Characterization and regulation of Ca2+-dependent K+ channels in human esophageal smooth muscle. (9/800)

We examined the properties of K+ channels in smooth muscle cells dissociated from human esophagus using patch-clamp recording in the cell-attached configuration. The predominant channel observed had a conductance of 224 +/- 4 pS, and current reversal was dependent on K+ concentration. Channel activity was voltage dependent and increased with elevation of intracellular free Ca2+ concentration ([Ca2+]i), consistent with this being the large-conductance Ca2+-dependent K+ (KCa) channel. ACh as well as caffeine caused transient increases in KCa channel activity, and the effects of ACh persisted in Ca2+-free solution, indicating that Ca2+ release from stores contributed to channel activation. Simultaneous patch clamp and fluorescence revealed that KCa channel activity was well correlated with elevation of [Ca2+]i. The functional role of KCa channels in esophagus was studied by measuring ACh-induced contraction of strips of muscle. Tetraethylammonium and iberiotoxin, blockers of KCa channels, increased ACh-induced contraction, consistent with a role for K+ channels in limiting excitation and contraction. These studies are the first to characterize KCa channels and their regulation in human esophageal smooth muscle.  (+info)

A toxin to nervous, cardiac, and endocrine ERG K+ channels isolated from Centruroides noxius scorpion venom. (10/800)

Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether-a-go-go-related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long-QT syndrome and Drosophila 'seizure' mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50=16+/-1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure-function relationships and novel pharmacological tools with potential therapeutic efficacy.  (+info)

Peptidyl inhibitors of shaker-type Kv1 channels elicit twitches in guinea pig ileum by blocking kv1.1 at enteric nervous system and enhancing acetylcholine release. (11/800)

Potent and selective peptidyl blockers of the Shaker-type (Kv1) voltage-gated potassium channels were used to determine the role of these channels in regulating the spontaneous motility of smooth muscle preparations. Margatoxin (MgTX), kaliotoxin, and agitoxin-2 at 1 to 10 nM and agitoxin-1 at 50 to 100 nM induce twitches in guinea pig ileum strips. These twitches are abolished by tetrodotoxin (TTX, 0.5 microM), atropine (1 microM), hexamethonium (10 microM), or nifedipine (0.1 microM). It is proposed that blockade of Kv1 channels by MgTX, kaliotoxin, or the agitoxins increases excitability of intramural nerve plexuses in the ileum, promoting release of acetylcholine from excitatory motor nerve terminals. This, in turn, leads to Ca2+-dependent action potentials and twitching of the muscle fibers. MgTX does not induce twitches in several other guinea pig and/or rat vascular, genitourinary, or gastrointestinal smooth muscles, although small increases in spontaneous myogenic activity may be seen in detrusor muscle exposed to >30 nM MgTX. This effect is not reversed by TTX or atropine. The TTX- and atropine-sensitive twitches of guinea pig ileum are also induced by nanomolar concentrations of alpha-dendrotoxin, a selective blocker of Shaker Kv1.1 and 1.2 subtypes, or stichodactylatoxin, a peptide isolated from sea anemone that displays high affinity for Kv1.1 and 1.3, but not by charybdotoxin, which blocks Kv1.2 and 1.3 but not 1.1. The data taken together suggest that high-affinity blockade of Kv1.1 underlies the ability of MgTX, kaliotoxin, agitoxin-1, agitoxin-2, alpha-dendrotoxin, and stichodactylatoxin to elicit TTX-sensitive twitches in guinea pig ileum.  (+info)

A K+ channel-blocking peptide from venom of Chinese scorpion Buthus martensii Karsch. (12/800)

AIM: To purify and characterize a potassium channel blocker (BmP-3) from the venom of Chinese scorpion Buthus martensii Karsch. METHODS: 1. Purification was carried out by gel-filtration, cation-exchange, and reversed-phase chromatographies. N-terminal was directly sequenced by double-coupling manual method. Molecular weight was determined on an electrospray ionization mass spectrometer. Amino acid composition was analyzed after acidic hydrolysis for 20 h in HCl 6 mol.L-1 at 110 degrees C. 2. Toxicity tests were conducted in mice and cockroaches. 3. The inhibitory effects of BmP-3 on K+ channels were tested in acutely dissociated rat hippocampal pyramidal neurons using whole-cell patch-clamp configuration. RESULTS: 1. A pure peptide (BmP-3, 8.1 mg) was obtained, about 0.08% of total proteins of the venom. The N-terminal sequences were VGCEE and the molecular weight was 2938 in ESI-mass spectra. 2. No death occurred at the dosage of 200 micrograms in mice and 8 micrograms in cockroaches. 3. The peptide at 10 mumol.L-1 reduced the peak outward K+ currents by 63% +/- 4% in vitro. CONCLUSION: BmP-3 inhibited K+ channels.  (+info)

Genomic organization of three neurotoxins active on small conductance Ca2+-activated potassium channels from the scorpion Buthus martensi Karsch. (13/800)

According to the known primary sequences of three neurotoxins active on small conductance Ca2+-activated potassium channels from the scorpion Buthus martensi Karsch, their corresponding cDNAs were cloned and sequenced using 3'- and 5'-RACE. All of them encoded a signal peptide composed of 28 residues and a mature toxin of 29, 28 and 33 residues, respectively. Their cDNA deduced sequences were totally consistent with those determined, and the C-terminal amidation of one neurotoxin was confirmed. The genomic DNAs of these three toxins were also amplified by PCR, cloned and sequenced. They all consisted of two exons disrupted by a small single intron. All of these introns were inserted within the signal peptide at the same -10 position upstream from the mature toxin, consisting of 94, 78 and 87 bp, respectively.  (+info)

O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase. (14/800)

The rapid response to hypoxia in the pulmonary artery (PA), carotid body, and ductus arteriosus is partially mediated by O2-responsive K+ channels. K+ channels in PA smooth muscle cells (SMCs) are inhibited by hypoxia, causing membrane depolarization, increased cytosolic calcium, and hypoxic pulmonary vasoconstriction. We hypothesize that the K+ channels are not themselves "O2 sensors" but rather respond to the reduced redox state created by hypoxic inhibition of candidate O2 sensors (NADPH oxidase or the mitochondrial electron transport chain). Both pathways shuttle electrons from donors, down a redox gradient, to O2. Hypoxia inhibits these pathways, decreasing radical production and causing cytosolic accumulation of unused, reduced, freely diffusible electron donors. PASMC K+ channels are redox responsive, opening when oxidized and closing when reduced. Inhibitors of NADPH oxidase (diphenyleneiodonium) and mitochondrial complex 1 (rotenone) both inhibit PASMC whole-cell K+ current but lack the specificity to identify the O2-sensor pathway. We used mice lacking the gp91 subunit of NADPH oxidase [chronic granulomatous disease (CGD) mice] to assess the hypothesis that NADPH oxidase is a PA O2-sensor. In wild-type lungs, gp91 phox and p22 phox subunits are present (relative expression: macrophages > airways and veins > PASMCs). Deletion of gp91 phox did not alter p22 phox expression but severely inhibited activated O2 species production. Nonetheless, hypoxia caused identical inhibition of whole-cell K+ current (in PASMCs) and hypoxic pulmonary vasoconstriction (in isolated lungs) from CGD vs. wild-type mice. Rotenone vasoconstriction was preserved in CGD mice, consistent with a role for the mitochondrial electron transport chain in O2 sensing. NADPH oxidase, though a major source of lung radical production, is not the pulmonary vascular O2 sensor in mice.  (+info)

A marine snail neurotoxin shares with scorpion toxins a convergent mechanism of blockade on the pore of voltage-gated K channels. (15/800)

kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. kappa-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in kappa-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, kappa-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, zdelta, is approximately 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) M-1 s-1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced kappa-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the alpha-KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.  (+info)

Modulation of glioma cell migration and invasion using Cl(-) and K(+) ion channel blockers. (16/800)

Human malignant gliomas are highly invasive tumors. Mechanisms that allow glioma cells to disseminate, migrating through the narrow extracellular brain spaces are poorly understood. We recently demonstrated expression of large voltage-dependent chloride (Cl(-)) currents, selectively expressed by human glioma cells in vitro and in situ (Ullrich et al., 1998). Currents are sensitive to several Cl(-) channel blockers, including chlorotoxin (Ctx), (Ullrich and Sontheimer; 1996; Ullrich et al; 1996), tetraethylammonium chloride (TEA), and tamoxifen (Ransom and Sontheimer, 1998). Using Transwell migration assays, we show that blockade of glioma Cl(-) channels specifically inhibits tumor cell migration in a dose-dependent manner. Ctx (5 microM), tamoxifen (10 microM), and TEA (1 mM) also prevented invasion of human glioma cells into fetal rat brain aggregates, used as an in vitro model to assess tumor invasiveness. Anion replacement studies suggest that permeation of chloride ions through glioma chloride channel is obligatory for cell migration. Osmotically induced cell swelling and subsequent regulatory volume decrease (RVD) in cultured glioma cells were reversibly prevented by 1 mM TEA, 10 microM tamoxifen, and irreversibly blocked by 5 microM Ctx added to the hypotonic media. Cl(-) fluxes associated with adaptive shape changes elicited by cell swelling and RVD in glioma cells were inhibited by 5 microM Ctx, 10 microM tamoxifen, and 1 mM TEA, as determined using the Cl(-)-sensitive fluorescent dye 6-methoxy-N-ethylquinolinium iodide. Collectively, these data suggest that chloride channels in glioma cells may enable tumor invasiveness, presumably by facilitating cell shape and cell volume changes that are more conducive to migration and invasion.  (+info)