Charybdotoxin Research Articles

Ionic mechanisms involved in the regulation of insulin secretion by muscarinic agonists

The effects of the muscarinic agonist oxotremorine-m (oxo-m) on insulin secretion, K(+)-permeability and electrical activity from isolated mouse pancreatic islets were studied. Oxo-m potentiated glucose-induced insulin secretion in a dose-dependent manner, saturating at ca. 10 microM. At 11.2 mM glucose, oxo-m (0.1 and 10 microM) had two distinct effects on beta-cell electrical activity. Both concentrations increased the steady-state burst frequency, however, at 10 microM an initial and transient polarization was measured, and the subsequent activity was accompanied by a slight depolarization. The polarizing effect of oxo-m was almost completely suppressed by charybdotoxin (ChTX), a blocker of the large conductance (maxi) [Ca2+]i-activated potassium channel (K(Ca)). In the presence of 11.2 mM glucose, oxo-m (50 microM) provoked a significant and transient increase in the 86Rb efflux from perifused islets. This effect was inhibited by ChTX. ChTX also potentiated oxo-m stimulated insulin secretion in the presence of glucose. Finally, the balance between the polarizing and depolarizing effects of oxo-m was variable in different islets and depended on glucose concentration. Insulin secretion stimulated by oxo-m in the presence of glucose was more closely correlated to the agonist induced increase in burst frequency than to an increase in plateau fraction. We conclude that muscarinic stimulation has at least two effects on beta-cell electrical activity, an initial hyperpolarization, owing to activation of K(Ca) channels, followed by depolarization and high-frequency bursts, proposed to reflect the activation of a current sensitive to the depletion of intracellular Ca2+ stores (CRAC).

Polarized ion transport during migration of transformed Madin-Darby canine kidney cells.

Epithelial cells lose their usual polarization during carcinogenesis. Although most malignant tumours are of epithelial origin little is known about ion channels in carcinoma cells. Previously, we observed that migration of transformed Madin-Darby canine kidney (MDCK-F) cells depended on oscillating K+ channel activity. In the present study we examined whether periodic K+ channel activity may cause changes of cell volume, and whether K+ channel activity is distributed in a uniform way in MDCK-F cells. After determining the average volume of MDCK-F cells (2013+/-270 microm3; n=8) by means of atomic force microscopy we deduced volume changes by calculating the K+ efflux during bursts of K+ channel activity. Therefore, we measured the membrane conductance of MDCK-F cells which periodically rose by 22.3+/-2.5 nS from a resting level of 6.5+/-1.4 nS (n=12), and we measured the membrane potential which hyperpolarized in parallel from -35.4+/-1.2 mV to -71.6+/-1.8 mV (n=11). The distribution of K+ channel activity was assessed by locally superfusing the front or rear end of migrating MDCK-F cells with the K+ channel blocker charybdotoxin (CTX). Only exposure of the rear end to CTX inhibited migration providing evidence for "horizontal" polarization of K+ channel activity in transformed MDCK-F cells. This is in contrast to the "vertical" polarization in parent MDCK cells. We propose that the asymmetrical distribution of K+ channel activity is a prerequisite for migration of MDCK-F cells.

Identification of Kv1.1 Expression by Murine CD4−CD8− Thymocytes: A ROLE FOR VOLTAGE-DEPENDENT K+ CHANNELS IN MURINE THYMOCYTE DEVELOPMENT

The patch-clamp recording technique and RNA-polymerase chain reaction were used to identify the voltage-dependent K+ channels expressed by murine fetal and adult CD4-CD8- thymocytes. Two distinct currents, encoded by the genes Kv1.1 and Kv1.3 were identified based upon their biophysical and pharmacologic characteristics and confirmed with RNA-polymerase chain reaction. Peptide blockers of Kv1.1 and Kv1.3 gene products were also applied to a murine fetal thymic organ culture system to investigate the developmental role of these K+ channels. Dendrotoxin (DTX) and charybdotoxin (CTX), antagonists of Kv1.1 and Kv1.3 channels, respectively, decreased thymocyte yields in organ culture without affecting thymocyte viability. DTX-treated thymi contained 56 +/- 8% (n = 8 experiments), and CTX-treated thymi contained 74 +/- 4% (n = 7 experiments) as many thymocytes as untreated lobes. DTX and CTX also altered the developmental progression of thymocytes in fetal organ culture. These data provide the first evidence of Kv1.1 expression in a lymphoid cell and indicate that thymocyte voltage-dependent K+ channels are critical to thymocyte preclonal expansion and/or maturation.

Effects of Ca2+ channel blockers on transmitter release and presynaptic currents at the frog neuromuscular junction.

1. The effects of the calcium channel blockers, funnel-web spider toxin (FTX), omega-agatoxin IVA (omega-Aga IVA) and omega-conotoxin GVIA (omega-CgTX), were tested on transmitter release and presynaptic currents in frog motor nerve endings. 2. Evoked transmitter release was blocked by FTX (IC50 = 0.02 microliter ml-1) and omega-CgTX (1 microM) but was not affected by omega-Aga IVA (0.5 microM). When FTX (0.1 microliter ml-1) was assayed on spontaneous release either in normal Ringer solution or in low Ca(2+)-high Mg2+ solution, it was found not to affect miniature endplate potential (MEPP) amplitude but to increase MEPP frequency by approximately 2-fold in both conditions. 3. Presynaptic calcium currents (ICa), measured by the perineurial technique in the presence of 10 mM tetraethylammonium chloride (TEA) and 200 microM BaCl2 to block K+ currents, were blocked by omega-CgTX (5 microM), partially blocked by FTX (1 microliter ml-1) and not affected by omega-Aga IVA (0.5 microM). 4. The presynaptic calcium-activated potassium current (IK(Ca)) measured by the perineurial technique in the presence of 0.5 microM 3,4-aminopyridine (DAP) to block voltage-dependent K+ currents, was strongly affected by charybdotoxin (ChTX) (300 nM) and completely abolished by BaCl2 (200 microM). This current was also blocked by omega-CgTX (5 microM) and by CdCl2 (200 microM) but was not affected by FTX (1 microliter ml-1). The blockade by omega-CgTX could not be reversed by elevating [Ca]o to 10 mM. 5. The results suggest that in frog synaptic terminals two omega-CgTX-sensitive populations might coexist. The transmitter release process seems to be mediated by calcium influx through a omega-CgTX- and FTX-sensitive population.

Fast, persistent, Ca(2+)-dependent K+ current controls graded electrical activity in crayfish muscle.

The early outward current in opener muscle fibres of crayfish (Procambarus clarkii) was studied using the two-electrode voltage-clamp technique. This current was abolished in Ca(2+)-free and 5 mM Cd2+ solutions, and was blocked by extra- or intracellular tetraethylammonium, indicating that it was a Ca(2+)-dependent K+ current [IK(Ca)]. IK(Ca) was voltage dependent, apamin insensitive and sensitive to charybdotoxin (CTX), which, in addition to its tetraethylammonium sensitivity, suggests that the channels mediating IK(Ca) behave in a BK type manner. IK(Ca) activation was extremely fast, reaching a maximum within 5 ms, and the inactivation was incomplete, stabilizing at a persistent steady-state. IK(Ca) was insensitive to intracellular ethylenebis(oxonitrilo)tetraacetate (EGTA), but was abolished by injection of the faster Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that voltage-dependent Ca2+ channels and those mediating IK(Ca) should be clustered closely on the membrane. Under two-electrode current-clamp recording mode, low amplitude, graded responses were evoked under control conditions, whereas repetitive all-or-none spikes were elicited by application of CTX or after loading the cells with BAPTA. We conclude that IK(Ca) activates extremely quickly, is persistent and is responsible for the generation and control of the low amplitude, graded, active responses of opener muscle fibres.

A highly calcium-selective cation current activated by intracellular calcium release in MDCK cells.

1. The whole-cell patch clamp technique and fluorescence microscopy with the Ca2+ indicators fura-2 and fluo-3 were used to measure the whole-cell current and the free intracellular Ca2+ concentration ([Ca2+]i) in Madin-Darby canine kidney (MDCK) cells. 2. In a Ca(2+)-free bath solution, thapsigargin (TG) caused a transient increase of [Ca2+]i. Subsequent addition of Ca2+ caused a long lasting elevation of [Ca2+]i. 3. In a Ca(2+)-free bath solution, extracellular application of TG, ATP or ionomycin, or intracellular application of inositol 1,4,5-trisphosphate (IP3), caused a small but significant inward current (Iin) and a transient outward Ca(2+)-dependent K+ current (IK(Ca)), consistent with intracellular Ca2+ release. Subsequent addition of Ca2+ induced a prominent Iin with a current density of -4.2 +/- 0.7 pA pF-1. This Iin was unaffected by inositol 1,3,4,5-tetrakisphosphate (IP4). 4. Na+ replacement by mannitol, N-methyl-D-glucamine+ (NMG+), aminomethylidin-trimethanol+ (Tris+) or choline+ reduced Iin by 54, 65, 52 and 56%, respectively. This indicates an apparent Ca2+ selectivity over Na+ of 26:1. Iin was, however, unaffected by replacing Cl- with gluconate- or by the K+ channel blocker charybdotoxin (CTX). 5. Iin was completely blocked by La3+ (IC50 = 0.77 microM). Consistently, La3+ completely reversed the TG-induced elevation of [Ca2+]i. SK&F 96365 (1-[3-(4-methoxyphenyl)-propoxyl]-1-(4-methoxy-phenyl)-ethyl-1H-im idazole) HCl did not inhibit the TG-induced Iin. It did, however, exhibit a biphasic effect on [Ca2+]i, consisting of an initial Ca2+ decay and a subsequent Ca2+ elevation. La3+ completely reversed the SK&F 96365-induced elevation of [Ca2+]i. 6. In the absence of Na+, Iin was dependent on the bath Ca2+ concentration (EC50 = 1.02 mM). Ca2+ replacement by Ba2+ or Mn2+ resulted in a reduction of Iin by 95 and 94%, respectively. 7. From these experiments we conclude that Ca2+ release from intracellular Ca2+ stores, induced by different independent methods, stimulates La(3+)-inhibitable Ca2+ entry in MDCK cells. Ca2+ entry is at least, in part, mediated by a cation current, which is highly, but not exclusively, selective for Ca2+ over Na+ and insensitive to SK&F 96365.

Charybdotoxin and its effects on potassium channels

Over the last few years, a considerable amount of information has been obtained regarding K+ channels. Different areas of research have contributed to knowledge in this field. Charybdotoxin (ChTX), a 37-amino acid peptide isolated from venom of the scorpion Leiurus quinquestriatus var. hebraeus, represents a remarkable tool for studying K+ channels. With its use, it has been possible to purify the high-conductance Ca(2+)-activated K+ (maxi-K) channel to homogeneity and determine the subunit composition of this channel. This has led to the discovery of an auxiliary beta-subunit that, when coexpressed with the pore-forming subunit, mSlo, alters the biophysical and pharmacological properties of this latter subunit. With the feasibility of producing large amounts of ChTX by recombinant techniques and the knowledge of the three-dimensional structure of the peptide, it has been possible to carry out site-directed mutagenesis studies and obtain a picture of the interaction surface of the toxin with two channels, maxi-K and Shaker, and to derive a picture of the complementary surface of the receptor in these two channels. Finally, ChTX, and the more selective K+ channel toxins that were subsequently discovered, have provided us with unique tools not only to determine the functional role that K+ channels play in target tissues but also to develop the molecular pharmacology of these channels.

Structure-activity studies on scorpion toxins that block potassium channels

Scorpion venoms contain toxins that block different types of potassium channels. Some of these toxins have affinity for high conductance Ca 2+-activated K + channels and for dendrotoxin-sensitive voltage-dependent K + channels. The structural features that determine the specificity of binding to different channel types are not known. We investigated this using natural and synthetic scorpion toxins. We have tested the effects of charybdotoxin (CTX) and two homologues (Lqh 15-1 and Lqh 18-2), iberiotoxin (IbTX), and kaliotoxin (KTX) from the scorpions Leiurus quinquestriatus hebreus, Buthus tamulus and Androctonus mauretanicus mauretanicus, respectively, and synthetic variants of CTX, namely CTX 2–37, CTX 3–37, CTX 4–37, and CTX 7–37, on a Ca 2+-activated K + current ( i kca) at a mammalian motor nerve terminal, and on the binding of a radiolabelled dendrotoxin, 125I-DpI, to voltage-dependent K + channels on rat brain synaptosomal membranes. The native toxins contain 37–38 amino acid residues, they are over 30% identical in sequence (CTX and IbTX are 68% identical), and they have similar three-dimensional conformations. All toxins, except IbTX, displaced 125I-DpI from its synaptosomal binding sites: Lqh 18-2 ( K i = 0.25 nM), KTX ( K i = 2.1 nM), CTX ( K i = 3.8 nM), CTX 2–37, ( K i = 30nM), Lqg 15-1 ( K i = 50nM), CTX 3–37 ( K i = 60 nM), CTX 4–37 ( K i= 50 nM), CTX 7–37 ( K i = 105 nM). IbTX had no effect at 3μM. When variants of CTX with deletions at the N-terminal portion were tested for their activity on I KCa on motor nerve terminals in mouse triangularis sterni nerve-muscle preparations, CTX 3–37 and CTX 4–37 were ineffective at 100nM; and CTX 7–37 was ineffective at up to 1 μM. IbTX and CTX (100 nM) completely blocked I KCa but KTX (100 nM) did not affect the nerve terminal I KCa. Different residues appear to be important for interactions of the toxins with different K + channels. IbTX did not displace dendrotoxin binding, but it did block I KCa, whereas KTX was as active as CTX against dendrotoxin binding but it did not affect the I KCa of the motor nerve terminals. The N-terminal section of the toxins appears to be particularly involved in block of I KCaat the motor nerve terminal: it is truncated in the inactive synthetic CTX variants; and it is positively charged at lysine-6 in KTX (which is inactive), but negatively charged in IbTX and neutral in CTX. Phenylalanine at position 2 seems to be essential: there is a marked loss in activity between CTX 2–37 and CTX 3–37, and KTX has valine-2. Phenylalanine may be important in the β-sheet region of charybdotoxin and iberiotoxin. For binding to dendrotoxin sites, the inactive IbTX lacks the conserved asparagine residues at positions 4 and 30, and contains additional negatively charged residues at positions 4, 6 and 24. The side-chains of these residues are on the opposite face of the molecule from the positively charged residues in the β -sheet region (namely Arg 25, Lys 27 and Arg 34).

Modulation of acetylcholine release at mouse neuromuscular junctions by interaction of three homologous scorpion toxins with K+ channels

1. The effects of three scorpion toxins, charybdotoxin (CTX), iberiotoxin (IbTX), and noxiustoxin (NTX) have been studied on acetylcholine release and on K+ channels by means of twitch tension and electrophysiological recording techniques using isolated skeletal muscle preparations and by a radioligand binding assay using 125I-labelled dendrotoxin I (DpI) and rat brain synaptosomal membranes. 2. On chick biventer cervicis preparations, CTX and IbTX (125 nM) augmented the twitch responses to indirect muscle stimulation. Further, the increase (about 70-80% of control twitch height) was fast in onset, reaching a maximum within 25-30 min. NTX at 125 nM produced a slower augmentation of the twitch responses to indirect muscle stimulation, with the maximum response being seen after 40-50 min. 3. On mouse triangularis sterni preparations, CTX (300 nM after 35-40 min) and IbTX (100 nM after 15 min) increased quantal content of the evoked endplate potentials (e.p.p.) by about two fold. However, NTX (300 nM) caused only a small increase in e.p.p. amplitude, which was followed by repetitive e.p.ps in response to single shock nerve stimulation after 40-50 min. 4. Extracellular recording of nerve terminal current waveforms in triangularis sterni preparations revealed that CTX and IbTX (3-100 nM), but not NTX (100 nM), blocked the Ca(2+)-activated K+ current, IK-Ca. However, there was no major change in the portion of the nerve terminal waveform associated with voltage-dependent K+ currents, IKv. 5. In the radioligand binding assay, NTX potently displaced labelled [125I]-DpI, whereas CTX produced only partial displacement. However, IbTX did not displace [125I]-DpI from its binding sites on rat brain synaptosomal membranes.6. We conclude that these three structurally homologous scorpion toxins act on different K+ channels and that this leads to different patterns of facilitation of acetylcholine release. IbTX acts selectively on high conductance Ca2+-activated K+ channels, leading to an increase in the amplitude of e.p.ps without any other changes. NTX acts on voltage-dependent K+ channels that are sensitive to dendrotoxin and causes repetitive e.p.ps. CTX shares amino acid residues that exist in the structures of IbTX and NTX;CTX acts on both Ca2+- and voltage-dependent K+ channels.

Role of Ca(2+)-activated K+ channels in electrical activity of longitudinal and circular muscle layers of canine colon

The role of Ca(2+)-activated K+ channels (BK channels) in the canine colon was evaluated by testing the effects of charybdotoxin (ChTX) and tetraethylammonium on K+ currents of isolated myocytes and on electrical and mechanical activity of tissue strips. ChTX blocked Ca(2+)-activated outward current [IK(Ca)] in a dose- and voltage-dependent manner. No significant differences in IK(Ca) density, ChTX block, or Ca2+ sensitivity of BK channels were observed between circular and longitudinal myocytes. ChTX (100 nM) blocked 60% of current at +80 mV. Delayed rectifier current was not inhibited by 100 nM ChTX. In the absence of agonists, ChTX did not affect electrical or mechanical activity of circular muscle strips. In the presence of 10(-6) M BAY K 8644 or 10(-6) M acetylcholine, ChTX increased slow-wave duration and amplitude, induced membrane potential oscillations, and potentiated contraction. In unstimulated longitudinal muscle strips, ChTX depolarized the tissue, increased burst duration and spiking frequency, and resulted in an increase in contractions. These results indicate that BK channels are important regulators of colonic motility. In the longitudinal layer, BK channels are involved in setting membrane potential and determine excitability. In the circular layer, ChTX-sensitive channels do not participate in the in vitro basal electrical activity but limit the responses to excitatory agonists.

Role of G proteins and KCa channels in the muscarinic and beta-adrenergic regulation of airway smooth muscle

We have examined the functional consequences of G protein coupling to calcium-activated potassium (KCa) channels using isometric tension records from guinea pig tracheal smooth muscle. After incubation with 1 microgram/ml pertussis toxin (PTX) for 6 h, the contraction response to 1 microM methacholine (MCh) was suppressed by 31.7 +/- 5.0% (n = 10). Similarly, the contraction was inhibited by 29.1 +/- 5.0% (n = 6) after application of 0.1 microM AF-DX 116, an M2-selective muscarinic receptor antagonist. Cholera toxin (CTX, 2.0 micrograms/ml for 6 h), which activates the stimulatory G protein of adenylyl cyclase (Gs), also suppressed contraction by 43.9 +/- 3.3% (n = 11). The inhibitory effects of PTX, AF-DX 116, or CTX were reversed in the presence of 100 nM charybdotoxin (ChTX), a selective KCa channel inhibitor. These findings suggest that disruption of inhibitory coupling between muscarinic receptor and KCa channels mediated by PTX-sensitive G proteins, or KCa channel activation induced by Gs/adenylyl cyclase-linked processes, antagonizes muscarinic contraction. The isoproterenol concentration-inhibition curves for precontracted trachea (1 microM MCh) were shifted to the left after perfusion with PTX or AF-DX 116, and the leftward shift of the curve was blocked by ChTX. Thus direct or indirect regulation of KCa channels mediated by the inhibitory guanine nucleotide binding protein (Gi) and Gs may play a functionally important role in the mechanical antagonism by the two receptor agonists.

Separation and identification of multiple potassium currents regulating the pacemaker activity of insect neurosecretory cells (DUM neurons)

1. Whole cell voltage-clamp studies performed in isolated adult neurosecretory cells identified as dorsal unpaired median (DUM) neurons of the terminal abdominal ganglion of the cockroach Periplaneta americana have allowed us to reveal a complex voltage-dependent outward current regulating the pacemaker activity. 2. The global outward current remaining after tetrodotoxin treatment was activated by depolarization above -50 mV, showing steep voltage dependence and outward rectification. 3. We used tail current analysis to determine the ionic selectivity of this outward current. The reversal potentials for two extracellular potassium concentrations (-92.7 and -65.4 mV for 3.1 and 10 mM, respectively) is consistent with the expected equilibrium potential for potassium ions. 4. Both peak and sustained components of the global outward K+ current were reduced by external application of 20 mM tetraethylammonium chloride, 10 nM iberiotoxin, 1 nM charybdotoxin (CTX) and 1 mM cadmium chloride. Subtraction of current recorded in CTX solution from that in control solution revealed an unusual biphasic Ca(2+)-dependent K+ current. The fast transient current resistant to 5 mM 4-aminopyridine (4-AP) is distinguished by its dependence on holding potential and time course from the late sustained current. 5. In addition, two other components of CTX-resistant outward K+ current could be separated by sensitivity to 4-AP, time course, and voltage dependence. Beside a calcium-independent delayed outwardly rectifying current, a 4-AP-sensitive fast transient current resembling the A-current has been also identified. It activates at negative potential (about -65 mV) and unlike the A-current of other neurons, it inactivates rapidly with complex inactivation kinetics. A-like current is half-inactivated at -63.5 mV and half-activated at -35.6 mV. 6. Our findings demonstrate for the first time in DUM neuron cell bodies the existence of multiple potassium currents underlying the spontaneous electrical activity. Their identification and characterization represent a fundamental step in further understanding the pacemaker properties of these insect neurosecretory cells.

Heterotetramer formation and charybdotoxin sensitivity of two K+ channels cloned from smooth muscle.

Delayed rectifier K+ channels are involved in the electrical activity of all excitable cells. The relationship between native K+ currents recorded from these cells and cloned K+ channel cDNAs has been difficult to ascertain partly because of contradictions in pharmacological characteristics between native and expressed currents. Through the study of the charybdotoxin (CTX) pharmacology of two cloned smooth muscle delayed rectifier K+ channels (cKv 1.2 and cKv1.5) expressed in oocytes, evidence for heterotetramer formation was obtained. We have shown that the presence of even a single CTX-insensitive subunit renders the heterotetrameric channel insensitive to CTX. The two K+ channel clones differ in an amino acid at the mouth of the pore region, which may be in a position to block the access of CTX to its binding site and hence determine CTX sensitivity of the heterotetrameric channel. These results may explain discrepancies reported between native and cloned smooth muscle K+ channels.

Pb 2+ activates potassium currents in bovine adrenal chromaffin cells

Inorganic lead (Pb 2+) is a potent environmental toxin which adversely affects several aspects of neuronal and secretory cell function. In this report, we provide evidence that at subnanomolar concentrations, Pb 2+ activates the outward K + currents in bovine adrenal chromaffin cells. Whole-cell patch clamp combined with intracellular perfusion was employed to monitor outward K + currents in bovine chromaffin cells before and after intracellular application of EGTA-Pb buffers. Intracellular Pb 2+ ≥ 10 −10 M enhanced the K +-currents in a concentration dependent manner, with apparent K 0.5 ≊ 5 × 10 −10 M . Extracellular application of 40 nM Charybdotoxin (ChTX) blocked the Pb 2+-dependent component of outward currents, suggesting that Pb 2+ activates the large conductance Ca 2+-dependent K + channels.

High-level expression and functional reconstitution of Shaker K+ channels.

Voltage-gated K+ channels were expressed in COS cells transiently transfected with a plasmid carrying a cDNA for an inactivation-removed Shaker K+ channel driven by an adenovirus promoter. Channel expression was followed by immunological detection, binding of radioactive charybdotoxin (CTX), and functional reconstitution into planar lipid bilayers. About 10(7) channels per transfected cell are expressed on the plasma membrane. The expressed channels are glycosylated and competent to bind CTX with the expected characteristics. Channels observed after insertion into planar lipid bilayers displayed the voltage-dependent gating, conduction, and ion selectivity behavior expected for this channel. Channels were solubilized in several detergents without loss of CTX binding activity. The results make plausible a systematic attack on the purification of milligram-level amounts of functional K+ channels from a heterologous expression system.

Evidence in support of a role for Ca(2+)-activated K+ channels in the hamster sperm acrosome reaction.

The sperm acrosome reaction (AR) is a crucial step for mammalian fertilization. This work describes experiments to test the effect of the cesium ion (Cs+) and charybdotoxin (ChTX) on the Ca2+ or Na+/K+ ionophores stimulated hamster sperm AR in vitro. Cs+ and ChTX, a polypeptide toxin from the venom of the scorpion Leirus quinquestriatus, are considered blockers of Ca(2+)-activated K+ channels in several somatic cells. Both agents inhibited the AR by 55-66%. The inhibition was completely reversed by the Na+/K+ ionophore nigericin, but not by the Ca2+ ionophore A-23187. Results give evidence in support of a role for Ca(2+)-activated K+ channels in K+ influx required for the occurrence of the hamster sperm acrosome reaction.

The charybdotoxin receptor of a Shaker K + channel: Peptide and channel residues mediating molecular recognition

Charybdotoxin (CTX) is a peptide of known structure that inhibits Shaker K + channels by a pore-blocking mechanism. Point mutagenesis of all 30 solvent-exposed residues identified the part of the CTX molecular surface making contact with the receptor in the K + channel. All close-contact residues are clustered in a well-defined interaction surface; the shape of this surface implies that the outer opening of the Shaker channel conduction pore abruptly widens to a 25 × 35 Å plateau. A mutagenic scan of the S5–S6 linker sequence of the Shaker K + channel identified those channel residues influencing CTX binding affinity. The Shaker residues making the strongest contribution to toxin binding are located close to the pore-lining sequence, and more distant residues on both sides of this region influence CTX binding weakly, probably by an electrostatic mechanism. Complementary mutagenesis of both CTX and Shaker suggests that Shaker-F425 contacts a specific area near T8 and T9 on the CTX molecular surface. This contact point constrains Shaker-F425 to be located at a 20 Å radial distance from the pore axis and 10–15 Å above the “floor” of the CTX receptor.

Selective Enhancement of an A Type Potassium Current by Dexamethasone in a Corticotroph Cell Line

Perforated patch recording was used to examine the effect of the synthetic steroid dexamethasone on the whole cell potassium (K+) current, in the mouse corticotroph tumour cell line AtT20/D16-16. In 15 out of 52 control cells (29%) there was a rapidly-activating, rapidly-inactivating K+ current of the A type, the amplitude of which was strongly dependent on the holding potential in use prior to its activation by depolarising voltage pulses, and which was blocked by 1 mM 4-aminopyridine (4-AP, n = 5). The effect of dexamethasone (100 nM, 2 h, 37 degrees C) was that the A current increased in prevalence (24 out of 31 cells, 77%), lost its dependence on holding potential (over the range studied), and as a result became significantly larger than in controls, for certain voltage steps (peak A current density was 18.5 +/- 2.4 pA/pF (n = 12) for control cells and 26.3 +/- 3.9 pA/pF (n = 18) for dexamethasone treated cells, for a step to +30 mV from -60 mV, values are mean +/- SEM). All cells exhibited a slowly-activating, sustained K+ current, which was unaffected by changes in the holding potential, unaffected by 4-AP and consisted of at least 3 components: one blocked by 30 mM tetraethylammonium(TEA) or 100 nM charybdotoxin (CTX); a second blocked by 100 nM apamin; and a third not blocked by TEA, CTX, apamin, clofilium (100 nM) or niflumic acid (0.1 mM). Dexamethasone produced no change in the slowly-activating, sustained current nor in any of its individual components. The effect of dexamethasone on the A current was completely blocked by 0.1 mM puromycin, a protein synthesis blocker, while puromycin alone did not affect the size or frequency of the A current, nor alter the slowly-activating, sustained current. Secretion studies using 4-AP confirmed that the A current has a role in stimulated adrenocorticotrophic hormone (ACTH) secretion. In summary, AtT20 cells contain at least four types of K+ current: an A current and 3 currents contributing to the slowly-activating current. Selective enhancement of the A current by dexamethasone, shown here to require synthesis of new protein, is one of the mechanisms whereby glucocorticoids exert inhibitory control on ACTH secretion.

Functional unit size of the charybdotoxin receptor in smooth muscle.

Target inactivation analysis was used to determine the functional size of the charybdotoxin (ChTX) receptor in aortic and tracheal sarcolemmal membrane vesicles. This receptor has previously been shown to be an integral component of the high-conductance Ca2+-activated K+ (Maxi-K) channel in these smooth muscles. Exposure of either bovine aortic or bovine tracheal sarcolemma to high-energy irradiation results in disappearance of 125I-labeled ChTX binding activity as a monoexponential function of radiation dose; from these functions molecular masses of 88 +/- 10 kDa and 89 +/- 6 kDa, respectively, can be calculated. Similar results were obtained from radiation inactivation studies with the detergent-solubilized ChTX receptor from aortic sarcolemmal membranes. The effect of radiation on 125I-labeled ChTX binding is to decrease the number of functional ChTX receptors without affecting the affinity of receptors for the toxin, indicating that radiation is destroying, rather than altering, the binding site. The validity of the radiation inactivation technique in these membrane preparations is supported by data obtained in parallel experiments in which target sizes of the alpha 1 subunit of the L-type Ca2+ channel and 5'-nucleotidase were measured. The molecular masses determined for these entities are in excellent agreement with those expected from previous studies. The present data are discussed in terms of the recently determined subunit composition of the smooth muscle Maxi-K channel. In light of the target size, a single alpha beta subunit heterodimer complex could serve as the ChTX receptor.

Fractal analysis of role of smooth muscle Ca2+ fluxes in genesis of chaotic arterial pressure oscillations.

We have investigated the role of vascular smooth muscle Ca2+ fluxes in the genesis of chaotic pressure oscillations induced by histamine in isolated resistance arteries from the rabbit ear. The responses exhibited distinct "fast" and "slow" components, with periods of 5-20 s and 1-5 min, respectively, which could be dissociated pharmacologically. The fast subsystem involved ion movements at the cell membrane and was inhibited by both low (< 2 mM) and high (> 5 mM) extracellular Ca2+ concentration ([Ca2+]o) by verapamil (which inhibits voltage-dependent Ca2+ influx) and by charybdotoxin (ChTX) and apamin (which block Ca(2+)-activated K+ channels). In contrast, the slow subsystem was intracellular and was selectively attenuated by ryanodine, which inhibits Ca(2+)-induced Ca2+ release from sarcoplasmic reticulum. The effects of these interventions on the complexity of the responses were quantified by calculating their fractal dimension, a parameter that estimates the minimum number of independent variables contributing to an irregular time series. Its mean value was generally > 2 under control conditions but decreased to < 2 in a concentration-dependent fashion in the presence of verapamil, ChTX, apamin, or ryanodine and when [Ca2+]o was outside the range of 2-3 mM. Each intervention thus removed one dimension of complexity from the mechanisms generating the rhythmic activity. We conclude that the interaction of a fast membrane oscillator, which involves Ca2+ influx, Ca(2+)-activated K+ efflux, and therefore presumably changes in membrane potential, and a slow intracellular oscillator involving Ca2+ sequestration and release from stores is responsible for vascular chaos in our model. The coupling between these subsystems is likely to be mediated by cytosolic [Ca2+].