Isolation of two saxitoxin-sensitive sodium channel subtypes from rat brain with distinct biochemical and functional properties

Summary Two different³H-saxitoxin-binding proteins, with distinct biochemical and functional properties, were isolated from rat brain using a combination of anion exchange and lectin affinity chromatography as well as high resolution size exclusion and anion exchange HPLC. The alpha subunits of the binding proteins had different apparent molecular weights on SDS-PAGE (Type A: 235,000; Type B: 260,000). When reconstituted into planar lipid bilayers, the two saxitoxin-binding proteins formed sodium channels with different apparent single-channel conductances in the presence of batrachotoxin (Type A: 22 pS; Type B: 12 pS) and veratridine (Type A: 9 pS; Type B: 5 pS). The subtypes were further distinguished by scorpion (Leiurus quinquestriatus) venom which had different effects on single-channel conductance and gating of veratridine-activated Type A and Type B channels. Scorpion venom caused a 19% increase in single-channel conductance of Type A channels and a 35-mV hyperpolarizing shift in activation. Scropion venom double the single-channel conductance of Type B channels and shifted activation by at least 85 mV.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris andbis-Tris at millimolar concentrations. HEPES (pK_a=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES _i ) and this effect increased with [HEPES _i ] so that conductance at the reversal potential was diminished 25% with 10mm HEPES _i )and 70% at very high [HEPES _i ]. HEPES _i block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES _i concentration at constant [HEPES^–] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK_a=7.54), substituted glycine buffers (pK_a=8.1–8.4) andbis-Tris (pK_a=6.46) had no measurable effect on conductance and appear suitable for use with this channel.     

Purinergic Modulation of Hippocampal Acetylcholine Release Involves α-Dendrotoxin-Sensitive Potassium Channels

Modulation of acetylcholine (ACh) release from superfused hippocampal slices was examined when the release of ACh was stimulated by exposure of slices to elevated K+ concentration. Evoked release was not sensitive to inhibition by 0.1 microM tetrodotoxin, but it could be inhibited in a dose-dependent manner by a muscarinic agonist (10-100 nM oxotremorine) and a purinergic agonist (10-100 nM 2-chloroadenosine). The alpha-dendrotoxin (100 nM), which selectively blocks voltage-gated inactivating K+ channels in nerve endings, did not affect the release of ACh under resting or depolarized conditions. However, alpha-dendrotoxin reduced the 2-chloroadenosine-induced inhibition of release, but did not alter the oxotremorine-induced inhibition. These results suggest that an alpha-dendrotoxin-sensitive K+ channel may be activated as an obligatory step in the modulation of ACh release by presynaptic purinergic receptor activation, but not in the modulation by presynaptic muscarinic receptors.     

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H⁺ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl^– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl^–=Br^–=I^–>SO ₄ ^2– F^–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl^–-flux measurements and studies on the H⁺ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H⁺ uptake into the endosomes.     

A nonselective cation channel activated by membrane deformation in oocytes of the ascidianBoltenia villosa

Summary Cell-attached patch clamp recordings from unfertilized oocytes of the ascidianBoltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. The estimated density of these stretch-activated channels is about 1.5/m², a figure equal to or greater than the density of known voltage-dependent channels in the oocyte. Ion substitution experiments done with combined whole-cell and attached patch recording, so absolute potentials are known, indicate that the channel passes Na⁺, Ca²⁺ and K⁺, but not Cl^–. The channel has at least two open and two closed states, with the rate constant that leaves the longer-lived closed state being the primary site of stretch sensitivity. External Ca²⁺ concentration affects channel kinetics: at low calcium levels, long openings predominate, whereas at high calcium virtually all openings are to the short-lived open state. In multiple channel patches, the response to a step change in suction is highly phasic, with channel open probability decreasing over several hundred milliseconds to a nonzero steady-state level after an initial rapid increase. This channel may play a role in the physiological response of cells of the early embryo to the membrane strains associated with morphogenetic events.     

Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers

Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k _d 3nm.B _max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl^– and Ca²⁺, respectively. The reconstituted Ca²⁺ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca²⁺ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca²⁺/TRIS⁺ permeability ratio of 11.0. They also conducted other divalent cations (Ba²⁺>Ca²⁺>Sr²⁺>Mg²⁺) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca²⁺ channels, sharing some of the characteristics of Ca²⁺ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca²⁺ release from internal membrane stores.     

Modulation of gating of a metabolically regulated,ATP-dependent K+ channel by intracellular pH in B cells of the pancreatic islet

Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K⁺ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH _i . (1) The time course of K⁺(ATP) channel activity during exposure to and washout of NH₄Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH₄Cl entry and inhibition of the Na _o ⁺ H _i ⁺ exchanger, resembles the time course of pH _i measured in other cell types that have been similarly treated. (2) Increasing pH _o over the range 6.25–7.9 increases K⁺(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H⁺ by the application of the K⁺/H⁺ exchanger nigericin. (3) Increasing pH _i over a similar range produces similar effects on K⁺(ATP) channels in inside-out excised patches exposed to small concentrations of ATP _i . The physiological role of pH _i in the metabolic gating of this channel remains to be explored.     

Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

Isoforms of Na,K-ATPase inArtemia salina: II. Tissue distribution and kinetic characterization

Summary To characterize the molecular properties conveyed by the isoforms of the subunit of Na,K-ATPase, the two major transepithelial transporting organs in the brine shrimp (Artemia salina), the salt glands and intestines, were isolated in pure form. The isoforms were quantified by ATP-sensitive fluorescein isothiocyanate (FITC) labeling. The salt gland enzyme exhibits only the 1 isoform, whereas the intestinal enzyme exhibits both the 1 and the 2 isoforms. After 32 hours of development, Na,K-ATPase activity [in mol P_i/mg protein/hr (1u)] in whole homogenates was 32±6 in the salt glands and 12±3 in the intestinal preparations (mean±sem). The apparent half-maximal activation constants (K _1/2) of the salt gland enzyme as compared to the intestinal enzyme were 3.7±0.6mm vs. 23.5±4mm (P<0.01) for Na⁺, 16.6±2.2mm vs. 8.29±1.5mm for K⁺ (P<0.01), and 0.87±0.8mm vs. 0.79±1.1mm for ATP (NS). The apparentK _i's for ouabain inhibition were 1.1×10^–4 m vs. 2×10^–5 m, respectively. Treatment of whole homogenates with deoxycholic acid (DOC) produced a maximal Na,K-ATPase activation of 46% in the salt gland as compared to 23% in the intestinal enzyme. Similar differences were found with sodium dodecyl sulfate (SDS). The two distinct forms of Na,K-ATPase isolated from the brine shrimp differed markedly in three kinetic parameters as well as in detergent sensitivity. The differences inK _1/2 for Na⁺ and K⁺ are more marked than those reported for the mammalian Na,K-ATPase isoforms. These differences may be attributed to the relative abundances of the subunit isoforms; other potential determinants (e.g. differences in membrane lipids), however, have not been investigated.During the tenure of an Educational Commission For Foreign Medical Graduates Visiting Associate Professorship.