K(+) channels is mediated by the distal N-terminal structure (ball) occluding the ion permeation pathway. Here we show that Kv1.4 K(+) channels are potently regulated by intracellular free heme; heme binds to the N-terminal inactivation domain and thereby impairs the inactivation process, thus enhancing the K(+) current with an apparent EC50 value of ∼20 nM. Functional studies on channel mutants and structural investigations on recombinant inactivation ball domain peptides encompassing the first 61 residues of Kv1.4 revealed a heme-responsive binding motif involving Cys13:His16 and a secondary histidine at position 35. Heme binding to the N-terminal inactivation domain induces a conformational constraint that prevents it from reaching its receptor site at the vestibule of the channel pore.

The Cole-Moore Effect: Still Unexplained?

In the first issue, on the first page of the Biophysical Journal in 1960, Cole and Moore provided the first confirmation of the Hodgkin and Huxley formulation of the sodium and potassium conductances that underlie the action potential. In addition, working with the squid giant axon, Cole and Moore noted that strong hyperpolarization preceding a depolarizing voltage-clamp pulse delayed the rise of the potassium conductance: once started, the time course of the rise was always the same but after significant hyperpolarization there was a long lag before the rise began. This phenomenon has come to be known as the Cole-Moore effect. Their article examines and disproves the hypothesis that the lag reflects the time required to refill the membrane with potassium ions after the ions are swept out of the membrane into the axoplasm by hyperpolarization. The work by Cole and Moore indirectly supports the idea of a membrane channel for potassium conductance. However, the mechanism of the Cole-Moore effect remains a mystery even now, buried in the structure of the potassium channel, which was completely unknown at the time.

Sodium channels as gateable non-photonic sensors for membrane-delimited reactive species.

Reactive oxygen species (ROS) and reactive oxygen intermediates (ROI) play crucial roles in physiological processes. While excessive ROS damages cells, small fluctuations in ROS levels represent physiological signals important for vital functions. Despite the physiological importance of ROS, many fundamental questions remain unanswered, such as which types of ROS occur in cells, how they distribute inside cells, and how long they remain in an active form. The current study presents a ratiometric sensor of intracellular ROS levels based on genetically engineered voltage-gated sodium channels (roNaV). roNaV can be used for detecting oxidative modification that occurs near the plasma membrane with a sensitivity similar to existing fluorescence-based ROS sensors. Moreover, roNaV has several advantages over traditional sensors because it does not need excitation light for sensing, and thus, can be used to detect phototoxic cellular modifications. In addition, the ROS dynamic range of roNaV is easily manipulated in real time by means of the endogenous channel inactivation mechanism. Measurements on ROS liberated from intracellular Lucifer Yellow and genetically encoded KillerRed have revealed an assessment of ROS lifetime in individual mammalian cells. Flashlight-induced ROS concentration decayed with two major time constants of about 10 and 1000 ms.

Identification and Characterization of GAL-021 as a Novel Breathing Control Modulator.

BACKGROUND: The authors describe the preclinical pharmacological properties of GAL-021, a novel peripheral chemoreceptor modulator. METHODS: The ventilatory effects of GAL-021 were characterized using tracheal pneumotachometry (n = 4 to 6), plethysmography (n = 5 to 6), arterial blood gas analyses (n = 6 to 11), and nasal capnography (n = 3 to 4) in naive animals and those subjected to morphine-induced respiratory depression. Morphine analgesia in rats was evaluated by tail-flick test (n = 6). Carotid body involvement in GAL-021 ventilatory effects was assessed by comparing responses in intact and carotid sinus nerve-transected rats. Hemodynamic effects of GAL-021 were evaluated in urethane-anesthetized rats (n = 7). The pharmacological profile of GAL-021 in vitro was investigated using radioligand binding, enzyme inhibition, and cellular electrophysiology assays. RESULTS: GAL-021 given intravenously stimulated ventilation and/or attenuated opiate-induced respiratory depression in rats, mice, and nonhuman primates, without decreasing morphine analgesia in rats. GAL-021 did not alter mean arterial pressure but produced a modest increase in heart rate. Ventilatory stimulation in rats was attenuated by carotid sinus nerve transection. GAL-021 inhibited KCa1.1 in GH3 cells, and the evoked ventilatory stimulation was attenuated in Slo1 mice lacking the pore-forming α-subunit of the KCa1.1 channel. CONCLUSIONS: GAL-021 behaved as a breathing control modulator in rodents and nonhuman primates and diminished opioid-induced respiratory depression without compromising opioid analgesia. It acted predominantly at the carotid body, in part by inhibiting KCa1.1 channels. Its preclinical profile qualified the compound to enter clinical trials to assess effects on breathing control disorders such as drug (opioid)-induced respiratory depression and sleep apnea.

Initial steps in the opening of a Shaker potassium channel.

The structural model of a K(V) (K(+)-selective, voltage-gated) channel in the open state is known (Protein Data Bank ID code 2R9R). Each subunit of the channel has four negatively charged residues distributed in the transmembrane segments S1, S2, and S3 that bind to and facilitate the movement within the membrane of the positively charged, voltage-sensing residues of S4. When extrapolated to the closed state, the two outermost negatively charged residues are exposed to extracellular fluid and not bound to S4 residues, all of which have theoretically been driven inward by voltage. If this closed state model is correct, these residues are available to bind external cations. We examined the effects of La(3+) on voltage-gated Shaker K(+) channels. Addition of the trivalent cation La(3+) (50 µM) extracellularly markedly prolongs the lag that precedes channel opening and slows the subsequent rise of K(+) current (I(K)) at all voltages. Decay kinetics of I(K) at negative voltages are unaltered. Gating current (I(g)) recorded from a nonconducting mutant shows that La(3+) reduces the initial amplitude of I(g) nearly twofold. We postulate that, in the resting state, La(3+) binds to the unoccupied, outermost negative residues, hindering outward S4 motion, thus increasing the lag on activation and slowing the rise of I(K). In the activated state, La(3+) is displaced by outward movement of arginine residues in S4; La(3+), therefore, is not present to affect channel closing. The results give strong support to the closed state model of the K(V) channel and a clear explanation of the effect of multivalent cations on cellular excitability.

Molecular mechanism of pharmacological activation of BK channels.

Large-conductance voltage- and Ca(2+)-activated K(+) (Slo1 BK) channels serve numerous cellular functions, and their dysregulation is implicated in various diseases. Drugs activating BK channels therefore bear substantial therapeutic potential, but their deployment has been hindered in part because the mode of action remains obscure. Here we provide mechanistic insight into how the dehydroabietic acid derivative Cym04 activates BK channels. As a representative of NS1619-like BK openers, Cym04 reversibly left-shifts the half-activation voltage of Slo1 BK channels. Using an established allosteric BK gating model, the Cym04 effect can be simulated by a shift of the voltage sensor and the ion conduction gate equilibria toward the activated and open state, respectively. BK activation by Cym04 occurs in a splice variant-specific manner; it does not occur in such Slo1 BK channels using an alternative neuronal exon 9, which codes for the linker connecting the transmembrane segment S6 and the cytosolic RCK1 domain--the S6/RCK linker. In addition, Cym04 does not affect Slo1 BK channels with a two-residue deletion within this linker. Mutagenesis and model-based gating analysis revealed that BK openers, such as Cym04 and NS1619 but not mallotoxin, activate BK channels by functionally interacting with the S6/RCK linker, mimicking site-specific shortening of this purported passive spring, which transmits force from the cytosolic gating ring structure to open the channel's gate.

Critical role of large-conductance calcium- and voltage-activated potassium channels in leptin-induced neuroprotection of N-methyl-d-aspartate-exposed cortical neurons.

In the present study, the neuroprotective effects of the adipokine leptin, and the molecular mechanism involved, have been studied in rat and mice cortical neurons exposed to N-methyl-d-aspartate (NMDA) in vitro. In rat cortical neurons, leptin elicited neuroprotective effects against NMDA-induced cell death, which were concentration-dependent (10-100 ng/ml) and largest when the adipokine was preincubated for 2h before the neurotoxic stimulus. In both rat and mouse cortical neurons, leptin-induced neuroprotection was fully antagonized by paxilline (Pax, 0.01-1 µM) and iberiotoxin (Ibtx, 1-100 nM), with EC50s of 38 ± 10 nM and 5 ± 2 nM for Pax and Ibtx, respectively, close to those reported for Pax- and Ibtx-induced Ca(2+)- and voltage-activated K(+) channels (Slo1 BK channels) blockade; the BK channel opener NS1619 (1-30 µM) induced a concentration-dependent protection against NMDA-induced excitotoxicity. Moreover, cortical neurons from mice lacking one or both alleles coding for Slo1 BK channel pore-forming subunits were insensitive to leptin-induced neuroprotection. Finally, leptin exposure dose-dependently (10-100 ng/ml) increased intracellular Ca(2+) levels in rat cortical neurons. In conclusion, our results suggest that Slo1 BK channel activation following increases in intracellular Ca(2+) levels is a critical step for leptin-induced neuroprotection in NMDA-exposed cortical neurons in vitro, thus highlighting leptin-based intervention via BK channel activation as a potential strategy to counteract neurodegenerative diseases.

Cysteines control the N- and C-linker-dependent gating of KCNH1 potassium channels.

KCNH1 (EAG1) is a member of the Kv family of voltage-gated potassium channels. However, KCNH1 channels also show some amino-acid sequence similarity to cyclic-nucleotide-regulated channels: they harbor an N-terminal PAS domain, a C-terminal cyclic nucleotide binding homology domain (cNBHD), and N- and C-terminal binding sites for calmodulin. Another notable feature is the channels' high sensitivity toward oxidative modification. Using human KCNH1 expressed in Xenopus oocytes and HEK 293 cells we investigated how oxidative modification alters channel function. Intracellular application of H(2)O(2) or cysteine-specific modifiers potently inhibited KCNH1 channels in two phases. Our systematic cysteine mutagenesis study showed that the rapid and dominant phase was attributed to a right-shift in the voltage dependence of activation, caused by chemical modification of residues C145 and C214. The slow component depended on the C-terminal residues C532 and C562. The cysteine pairs are situated at structural elements linking the transmembrane S1 segment with the PAS domain (N-linker) and the transmembrane channel gate S6 with the cNBH domain (C-linker), respectively. The functional state of KCNH1 channels is determined by the oxidative status of these linkers that provide an additional dimension of channel regulation.

Drosophila QVR/SSS modulates the activation and C-type inactivation kinetics of Shaker K(+) channels.

The quiver/sleepless (qvr/sss) gene encodes a small, glycosylphosphatidylinositol-anchored protein that plays a critical role in the regulation of sleep in Drosophila. Loss-of-function mutations in qvr/sss severely suppress sleep and effect multiple changes in in situ Shaker K(+) currents, including decreased magnitude, slower time-to-peak, and cumulative inactivation. Recently, we demonstrated that SLEEPLESS (SSS) protein modulates Shaker channel activity, possibly through a direct interaction at the plasma membrane. We show here that SSS accelerates the activation of heterologously expressed Shaker channels with no effect on deactivation or fast N-type inactivation. Furthermore, this SSS-induced acceleration is sensitive to the pharmacological disruption of lipid rafts and sufficiently accounts for the slower time-to-peak of in situ Shaker currents seen in qvr/sss mutants. We also find that SSS decreases the rate of C-type inactivation of heterologously expressed Shaker channels, providing a potential mechanism for the cumulative inactivation phenotype induced by qvr/sss loss-of-function mutations. Kinetic modeling based on the in vitro results suggests that the SSS-dependent regulation of channel kinetics accounts for nearly 40% of the decrease in Shaker current magnitude in flies lacking SSS. Sleep duration in qvr/sss-null mutants is restored to normal by a qvr/sss transgene that fully rescues the Shaker kinetic phenotypes but only partially rescues the decrease in current magnitude. Together, these results suggest that the role of SSS in the regulation of sleep in Drosophila correlates more strongly with the effects of SSS on Shaker kinetics than current magnitude.

Trichostatin A abrogates airway constriction, but not inflammation, in murine and human asthma models.

Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (R(L)) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca(2+). The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in R(L) and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca(2+) in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca(2+) in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.

Blocking Kir6.2 channels with SpTx1 potentiates glucose-stimulated insulin secretion from murine pancreatic ß cells and lowers blood glucose in diabetic mice.

ATP-sensitive K+ (KATP) channels in pancreatic ß cells are comprised of pore-forming subunits (Kir6.2) and modulatory sulfonylurea receptor subunits (SUR1). The ATP sensitivity of these channels enables them to couple metabolic state to insulin secretion in ß cells. Antidiabetic sulfonylureas such as glibenclamide target SUR1 and indirectly suppress Kir6.2 activity. Glibenclamide acts as both a primary and a secondary secretagogue to trigger insulin secretion and potentiate glucose-stimulated insulin secretion, respectively. We tested whether blocking Kir6.2 itself causes the same effects as glibenclamide, and found that the Kir6.2 pore-blocking venom toxin SpTx1 acts as a strong secondary, but not a strong primary, secretagogue. SpTx1 triggered a transient rise of plasma insulin and lowered the elevated blood glucose of diabetic mice overexpressing Kir6.2 but did not affect those of nondiabetic mice. This proof-of-concept study suggests that blocking Kir6.2 may serve as an effective treatment for diabetes and other diseases stemming from KATP hyperactivity that cannot be adequately suppressed with sulfonylureas.