Interactions of piperidine derivatives with the nicotinic cholinergic receptor complex from Torpedo electric organ

The interactions of eight piperidine derivatives with nicotinic receptor complexes fromTorpedo californica electric organ were studied using [¹²⁵I]alpha-bungarotoxin ([¹²⁵I]BGT) as a probe for the acetylcholine binding site and [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) as a probe for a site associated with the receptor-gated ion channel.Cis- andtrans-2-methyl-6-n-undecanyl piperidines (MUP), major constituents of fire ant venom, had a high-affinity for [³H]H₁₂-HTX binding sites (K_i=0.08–0.24 M), but had no affect on receptor binding. MUP affinity for [³H]H₁₂-HTX binding sites was approximately doubled in the presence of 1 M carbamylcholine. Introduction of a 2-hydroxyl group to the undecanyl side channel had little effect on activity of the alkaloid. The analog 2,6- (but not 3,5-) dimethylpiperidine was a moderately active inhibitor of [³H]H₁₂-HTX binding (K _i-8.8 M). 2-Methylpiperidine was considerably less active (K _i=600 M), although it was more potent than either 3- or 4-methylpiperidine. The affinities of 2,6-dimethylpiperidine and 2-methylpiperidine for [³H]H₁₂-HTX binding sites were decreased in the presence of 1 M carbamylcholine. Carbamylcholine affinity for the receptor was increased by up to 7 fold in the presence of 10 and 32 M MUP, but was decreased in the presence of 2,6-dimethylpiperidine and 2-methylpiperidine. Thecis- andtrans-isomers of MUP were equipotent in producing each of its effects. In these actions, MUP resembles a variety of other compounds derived from 2,6-disubstituted piperidines, including histrionicotoxins, gephyrotoxins and pumiliotoxins. These studies establish the importance of alkyl substitutions in theortho position of the piperidine ring in conferring ion channel specificity, and the importance of substantial alkyl side chains in conferring the ability of channel blockers to stabilize the nicotinic receptor complex in high affinity, desensitized conformations.     

Ion currents associated with root tips, emerging laterals and induced wound sites in Nicotians tabacum: spatial relationship proposed between resulting electrical fields and phytophthoran zoospore infection

Abstract Growing roots of Nicotiana tabacum var. Havana generate transcellular ion currents which traverse developing and wounded tissues. Positive current of around 10 mA m^?2 enters meristematic and elongating cells at the tip of primary roots. The growing tips of first order laterals are also traversed by a similar positive current with a density of around 2.0 mA m^?2, as are immature laterals emerging at the primary root surface. These self-generated ion currents flow basipetally through developing tissues and leave from mature non-elongating tissue. A large positive current of around 70 mA m^?2 also enters induced wound sites on the primary root surface. Motile zoospores of the fungal pathogen Phytophthora parasitica var. nicotianae have been reported to associate preferentially with these regions of the root. This might suggest that electrotaxis may be part of the mechanism by which zoospores locate root regions susceptable to fungal infection.     

The synthetic precursor specific region of pre-pro-parathyroid hormone forms ion channels in lipid bilayers

We have used the chemically synthesized sequence of pre-pro-parathyroid hormone and several of its analogues to test the notion that the capacity of amphipathic peptides to aggregate in membranes and form ion-permeable channels correlates with their ability to function as signal sequences for secreted proteins. We found that pre-pro-parathyroid hormone (the signal sequence and pro-region of parathyroid hormone (M)), as well as some of its analogues, forms aggregates of monomers which are ion-permeable. The ion-permeable aggregates (2–3 monomers) formed by (M) are voltage-dependent and are more permeable for cations than for anions. The compounds which formed ion channels in bilayers also acted as potential signal sequences. We conclude that the ability of peptides to form ion-permeable pathways in bilayers may be correlated to their ability to function as signal peptides.     

The blocking effect of l-cis-diltiazem on the light-sensitive current of isolated rods of the tiger salamander

The effect of the organic compound l-cis-diltiazem on the light-sensitive current of isolated rods of the tiger salamander was analysed by rapidly changing the extracellular medium using the method of Hodgkin et al. (1985). Addition to the extracellular medium of small amounts of l-cis-diltiazem rapidly inhibits the photocurrent. Complete suppression of the current was observed with 1 mM l-cis-diltiazem. Half blockage of the photocurrent occurred with about 150 M l-cis-diltiazem. The blocking effect of l-cis-diltiazem was enhanced by light and by a reduction of extracellular Na. A concentration of l-cis-diltiazem of 140 M, which suppresses one third of the photocurrent, was able to completely suppress the photocurrent carried by Ba. It is suggested that l-cis-diltiazem blocks the light-sensitive channel, possibly competing with cyclic guanosine-3-5-monophosphate (cGMP) for an internal regulatory site.     

Kinetics of channelized membrane ions in magnetic fields

The cyclotron resonance model for channel ion transport in weak magnetic fields is extended to include damping losses. The conductivity tensor is obtained for different electric field configurations, including the circuital field E phi normal to the channel axis. The conductivity behavior close to the cyclotron resonance frequency omega c is compared to existing Ca2+-efflux data in the literature. A collision time of .023 s results from this comparison under the assumption that K+ ions are transiting in a 0.35 G field. We estimate a mean kinetic energy of 3.5 eV for this ion at resonance. This model leads to discrete modes of vibration (eigenfrequencies) in the ion-lattice interaction, such that omega n = n omega c. The presence of such harmonics is compatible with recent results by Blackman et al. [1985b] and McLeod et al. [1986] with the interesting exception that even modes do not appear in their observations, whereas the present model has no restriction on n. This harmonic formalism is also consistent with another reported phenomenon, that of quantized multiple conductances in single patch-clamped channels.     

The profile distribution of total and extractable boron in cacao-growing soils in southwestern Nigeria

The profile distribution of total and extractable B was determined in 16 Nigerian cacao-growing soil profiles formed from different parent materials. Total B for all soils ranged from 8 to 54μgg⁻¹ with a mean of 24μgg⁻¹. The soils formed from sandstones in the rainforest zone contained higher amounts of total B than soils derived from basement complex. Boron extractable in hot water, in 0.1% CaCl₂, and in 1N NH₄OAc varied from 0.13 to 1.38, 0.44 to 1.20 0.03 to 0.56μgg⁻¹ respectively. The corresponding means were 0.66, 0.75 and 0.27μgg⁻¹ B. Soils on metamorphic rocks gave the highest values. All extractable B values were related to organic matter while only CaCl₂-extractable B correlated with total B. Generally total and extractable B values were higher in the top soils than in the subsoils.     

Blockade of potassium channels in the plasmalemma ofChara corallina by tetraethylammonium,Ba2+, Na+ and Cs+

Summary The outer membranes of plant cells contain channels which are highly selective for K⁺. In the giant-celled green algaChara corallina, K⁺ currents in the plasmalemma were measured during the action potential and when the cell was depolarized to the K⁺ equilibrium potential in high external K⁺ concentrations. Currents in both conditions were reduced by externally added tetraethylammonium (TEA⁺), Ba²⁺, Na⁺ and Cs⁺. In contrast to inhibition by TEA⁺, the latter three ions inhibited inward K⁺ current in a voltage-dependent manner, and reduced inward current more than outward. Ba²⁺ and Na⁺ also appeared to inhibit outward current in a strongly voltage-dependent manner. The blockade by Cs⁺ is studied in more detail in the following paper. TEA⁺ inhibited both inward and outward currents in a largely voltage-independent manner, with an apparentK _D of about 0.7 to 1.1mm, increasing with increasing external K⁺. All inhibitors reduced current towards a similar linear leak, suggesting an insensitivity of the background leak inChara to these various K⁺ channel inhibitors. The selectivity of the channel to various monovalent cations varied depending on the method of measurement, suggesting that ion movement through the K⁺-selective channel may not be independent.     

Ion channels activated by osmotic and mechanical stress in membranes of opossum kidney cells

Summary For patch-clamp measurements cultured kidney (OK) cells were exposed to osmotic and mechanical stress. Superfusion of a cell in whole cell configuration with hypotonic media (190 mOsm) evokes strong depolarization, which is reversible by returning to the isotonic bath medium. In the cell-attached configuration the exposure to hypotonic media evokes up to six ion channels of homogeneous single-channel properties in the membrane patch. Subsequently, the channels became activated after a time lag of a few seconds. At an applied membrane potential of 0 mV, the corresponding membrane current is directed inward and shows a transient behavior in the time range of minutes. In the same membrane patch these ion channels can be activated by application of negative hydrostatic pressure. The channel has a single-channel conductance of about 22 pS and is permeable to Na⁺ and K⁺ as well as to Cl^–. It is suggested that volume regulation involves mechanoreceptor-operated ion channels.     

Coupled Na+/H+ exchange in rat parotid basolateral membrane vesicles

Summary pH gradient-dependent sodium transport in highly purified rat parotid basolateral membrane vesicles was studied under voltage-clamped conditions. In the presence of an outwardly directed H⁺ gradient (pH_in=6.0, pH_out=8.0)²²Na uptake was approximately ten times greater than uptake measured at pH equilibrium (pH_in=pH_out=6.0). More than 90% of this sodium flux was inhibited by the potassium-sparing diuretic drug amiloride (K ₁ =1.6 m) while the transport inhibitors furosemide (1mm), bumetanide (1mm) SITS (0.5mm) and DIDS (0.1mm) were without effect. This transport activity copurified with the basolateral membrane marker K⁺-stimulatedp-nitrophenyl phosphatase. In addition²²Na uptake into the vesicles could be driven against a concentration gradient by an outwardly directed H⁺ gradient. pH gradient-dependent sodium flux exhibited a simple Michaelis-Menten-type dependence on sodium concentration cosistent with the existence of a single transport system withK _M =8.0mm at 23°C. A component of pH gradient-dependent, amiloride-sensitive sodium flux was also observed in rabbit parotid basolateral membrane vesicles. These results provide strong evidence for the existence of a Na⁺/H⁺ antiport in rat and rabbit parotid acinar basolateral membranes and extend earlier less direct studies which suggested that such a transporter was present in salivary acinar cells and might play a significant role in salivary fluid secretion.     

Regulation of nicotinic acetylcholine receptors by protein phosphorylation

Neurotransmitter receptors and ion channels play a critical role in the transduction of signals at chemical synapses. The modulation of neurotransmitter receptor and ion channel function by protein phosphorylation is one of the major regulatory mechanisms in the control of synaptic transmission. The nicotinic acetylcholine receptor (nAcChR) has provided an excellent model system in which to study the modulation of neurotransmitter receptors and ion channels by protein phosphorylation since the structure and function of this receptor have been so extensively characterized. In this article, the structure of the nAcChR from the electric organ of electric fish, skeletal muscle, and the central and peripheral nervous system will be briefly reviewed. Emphasis will be placed on the regulation of the phosphorylation of nAcChR by second messengers and by neurotransmitters and hormones. In addition, recent studies on the functional modulation of nicotinic receptors by protein phosphorylation will be reviewed.