Potassium-39 NMR of K+ interaction with the gramicidin channel and NMR-derived conductance ratios for Na+, K+ and Rb+

Summary A potassium-39 NMR study of potassium ion interaction with the gramicidin transmembrane channel in phospholipid bilayers at high ion activity is reported which allows determination of a weak binding constant, K _b ^w 8.3/m, and an off-rate constant for the weak site,k _off ^w 2.6×10⁷/sec. These values are interpreted with the aid of additional NMR data as the binding constant for formation of the doubly occupied channel state and the rate constant for an ion leaving the doubly occupied state. Considering the singly occupied channel state for the potassium ion to be electrically silent at 1 molar ion activity, as with the sodium ion, the single-channel conductance for 100 mV and 30°C calculated to be 29 pS, and using the same approximation with previous NMR results on the sodium and rubidium ions, reasonable conductance ratios were calculated. Further experimental estimates of the other three constants with the experimental location of binding sites and Eyring rate theory to introduce voltage dependence allowed a more complete calculation of the two-site channel. The single-channel conductance for potassium ion is calculated to be 24 pS at 1m activity and 26 pS at 0.6m activity, which compares for diphytanoyl phosphatidylcholine membranes to an experimental most probable single-channel conductance of 25 pS and a mean channel conductance of 20 pS. The calculated conductance ratios using NMR-derived constants were (K)/(Na)=2.0 and (Rb)/(Na)=4.3. These results are close to the experimental values and provide further basis for the use of NMR of quadrupolar ions to provide information on the ionic mechanism of channel transport.     

Sodium-selective channels in membranes of rat macrophages

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na⁺ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na⁺ over K⁺ (as for Cs⁺, NH₄ ⁺, Ca²⁺, Ba²⁺) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li⁺-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722.     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

Regulation of K+ channels in the basolateral membrane ofNecturus oxyntic cells

Summary Patch-clamp methods were used to study single-channel events in isolated oxyntic cells and gastric glands fromNecturus maculosa. Cell-attached, excised inside-out and outside-out patches from the basolateral membrane frequently contained channels which had conductances of 67±21 pS in 24% of the patches and channels of smaller conductance, 33±6 pS in 56% of the patches. Channels in both classes were highly selective for K⁺ over Na⁺ and Cl^–, and shared linear current-voltage relations. The 67-pS channel was activated by membrane depolarization, whereas the activity of the 33-pS channel was relatively voltage independent. The larger conductance channels were activated by intracellular Ca²⁺ in the range between 5 and 500nm, but unaffected by cAMP. The smaller conductance channels were activated by cAMP, but not Ca²⁺. The presence of K⁺ channels in the basolateral membrane which are regulated by these known second messengers can account for the increase in conductance and the hyperpolarization of the membrane observed upon secretagogue stimulation.     

The influence of membrane potential on chloride channels activated by GABA in rat cultured hippocampal neurons

Chloride currents were activated by a low concentration of GABA (0.5 m) in neonatal rat hippocampal neurons cultured for up to 14 days. Currents elicited by 0.5 m GABA in neurons, voltage-clamped using the whole-cell technique with pipettes containing 149 mm Cl^–, reversed close to 0 mV whether pipettes contained 144 mm Na⁺ or 140 mm Cs⁺, and were blocked by 100 m bicuculline. Current-voltage curves showed outward rectification. Single channel currents appeared in cell-attached patches when the pipette tip was perfused with pipette solution containing 0.5 m GABA and disappeared when a solution containing 100 m bicuculline plus 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification and, in some patches, had a much lower probability of opening at hyperpolarized potentials. The average chord conductance in 10 patches hyperpolarized by 80 mV was 7.8±1.6 pS (sem) compared with a chord conductance of 34.1±3.5 pS (sem) in the same patches depolarized by 80 mV. Similar single channel currents were also activated in cell-free, inside-out patches in symmetrical chloride solutions when 0.5 m GABA was injected into the pipette tip. The channels showed outward rectification similar to that seen in cell-attached patches, and some channels had a lower probability of opening at hyperpolarized potentials. The average chord conductance in 13 patches hyperpolarized by 80 mV was 11.8±2.3 pS (sem) compared with 42.1±3.1 pS (sem) in the same patches depolarized by 80 mV.We are grateful to B. McLachlan and M. Robertson for their general assistance, to C. McCulloch and M. Smith for writing computer programs and to W. O'Hare for making the pipette injection device.     

Characterization of the acetylcholine-sensitive muscarinic K+ channel in isolated feline atrial and ventricular myocytes

M₂-cholinergic receptor activation by acetylcholine (ACh) is known to cause a negative inotropic and chronotropic action in atrial tissues. This effect is still controversial in ventricular tissues. The ACh-sensitive muscarinic K⁺ channel (I _K(ACh)) activity was characterized in isolated feline atrial and ventricular myocytes using the patch-clamp technique. Bath application of ACh (1 m) caused shortening of action potential duration without prior stimulation with catecholamines in atrial and ventricular myocytes. Resting membrane potential was slightly hyperpolarized in both tissues. These effects of ACh were greater in atrium than in ventricle. ACh increased whole-cell membrane current in atrial and ventricular myocytes. The current-voltage (I-V) relationship of the ACh-induced current in ventricle exhibited inward-rectification whose slope conductance was smaller than that in atrium. In single channel recording from cell-attached patches, I _K(ACh) activity was observed when ACh was induced in the pipette solution in both tissues. The channel exhibited a slope conductance of 47 ±1 pS (mean ± sd, n=14) in atrium and 47 ±2 pS (n= 10) in ventricle (not different statistically; ns). The open times were distributed according to a single exponential function with mean open lifetime of 2.0±0.3 msec (n= 14) in atrium and 1.9±0.3 msec (n=10) in ventricle (ns); these conductance and kinetic properties were similar between the two tissues. However, the relationship between the concentration of ACh and single channel activity showed a higher sensitivity to ACh in atrium (IC ₅₀ =0.03 m) than in ventricle (IC ₅₀ =0.15 m). In excised inside-out patches, ventricular I _K(ACh) required higher concentrations of GTP to activate the channel compared to atrial channels. These results suggest a reduced I _K(ACh) channel sensitivity to M₂-cholinergic receptor-linked G protein (G_i) in ventricle compared to atrium in feline heart.     

Anion channels in giant liposomes made of endoplasmic reticulum vesicles from rat exocrine pancreas

Summary Using the method of dehydration and rehydration, rough endoplasmic reticulum (RER) vesicles, isolated by differential centrifugation, can be enlarged to giant liposomes with diameters ranging from 5 to 200 m. Patch-clamp studies on these giant RER liposomes revealed the existence of a channel with a mean conductance of 260±7 pS (n=23; 140 mmol/liter KCl on both sides). The channel is about four times more permeable for Cl^– than for K⁺. Its activity is strongly voltage regulated. At low potentials (±20 mV) the channel is predominantly in its open state with an open probability near 1.0, whereas it closes permanently at high positive and negative voltages (±70 mV). The channel activity is not influenced by changing the free Ca²⁺ concentration from 1 mmol/liter to less than 10^–9 mol/liter on either side, and is also not affected by typical Cl^–-channel blockers like diphenylamine-2-carboxylate (DPC, 1 mmol/liter) or 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS, 1 mmol/liter). Another chloride channel with a singlechannel conductance of 79±6 pS (n=4) was less frequently observed. In the potential range of –80 to +40 mV this channel displayed no voltage-dependent gating. We assume that these anion channels are involved in the maintenance of electroneutrality during Ca²⁺ uptake in the RER.     

Analysis of sodium current fluctuations in the cut-open squid giant axon

Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.     

Effects of D-600 on sodium current in squid axons

Summary The effects of the calcium antagonist D-600 (methoxyverapamil) on the excitatory inward sodium current,I _Na, of internally perfused squid giant axons were studied under voltageclamp conditions. We observed little or no effect of the drug when it was added to the external solution at concentrations of 10–200 M. Furthermore, it did not produce a frequency, or use-dependent block ofI _Na when repetitive voltage-clamp pulses were used at rates of 2–5Hz. However, it did produce use-dependent blockade ofI _Na when it was placed internally at a concentration of 200 M. These results in conjunction with other studies suggest that D-600 is a selective blocker of calcium channels in squid axons when the drug is placed in the external solution. Its effects, when placed in the internal solution, are similar to those of permanently charged local anesthetic derivatives, which also produce use-dependent block ofI _Na.     

Fluctuation analysis of short-circuit current in a warm-blooded sodium-retaining epithelium: Site current,density, and interaction with triamterene

Summary Density and conductance of the Na-site in hen coprodeum were studied by employing fluctuation analysis of shortcircuit current at sodium concentrations from 26 to 130mm. Fluctuations of current in the frequency range 2–800 Hz were induced by triamterene, a reversible blocker of conducting epithelial Na-sites. At 130mm Na the site density was 5.8±1.0 m^–2 and the site conductance was 4 pS. This conductance is equal to that of the frog skin (W. Van Driessche and B. Lindemann, 1979,Nature (London) 282:519–520). Extrapolation of site density to zero sodium renders a total of 38±28 sites m^–2, which is compared with other estimates for the coprodeum. The site-triamterene association and dissociation constants were 9.5±0.4 rad sec^–1 m ^–1 and 255±20 rad sec^–1 and they were independent of external sodium concentration. An analysis of the affinity constant for triamterene based on the DC-short-circuit current was found to be unrelated to the external sodium concentration and identical to that obtained from fluctuation analysis indicating a noncompetitive interaction between sodium and triamterene. Due to the oxygen demand of the epithelium we have developed an experimental method using short data processing times. A new analytical approach using integration of the power density spectrum proved necessary because of low signal-to-noise ratios.     

Fused cells of frog proximal tubule: I. Basic membrane properties

Summary Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca²⁺ and voltage activated, has a conductance of 221±7 pS, and can be inhibited by 10mm tetraethylammonium and by 1mm quinidine, but not by 4-aminopyridine, nor by 1mm Ba²⁺ added to the outer side. Using the whole-cell configuration, we find that most of the cationic conductance of the membrane is constituted by a K-specific one (maximum K conductance 32.1±3.9 nSvs. a leak conductance of 1.01±0.17 nS). Comparisons of the maximum K conductance with that of a single K channel indicates that an MDCK cell has an average of 145 such channels. The membrane capacity is 24.5±1.4 pF.     

Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon

We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.     

Studies on lithium transport across the red cell membrane

Summary Binding of³H-saxitoxin to Na⁺ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1nm; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30nm. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K⁺ concentrations, or by activation of the Na⁺ channels with batrachotoxin plus a purified polypeptide toxin from the scorpionLeiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70–80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60–70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2nm. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0° of 0.1 min^–1 for membrane bound and 0.2 min^–1 for detergent-solubilized binding sites. The measured association rate constant was 6×10⁸ m ^–1 min^–1 at 0° for membrane-bound saxitoxin binding sites.     

Anion and cation permeability of a large conductance anion channel in the T84 human colonic cell line

Summary A large conductance multi-state channel was identified and characterized in single channel recordings from cell-attached and excised patches of the human colonic tumor cell line, T84. The channel activity was dependent on the presence of both permeable cations and anions. In Na⁺-free symmetrical Cl^– solutions or Cl^–-free symmetrical Na⁺ solutions the channel was inactive. Addition of 5mm NaCl (Nal or KCl) induced channel activity. The selectivity sequence obtained from the shift in reversal potential was I^–(1.9) > Cl^–(1) > Na⁺(0.5) > K⁺(0.3). SO ₄ ^2– , SCN^– (thiocyanate) and NMDG⁺ were impermeant. Multiple subconductance states were identified at all voltages explored (±90 mV). The minimum conductance encountered in symmetrical 100mm NaCl was a 15 pS substate, the maximum, 210 pS. The channel appeared to be composed of multiples of the 15 pS subunits which were reversibly blocked by the loop diuretic bumetanide (5 m).The authors wish to thank Morris Priddy and Charley Roberson for excellent technical assistance and Linda Pai and Steve Valder for participation in the early experiments. This study was supported by UPSH R01-DK39617 to A. Beaudet. L.V. was supported by a one-year fellowship from the Cystic Fibrosis Foundation.     

The characterization of a monovalent cation-selective channel of mammalian cardiac muscle sarcoplasmic reticulum

Summary Rabbit cardiac muscle sarcoplasmic reticulum (SR) was isolated and separated into ryanodine-sensitive and-insensitive fractions (L.R. Jones and S.E. Cala,J. Biol. Chem. 256:11809–11818, 1981). Vesicles of cardiac SR were incorporated into planar phospholipid bilayers by fusion and the channel activity of the membrane studied under voltage-clamp conditions (C. Miller,J. Membrane Biol. 40: 1–23, 1978). Both fractions contain a monovalent cation-selective three-state channel. In the presence of 75mm K₂SO₄, the fully open state () conductance of this channel is 157.2±30 pS and the sub-state () conductance is 100.7±21 pS. Both open states display the same selectivity sequence for monovalent cations, i.e. K⁺>NH ₄ ⁺ >Rb⁺>Na⁺>Li⁺ and may be blocked by the skeletal muscle relaxants decamethonium and hexamethonium. Block occurs when the compounds are added to either side of the membrane. The properties of the cardiac SR cation channel are compared with those of the previously reported monovalent cation-selective channels of mammalian and amphibian skeletal muscle SR.     

Subconductance states of single sodium channels modified by chloramine-T and sea anemone toxin in neuroblastoma cells

Single channel currents of chloramine-T (Chl-T) and sea anemone toxin (ATX-II) modified sodium channels were studied in neuroblastoma cells. With both substances similar subconductance states have been observed. The conductances of the sublevels were multiples of the unit step which was about onefourth of the most frequently occurring main conductance. Thus, the current levels observed were one fourth, half and five-fourths of the main current size. Both substances caused a slower decay of the averaged current compared to the current of the native channels. The main single-channel conductance was 15.2 pS (T=16°C) for the Chl-T and 10.8 pS (T=12°C) for the ATX-II modified channels. The channel open time was doubled by ATX-II, but was not increased significantly by Chl-T. The existence of the subconductance states suggests that the native channels may also have multiple open conformations.     

Reconstitution of the native mitochondrial outer membrane in planar bilayers. Comparison with the outer membrane in a patch pipette and effect of aluminum compounds

Summary Detergent-free rat brain outer mitochondrial membranes were incorporated in planar lipid bilayers in the presence of an osmotic gradient, and studied at high (1 m KCl) and low (150 mm KCl) ionic strength solutions. By comparison, the main outer mitochondrial membrane protein, VDAC, extracted from rat liver with Triton X-100, was also studied in 150 mm KCl. In 1 m KCl, brain outer membranes gave rise to electrical patterns which resembled very closely those widely described for detergent-extracted VDAC, with transitions to several subconducting states upon increase of the potential difference, and sensitivity to polyanion. The potential dependence of the conductance of the outer membrane, however, was steeper and the extent of closure higher than that observed previously for rat brain VDAC. In 150 mm KCl, bilayers containing only one channel had a conductance of 700 ± 23 pS for rat brain outer membranes, and 890 ± 29 pS for rat liver VDAC. Use of a fast time resolution setup allowed demonstration of open-close transitions in the millisecond range, which were independent of the salt concentration and of the protein origin. We also found that a potential difference higher than approx. ± 60 mV induced an almost irreversible decrease of the single channel conductance to few percentages of the full open state and a change in the ionic selectivity. These results show that the behavior of the outer mitochondrial membrane in planar bilayers is close to that detected with the patch clamp (Moran et al., 1992, Eur. Biophys. J. 20:311–319).The neurotoxicological action of aluminum was studied in single outer membrane channels from rat brain mitochondria. We found that m concentrations of Al Cl₃ and aluminum lactate decreased the conductance by about 50%, when the applied potential difference was positive relative to the side of the metal addition.The authors thank Dr. O. Moran for helpful discussions, Dr. M. Colombini for a sample of polyanion, and the Sharing Company for financial support to Dr. T. M. This work was partly supported by funds from the Ministero dell' Universitá e della Ricerca Scientifica e Tecnologica of Italy.     

Channel formation in phospholipid bilayer membranes by the toxin ofHeminthosporium maydis,race T

Summary Southern Corn Leaf Blight is caused by a toxin produced by a virulent form ofHelminthosporium maydis (Race T). The toxin has been shown to uncouple oxidative phosphorylation and dissipate Ca²⁺ gradients in mitochondria isolated from susceptible, but not resistant, corn. The possibility that the toxin acted by increasing the permeability of membranes to ions was tested using a planar bilayer membrane system. Addition of the toxin to the bilayer system, under voltage-clamp conditions, resulted in stepwise increases in current across the phospholipid bilayer, a response characteristic for channel formers. Single-channel conductance in 1m KCl is 27 pS which corresponds to 1.7×10⁷ ions sec^–1 channel^–1 at 100 mV applied potential. The toxin channels are: (i) fairly uniform in conductance, (ii) ideally selective for K⁺ over Cl^–, and (iii) most conductive to H⁺. The channel showed the following selectivity for alkali metal cations: Rb⁺>K⁺>Cs⁺>Na⁺>Li⁺ (169731) based on the most frequently observed conductance in 1m chloride salts. The toxin showed no voltage dependence over the range of –100 to +100 mV. Channel formation was also a property of a synthetic analog (Cmpd IV) of the toxin. The ability of the native toxin to form channels may be a mode of toxin action on mitochondrial membranes from susceptible corn.     

Secretin-regulated chloride channel on the apical plasma membrane of pancreatic duct cells

Summary Using the patch clamp technique we have identified a small conductance ion channel that typically occurs in clusters on the apical plasma membrane of pancreatic duct cells. The cell-attached current/voltage (I/V) relationship was linear and gave a single channel conductance of about 4 pS. Since the reversal potential was close to the resting membrane potential of the cell, and unaffected by changing from Na⁺-rich to K⁺-rich pipette solutions, the channel selects for anions over cations in cell-attached patches. The open state probability was not voltagedependent. Adding 25mm-bicarbonate to the bath solution caused a slight outward rectification of theI/V relationship, but otherwise, the characteristics of the channel were unaffected. In excised, inside-out, patches theI/V relationship was linear and gave a single channel conductance of about 4 pS. A threefold chloride concentration gradient across the patch (sulphate replacement) shifted the single channel current reversal potential by –26 mV, indicating that the channel is chloride selective. Stimulation of duct cells with secretin (10nm), dibutyryl cyclic AMP (1mm) and forskolin (1 m) increased channel open state probability and also increased the number of channels, and/or caused disaggregation of channel clusters, in the apical plasma membrane. Coupling of this channel to a chloride/bicarbonate exchanger would provide a mechanism for electrogenic bicarbonate secretion by pancreatic duct cells.