Showing papers in "The Journal of Membrane Biology in 1991"

Liquid junction potentials and small cell effects in patch-clamp analysis.

Abstract: Since the early 1980s, the patch-clamp technique (Hamill et al., 1981) has been of particular value in investigating the properties of ion channels in cells. When used in either the intact or excised configurations, the properties of individual ionic channels can be directly measured. In addition, the whole-cell configuration can be used to investigate the total response of the full complement of channels in a cell. The whole-cell configuration is of particular value in exploring the properties of very small cells which are not readily accessible to conventional microelectrode techniques. in all of the above measurements, there are two potential sources of error, in every situation there may be significant errors due to uncompensated junction potentials, which m a y a p p e a r to be eliminated by the normal zeroing procedure whereby residual potentials between pipette and bath solutions are offset prior to patch formation. In addition, in the intact and whole-cell patch configurations, the effect of the cells being small can introduce radical errors in the measurement of single-channel and whole-cell properties. The aim of this review is firstly to outline the contribution of such junction potentials and the errors resulting from measurements on small cells and secondly to indicate how adequate junction potential corrections can be applied and the true values of underlying membrane parameters determined for such cells. Where necessary, appropriate equations have been presented. Much of the material is a review of published work. However, the review also seeks to extend the implications of that work and, in particular, it also includes (in an appendix) a timedependent solution of the current relaxation following a channel closure.

Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin.

Abstract: The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca2−, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF3 and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPΓs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.

Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth.

Abstract: Incorporation of the gene for connexin 43, a cell-cell channel protein of gap junction, into the genome of communication-deficient transformed mouse 10T1/2 cells restored junctional communication and inhibited growth. Growth was slowed, saturation density reduced and focus formation suppressed, and these effects were contingent on overexpression of the exogenous gene and the consequent enhancement of communication. In coculture with normal cells the growth of the connexin overexpressors was completely arrested, as these cells established strong communication with the normal ones. Thus, in culture by themselves or in coculture, the connexin overexpressor cells grew like normal cells. These results demonstrate that the cell-cell channel is instrumental in growth control; they are the expected behavior if the channel transmits cytoplasmic growth-regulatory signals.

Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: inhibition of fatty acid transport.

Abstract: Sulfo-N-succinimidyl derivatives of the long-chain fatty acids, oleic and myristic, were synthesized and covalently reacted with isolated rat adipocytes. The plasma membrane proteins labeled by these compounds and the effect of labeling on the transport of long-chain fatty acids were investigated. Sulfo-N-succinimidyl oleate (SSO) and myristate (SSM) inhibited the transport of fatty acids (by about 70%). Inhibition of fatty acid transport was not a result of alterations in cell integrity, as intracellular water volume was not changed. It did not reflect effects on fatty acid metabolism, since it was observed under conditions where greater than 90% of the fatty acid taken up was recovered in the free form. The inhibitory effect was specific to the fatty acid transport system, as the transport of glucose and the permeation of retinoic acid, a substance with structural similarities to long-chain fatty acids, were unaffected. Sulfosuccinimidyl oleate reacted exclusively with a plasma membrane protein with an apparent size of 85 kDa while sulfosuccinimidyl myristate also labeled a 75-kDa protein. These proteins were among the ones labeled by diisothiocyanodisulfonic acid (DIDS) which also inhibits fatty acid transport irreversibly. The data suggest that the 85-kDa protein, which is the only one labeled by all three inhibitors is involved in facilitating membrane permeation of long-chain fatty acids.

Recent advances in the molecular characterization of plasma membrane Ca2+ pumps.

Abstract: Since its discovery some 25 years ago (Schatzmann, 1966) the plasma membrane Ca 2+ pump (PMCA) has constantly gained in importance as a model membrane protein to study the structural, functional, regulatory and genetic basis of ATP-driven cation transport. The success in the characterization of this enzyme parallels that made in the molecular analysis of other complex low-abundance proteins spanning the lipid bilayer and reflects to a large extent the impressive technical progress made during the past two decades in all areas of modern biological research. It is not surprising, therefore, that several reviews have already been devoted mostly, if not exclusively, to the various aspects of the plasma membrane Ca 2+ pump. Rather than trying to resummarize the contents of previous reviews, the present contribution will focus on those developments concerning the characterization of the Ca 2 § pump which have only recently (i.e., during the past three to five years) been added to our existing views on this important enzyme. Accordingly, no attempt will be made to present a comprehensive picture of the subject; for more detailed information on different aspects of the plasma membrane Ca 2+ pump the reader is referred to previously published reviews and references found therein. A recent summary of the general properties, and particularly of ion transportrelated characteristics, of the pump can be found in Garrahan and Rega (1990), and a synopsis of the problems related to the mechanism of calcium pumping has been given by Jencks (1989). A limited selection of further reviews published during the past decade includes contributions by Sarkadi [1980 (emphasis on early results on the enzymatic properties

Intracellular free zinc and zinc buffering in human red blood cells.

Abstract: Zn2+ has been allowed to equilibrate across the red cell membrane using two agents that increase membrane permeability to this ion: the ionophore A23187 and the specific carrier ethylmaltol. Extracellular free Zn2+ was controlled with EGTA (1,2-di(2-aminoethoxy)ethane-NNN′N′tetra-acetic acid)) buffers, except in the case of ethylmaltol, which itself acts as a buffer. Measurement of cellular zinc content at different levels of free Zn2+ facilitated the study of intracellular Zn2+ binding. It was also possible to estimate intracellular free Zn2+ concentration in untreated cells using a “null-point” technique. Intracellular zinc was found to consist of an inexchangeable component of about 129 μmol/1013 cells and an exchangeable component of 6.7±1.5 μmol/1013 cells, with a free concentration of about 2.4×10−11 m. The main component of Zn2+ buffering is hemoglobin, with a dissociation constant of about 2×10−8 m.

Major myelin proteolipid: The 4-α-helix topology

Abstract: Several conflicting models have been proposed for the membrane arrangement of the major myelin proteolipid (PLP) We have compared features of the sequence of PLP with those of other eukaryotic integral membrane proteins, with the view of identifying the most likely transmembrane topology A new, simple model is suggested, which features four hydrophobic α-helices spanning the whole thickness of the lipid bilayer Its orientation may be such that both the N-and C-termini face the cytosol None of the biochemical, biophysical or immunological experiments hitherto reported provides incontrovertible evidence against the model The effect or absence thereof of various PLP mutations is discussed in the frame, of the proposed 4-helix topology

A negative slope in the current-voltage relationship of the Na+/K+ pump in Xenopus oocytes produced by reduction of external [K+].

Abstract: To investigate the voltage dependence of the Na−/K− pump, current-voltage relations were determined in prophasearrested oocytes ofXenopus laevis. All solutions contained 5mm Ba2− and 20mm tetraethylammonium (TEA) to block K− channels. If. in addition, the Na+/K+ pump is blocked by ouabain, K+-sensitive currents no larger than 50 nA/cm2 remain. Reductions in steady-state current (on the order of 700 nA/cm2) produced by 50 μm ouabain or dihydro-ouabain or by K+ removal, therefore, primarily represent current generated by the Na−/K− pump. In Na−-free solution containing 5mm K+, Na+/K+ pump current is relatively voltage independent over the potential range from −160 to +40 mV. If external [K+] is reduced below 0.5mm, negative slopes are observed over this entire voltage range. Similar results are seen in Na+- and Ca2+-free solutions in the presence of 2mm Ni2+, an experimental condition designed to prevent Na+/Ca2+ exchange. The occurrence of a negative slope can be explained by the voltage dependence of the apparent affinity for activation of the Na+/K+ pump by external K+, consistent with the existence of an external ion well for K− binding. In 90mm Na+, 5mm K+ solution, Na+/K+ pump current-voltage curves at negative membrane potentials have a positive slope and can be described by a monotonically increasing sigmoidal function. At an extracellular [K+] of 1.3mm, a negative slope was observed at positive potentials. These findings suggest that in addition to a voltage-dependent step associated with Na+ translocation, a second voltage-dependent step that is dependent on external [K+], possibly external K+ binding, participates in the overall reaction mechanism of the Na+/K+ pump.

Role of organic osmolytes in adaptation of renal cells to high osmolality.

Abstract: Kidney cells accumulate organic osmolytes in order to protect themselves from the high concentrations of NaCl and urea in the blood and interstitial fluid of the renal medulla The renal medullary organic osmolytes are sorbitol, inositol, betaine and GPC The concentrations of these solutes in renal medullary NaCl and urea concentration, as summarized in Fig 8 (the putative controlled steps are highlighted) Sorbitol accumulates by synthesis from glucose, catalyzed by aldose reductase Hypertonicity increases the transcription of the gene that encodes this enzyme GPC is synthesized from choline, and the amount retained apparently may be controlled by the activity of GPC diesterase, an enzyme that catabolizes GPC Inositol and betaine are taken up from the medium by sodium-dependent transport, and this transport is increased by hypertonicity Control of these processes is slow (hours to days), but a decrease in tonicity causes a transient, rapid efflux of the solutes, which prevents the cells from becoming overly distended Similar strategies are used by all types of cells, including bacteria and those in plants and animals, that can adapt to hyperosmotic stress

Analysis of kinetic data in transport studies: new insights from kinetic studies of Na(+)-D-glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus.

Abstract: Using the fast sampling, rapid filtration apparatus (FSRFA) recently developed in our laboratory (Berteloot et al., 1991, J. Membrane Biol. 122:111-125), we have studied the kinetic characteristics of Na(+)-D-glucose cotransport in brush-border membrane vesicles isolated from normal adult human jejunum. True initial rates of transport have been determined at both 20 and 35 degrees C using a dynamic approach which involves linear-regression analysis over nine time points equally spaced over 4.5 or 2.7 sec, respectively. When the tracer rate of transport was studied as a function of unlabeled substrate concentrations added to the incubation medium, a displacement curve was generated which can be analyzed by nonlinear regression using equations which take into account the competitive inhibition of tracer flux by unlabeled substrate. This approach was made imperative since at 20 degrees C, in the presence of high substrate concentrations or 1 mM phlorizin, no measurable diffusion was found and the resultant zero slope values cannot be expressed into a classical v versus S plot. All together, our results support the existence of a single Na(+)-D-glucose cotransport system in these membranes for which Na+ is mandatory for uptake. This conclusion is at variance with that of a recent report using the same preparation (Harig et al., 1989. Am J. Physiol. 256:8618-8623). Since the discrepancy seems difficult to resolve on the consideration of experimental conditions alone, we have determined the kinetic parameters of D-glucose transport using one time point measurements and linear transformations of the Michaelis-Menten equation, in order to investigate the potential problems of such a widely used procedure. Comparing these approaches, we conclude that: (i) the dynamic uptake measurements give a better understanding of the different uptake components involved: (ii) it does not matter whether a dynamic or a one time point approach is chosen to generate the uptake data provided that a nonlinear-regression analysis with proper weighting of the data points is performed; (iii) analytical procedures which rely on linearization of Michaelian process(es) are endowed with a number of difficulties which make them unsuitable to resolve multicomponent systems in transport studies. A more general procedure which uses a nonlinear-regression analysis and a displacement curve is proposed since we demonstrate that it is far superior in terms of rapidity, data interpretation, and visual information.

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements.

Abstract: Membrane fragments containing a high density of Na,K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na,K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.

Noninvasive recording of receptor cell action potentials and sustained currents from single taste buds maintained in the tongue: The response to mucosal NaCl and amiloride

Abstract: Apical membrane currents were recorded from the taste pore of single taste buds maintained in the tongue of the rat, using a novel approach. Under a dissection microscope, the 150-μm opening of a saline-filled glass pipette was positioned onto single fungiform papillae, while the mucosal surface outside the pipette was kept dry. Electrical responses of receptor cells to chemical stimuli, delivered from the pipette, were recorded through the pipette while the cells remained undamaged in their natural environment. We observed monophasic transient currents of 10-msec duration and 10–100 pA amplitude, apparently driven by action potentials arising spontaneously in the receptor cells. When perfusing the pipette with a solution of increased Na but unchanged Cl concentration, a stationary inward current (from pipette to taste cell) of 50–900 pA developed and the collective spike rate of the receptor cells increased. At a mucosal Na concentration of 250mm, the maximal collective spike rate of a bud was in the range of 6–10 sec−1. In a phasic/tonic response, the high initial rate was followed by an adaptive decrease to 0.5–2 sec−1. Buds of pure phasic response were also observed. Amiloride (30 μm) present in the pipette solution reversibly and completely blocked the increase in spike rate induced by mucosal Na. Amiloride also decreased reversibly the stationary current which depended on the presence of mucosal Na (inhibition constant near 1 μm). During washout of amiloride, spike amplitudes were first small, then increased, but always remained smaller than the amiloride-blockable stationary current of the bud. This is understandable since the stationary current of a bud arises from a multitude of taste cells, while each current spike is presumably generated by just one taste cell. We suggest that, in a Na-sensitive receptor cell, (i) the apical amiloride-blockable Na inward current serves as a generator current causing cell depolarization and firing of action potentials, and (ii) each current spike recorded from the taste pore arises mainly from a modulation of the apical Na inward current of this cell, because the action potential generated by the taste cell will transiently decrease or abolish the driving force for the apical Na inward current. The transients are indicators of receptor cell action potentials, which appear to be physiological responses of taste cellsin situ.

Charge translocation by the Na,K-pump: II. Ion binding and release at the extracellular face.

Abstract: In the first part of the paper, evidence has been presented that electrochromic styryl dyes, such as RH 421, incorporate into Na, K-ATPase membranes isolated from mammalian kidney and respond to changes of local electric field strength. In this second part of the paper, fluorescence studies with RH-421-labeled membranes are described, which were carried out to obtain information on the nature of charge-translocating reaction steps in the pumping cycle. Experiments with normal and chymotrypsin-modified membranes show that phosphorylation by ATP and occlusion of Na+ are electroneutral steps, and that release of Na+ from the occluded state to the extracellular side is associated with translocation of charge. Fluorescence signals observed in the presence of K+ indicate that binding and occlusion of K+ at the extracellular face of the pump is another major electrogenic reaction step. The finding that the fluorescence signals are insensitive to changes of ionic strength leads to the conclusion that the binding pocket accommodating Na+ or K+ is buried in the membrane dielectric. This corresponds to the notion that the binding sites are connected with the extracellular medium by a narrow access channel (“ion well”). This notion is further supported by experiments with lipophilic ions, such as tetraphenylphosphonium (TPP+) or tetraphenylborate (TPB−), which are known to bind to lipid bilayers and to change the electrostatic potential inside the membrane. Addition of TPP+ leads to a decrease of binding affinity for Na+ and K+, which is thought to result from the TPP−-induced change of electric field strength in the access channel.

Vasopressin alters the mechanism of apical Cl- entry from Na+:Cl- to Na+:K+:2Cl- cotransport in mouse medullary thick ascending limb.

Abstract: Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K+ dependence of the apical, furosemide-sensitive Na+:Cl− cotransporter and on transport-related oxygen consumption (QO2). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K+ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K+)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na+ and Cl−, but not K+, while in the presence of AVP the apical cotransporter required all three ions.86Rb+ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,22Na+ uptake was unaffected by extracellular K+ in the absence of AVP while after AVP exposure22Na+ uptake was strictly K+-dependent. The AVP-induced coupling of K+ to the Na+:Cl− cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular22Na+ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na+:Cl− cotransport to Na+:K+:2Cl− cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K+ to the apical Na+:Cl− cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.

Ion channel gating in plants : physiological implications and integration for stomatal function

Abstract: Primary evidence of ion channel activity in plant cells can be traced back to the first half of this century. Cole and Curtis (1938), in their pioneering studies of neuronal action potentials, made use of giant Characean algal cells in the winter months when squid axons were not available. They noted the parallel to the action potential of the nerve membrane; like the nerve, Characean action potentials were accompanied by a 30to 50-fold rise in membrane conductance. Subsequent work by Gaffey and Mullins (1958), Hope (1961), Findlay (1962) and Kishimoto (1965) established the ionic nature of the Characean action potential and fundamental features of the transient, especially its gating, the voltage dependence of which gives rise to the self-propagating wave of the action potential. Similar observations could be drawn from higher plants, including the insectivorous plant Dionaea, albeit in much less detail (see Sibaoka, 1969). These studies fostered a view (cf. Simons, 1981; Hill & Findlay, 1981) that in plants, as in animals, gated ion fluxes can serve to conduct and transduce signals between stimulus and response. Nonetheless, the plant kingdom offers relatively few examples of such excitability and, in the case of the Characean algae, its physiological significance (but not its scientific value) remains obscure. Thus, until recently, ion channels were generally seen as curiosities well removed from the dominant themes of transport relations in plant cells. The advent of the patch electrode, planar bilayer, and associated recording techniques has had as great (if not greater) an impact on our understanding of plant cell membranes as it has on that of animal cells. Now at the end of the decade which saw the

Skeletal muscle ATP-sensitive K+ channels recorded from sarcolemmal blebs of split fibers: ATP inhibition is reduced by magnesium and ADP.

Abstract: A new, nonenzymatically treated preparation of amphibian sarcolemmal blebs has been used to study the regulation of skeletal muscle ATP-sensitive K+ [K(ATP)] channels. When a frog skeletal muscle fiber is split in half in a Ca2+-free relaxing solution, large hemispherical membrane blebs appear spontaneously within minutes without need for Ca2+-induced contraction or enzymatic treatment. These blebs readily formed gigaseals with patch pipettes, and excised inside-out patches were found to contain a variety of K+ channels. Most prominent were K(ATP) channels similar to those found in the surface membrane of other muscle and nonmuscle cells. These channels were highly selective for K+, had a conductance of ≈ 53 pS in 140mmK+, and were blocked by internal ATP. The presence of these channels in most patches implies that split-fiber blebs are made up, at least in large part, of sarcolemmal membrane. In this preparation, K(ATP) channels could be rapidly and reversibly blocked by glibenclamide (0.1–10 μm) in a dose-dependent manner. These channels were sensitive to ATP in the micromolar range in the absence of Mg. This sensitivity was noticeably reduced in the presence of millimolar Mg, most likely because of the ability of Mg2+ ions to bind ATP. Our data therefore suggest that free ATP is a much more potent inhibitor of these channels than MgATP. Channel sensitivity to ATP was significantly reduced by ADP in a manner consistent with a competition between ADP, a weak inhibitor, and ATP, a strong inhibitor, for the same inhibitory binding sites. These observations suggest that the mechanisms of nucleotide regulation of skeletal muscle and pancreatic K(ATP) channels are more analogous than previously thought.

Calcium-dependent control of volume regulation in renal proximal tubule cells: I. Swelling-Activated Ca2+ entry and release

Abstract: The mechanism of Ca2+-dependent control of hypotonic cell volume regulation was investigated in the isolated, nonperfused renal proximal straight tubule. When proximal tubules were exposed to hypotonic solution with 1 mM Ca2+, cells swelled rapidly and then underwent regulatory volume decrease (RVD). This treatment resulted in an increase in intracellular free calcium concentration ([Ca2+]i) by a mechanism that had two phases: the first was a transient increase from baseline (136 nM) to a peak (413 nM) that occurred in the first 15–20 sec, but was followed by a rapid decay toward the pre-swelling levels. The second phase was characterized by a sustained elevation of [Ca2+]i above the baseline (269 nM), which was maintained over several minutes. The dependence of these two phases on extracellular Ca2+ was determined. Reduction of bath [Ca2+] to 10 or 1 μM partially diminished the transient phase, but abolished the sustained phase completely, such that [Ca2+]i fell below the base-line levels during RVD. It was concluded that the transient increase resulted predominantly from swelling-activated release of intracellular Ca2+ stores and that the sustained phase was due to swelling-activated Ca2+ entry across the plasma membrane. Ca2+ entry probably also contributed to the transient increase in [Ca2+]i. The time dependence of swelling-activated Ca2+ entry was also investigated, since it was previously shown that RVD was characterized by a “calcium window” period (<60 sec). during which extracellular Ca2+ was required. Outside of this time period, RVD would inactivate and could not be reactivated by subsequent addition of Ca2+. It was found that the Ca2+ permeability did not inactivate over several minutes, indicating that the temporal dependence of RVD on extracellular Ca2+ is not due to the transient activation of a Ca2+ entry pathway.

Chloride channels in the nuclear membrane.

Abstract: Chloride-selective ion channels were measured from isolated rat liver nuclei. Single ion channel currents were recorded in both "nuclear-attached" and in excised patches in the inside-out configuration of the patch-clamp technique. Two types of chloride conductance were defined, a large conductance (150 pS; iCl,N) channel with complex kinetics and multiple substates, and a second smaller conductance (58 pS;ICln) channel sensitive to block by ATP. The channels were inhibited by pharmacological agents known to block chloride channels and were insensitive to internal and external changes in calcium and magnesium. Presumably the channels reside in the external membrane of the nuclear double membrane and may mediate charge balance in the release and uptake of calcium from the perinuclear space.

Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity.

Abstract: Solution osmolarity is known to affect Na+ transport rates across tight epithelia but this variable has been relatively ignored in studies of cultured renal epithelia. Using electrophysiological methods to study A6 epithelial monolayers, we observed a marked effect of solution tonicity on amiloride-sensitive Na+ currents (I(sc)). I(sc) for tissues bathed in symmetrical hyposmotic (170 mOsm), isosmotic (200 mOsm), and hyperosmotic (230 or 290 mOsm) NaCl Ringer's solutions averaged 25 +/- 2, 9 +/- 2, 3 +/- 0.4, and 0.6 +/- 0.5 microA/cm2, respectively. Similar results were obtained following changes in the serosal tonicity: mucosal changes did not significantly affect I(sc). The changes in I(sc) were slow and reached steady-state within 30 min. Current fluctuation analysis measurements indicated that single-channel currents and Na+ channel blocker kinetics were similar for isosmotic and hyposmotic conditions. However, the number of conducting Na+ channels was approximately threefold higher for tissues bathed in hyposmotic solutions. No channel activity was detected during hyperosmotic conditions. The results suggest that Na+ channels in A6 epithelia are highly sensitive to relatively small changes in serosal solution tonicity. Consequently, osmotic effects may partly account for the large variability in Na+ transport rates for A6 epithelia reported in the literature.

Angiotensin II, vasopressin and GTP[γ-S] inhibit inward-rectifying K+ channels in porcine cerebral capillary endothelial cells

Abstract: Cerebral capillaries from porcine brain were isolated. and endothelial cells were grown in primary culture. The whole-cell tight seal patch-clamp method was applied to freshly isolated single endothelial cells, and cells which were held in culture up to one week. With high K+ solution in the patch pipette and in the bath we observed inward-rectifying K+ currents, showing a time-dependent decay in part of the experiments. Ba2+ (1–10mm) in the bath blocked this current, whereas outside tetraethylammonium (10mm) decreased the peak current but increased the steady-state current. Addition of 1 μm of angiotensin II or of arginine-vasopressin to the extracellular side caused a time-dependent inhibition of the inward-rectifying K+ current in part of the experiments. Addition of 100 μm GTP[γ-S] to the patch pipette blocked the K+ inward rectifier. In cell-attached membrane patches two types of single inward-rectifying K+ channels were observed, with single channel conductances of 7 and 35 pS. Cell-attached patches were also obtained at the antiluminal membrane of intact isolated cerebral capillaries. Only one type of K+ channel withg=30 pS was recorded. In conclusion, inwardly rectifying K+ channels, which can be inhibited by extracellular angiotensin II and arginine-vasopressin, are present in cerebral capillary endothelial cells. The inhibition of this K+ conductance by GTP[γ-S] indicates that G-proteins are involved in channel regulation. It is suggested that angiotensin II and vasopressin regulate K+ transport across the blood-brain barrier, mediating their effects via G-proteins.

Asymmetry of membrane fluidity in the lipid bilayer of blood platelets: fluorescence study with diphenylhexatriene and analogs.

Abstract: Membrane fluidity of bovine platelets was examined with diphenylhexatriene (DPH), its cationic trimethylammonium derivative (TMA-DPH) and anionic propionic acid derivative (DPH-PA). After addition of these probes to platelet suspensions at 37 degrees C, the fluorescence intensity of DPH-PA reached equilibrium within 2 min, whereas those of DPH and TMA-DPH increased gradually. With increase in the fluorescence intensity of TMA-DPH, its fluorescence anisotropy decreased significantly, but the fluorescence anisotropies of DPH-PA and DPH did not change during incubation. The gradual increase of fluorescence intensity of TMA-DPH was due to its penetration into the cytoplasmic side of the platelet membrane, as shown quantitatively by monitoring decrease in its extractability with albumin. Transbilayer movement of TMA-DPH was markedly temperature-dependent, and was scarcely observed at 15 degrees C. The fluorescence intensity of TMA-DPH was much higher in platelet membranes and vesicles of extracted membrane lipids than the initial intensity in intact platelets. Moreover, the fluorescence anisotropy of TMA-DPH was much lower in the former preparations than the initial value in intact platelets. These results suggest that binding sites for TMA-DPH in the cytoplasmic side of the platelet membrane are more fluid than those in the outer leaflet of the plasma membrane. Platelet activation by ionomycin induced specific change in the fluorescence properties of TMA-DPH without causing transbilayer incorporation of the probe.

Osmolarity-sensitive release of free amino acids from cultured kidney cells (MDCK).

Abstract: The amino acid pool of MDCK cells was essentially constituted by alanine, glycine, glutamic acid, serine, taurine, lysine, beta-alanine and glutamine. Upon reductions in osmolarity, free amino acids were rapidly mobilized. In 50% hyposmotic solutions, the intracellular content of free amino acids decreased from 69 to 25 mM. Glutamic acid, taurine and beta-alanine were the most sensitive to hyposmolarity, followed by glycine, alanine and serine, whereas isoleucine, phenylalanine and valine were only weakly reactive. The properties of this osmolarity-sensitive release of amino acids were examined using 3H-taurine. Decreasing osmolarity to 85, 75 or 50% increased taurine efflux from 0.6% per min to 1.6, 3.5 and 5.06 per min, respectively. The time course of 3H-taurine release closely follows that of the regulatory volume decrease in MDCK cells. Taurine release was unaffected by removal of Na+, Cl- or Ca2+, or by treating cells with colchicine or cytochalasin. It was temperature dependent and decreased at low pH. Taurine release was unaffected by bumetanide (an inhibitor of the Na+/K+/2Cl- carrier); it was inhibited 16 and 67 by TEA and quinidine (inhibitors of K+ conductances), unaffected by gadolinium or diphenylamine-2-carboxylate (inhibitors of Cl- channels) and inhibited 50% by DIDS. The inhibitory effects of DIDS and quinidine were additive. Quinidine but not DIDS inhibited taurine uptake by MDCK cells.

Ca2(+)-activated K+ channel from human erythrocyte membranes: single channel rectification and selectivity.

Abstract: The Ca2+-activated K+ channel of the human red cell membranes was characterized with respect to rectification and selectivity using the patch-clamp technique. In inside-out patches exposed to symmetric solutions of K+, Rb+, and NH4+, respectively, inward rectifyingi-V curves were obtained. The zero current conductances were: K+ (23.5 pS±3.2)>NH4+ (14.2 pS±1.2)>Rb+ (11.4 pS±1.8). With low extracellular K+ concentrations (substitution with Na+) the current fluctuations reversed close to the Nernst potential for the K ion and the rectification as well as thei-V slopes decreased. With mixed intracellular solutions of K+ and Na+ enhanced rectification were observed due to a Na+ block of outward currents. From bi-ionic reversal potentials the following permeability sequence (PK/PX) was calculated: K+ (1.0)>Rb+ (1.4±0.1)>NH4+(8.5±1.3)>Li+(>50); Na+ (>110); Cs+ (≫5). Li+, Na+, and Cs+ were not found to carry any current, and only minimum values of the permeability ratios were estimated. Tl+ was permeant, but the permeability and conductance were difficult to quantify, since with this ion the single channel activity was extremely low and the channels seemed to inactivate. The inward rectification in symmetric solutions indicate an asymmetric open channel structure, and the different selectivity sequences based on conductances and permeabilities reflect interionic interactions in the permeation process.

Single-channel study of the cGMP-dependent conductance of retinal rods from incorporation of native vesicles into planar lipid bilayers.

Abstract: Unitary currents through cGMP-dependent channels of retinal rods are observed following incorporation into planar lipid bilayers of native vesicles from purified rod outer segment membranes washed free of soluble and peripheral proteins. The influence of the concentration of cGMP, inhibitors (cis-diltiazem, tetracaine and Ag+) and divalent cations (Ca2+, Mg2+, and Co2+) on the conductance and open probability of the channel is described, as well as the voltage dependence of these effects. The cGMP dependence suggests the existence of four binding sites for cGMP and reveals that sequential binding of four cGMP molecules corresponds to the opening of four discrete conductance levels. Finally, we provide conclusive evidence that activated G-protein does not directly inactivate the cGMP-dependent channels of bovine retinal rods.

Charybdotoxin-sensitive K (Ca) channel is not involved in glucose-induced electrical activity in pancreatic β-cells

Abstract: The effects of charybdotoxin (CTX) on single [Ca2+] i -activated potassium channel (K (Ca)) activity and whole-cell K+ currents were examined in rat and mouse pancreatic β-cells in culture using the patch-clamp method. The effects of CTX on glucose-induced electrical activity from both cultured β-cells and β-cells in intact islets were compared. K(Ca) activity was very infrequent at negative patch potentials (−70

Effects of the anesthetics heptanol, halothane and isoflurane on gap junction conductance in crayfish septate axons: A calcium- and hydrogen-independent phenomenon potentiated by caffeine and theophylline, and inhibited by 4-aminopyridine

Abstract: This study has monitored junctional and nonjunctional resistance. [Ca2+] i and [H−] i , and the effects of various drugs in crayfish septate axons exposed to neutral anesthetics. The uncoupling efficiency of heptanol and halothane is significantly potentiated by caffeine and theophylline. The modest uncoupling effects of isoflurane, described here for the first time, are also enhanced by caffeine. Heptanol causes a decrease in [Ca2+] i and [H+] i both in the presence and absence of either caffeine or theophylline. A similar but transient effect on [Ca2+] i is observed with halothane. 4-Aminopyridine strongly inhibits the uncoupling effects of heptanol. The observed decrease in [Ca2−] i with heptanol and halothane and negative results obtained with different [Ca2+] o , Ca2+-channel blockers (nisoldipine and Cd2+) and ryanodine speak against a Ca2+ participation. Negative results obtained with 3-isobutyl-l-methylxanthine, forskolin, CPT-cAMP, 8Br-cGMP, adenosine, phorbol ester and H7, superfused in the presence and absence of caffeine and/or heptanol. indicate that neither the heptanol effects nor their potentiation by caffeine are mediated by cyclic nucleotides, adenosine receptors and kinase C. The data suggest a direct effect of anesthetics. possibly involving both polar and hydrophobic interactions with channel proteins. Xanthines and 4-aminopyridine may participate by influencing polar interactions. The potentiating effect of xanthines on cell-to-cell uncoupling by anesthetics may provide some clues on the nature of cardiac arrhythmias in patients treated with theophylline during halothane anesthesia.

Interaction of caffeine-, IP3- and vanadate-sensitive Ca2+ pools in acinar cells of the exocrine pancreas.

Abstract: Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP3) sensitive and IP3-insensitive nonmitochondrial intracellular Ca2+ pools in acinar cells of the exocrine pancreas. For further characterization of Ca2+ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designated P1 to P5 from the top to the bottom of the gradient. Measuring Ca2+ uptake and Ca2+ release with a Ca2+ electrode, we could differentiate three nonmitochondrial intracellular Ca2+ pools: (i) an IP3-sensitive Ca2+ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca2+ pool (CasCaP), vanadate- and IP3-insensitive, and (iii) a vanadate-sensitive Ca2+ pool (VasCaP), neither IP3- nor caffeine-sensitive, into which Ca2+ uptake is mediated via a Ca2+ ATPase sensitive to vanadate at 10(-4) mol/liter. A fourth Ca2+ pool is neither IP3- nor caffeine- or vanadate-sensitive. Percoll fraction P1 contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca2+ uptake and Ca2+ release. When membrane vesicles were incubated in the presence of caffeine (2 x 10(-2) mol/liter), Ca2+ uptake up to the steady state [Ca2+] did not appear to be altered as compared to the control Ca2+ uptake. However, in control vesicles spontaneous Ca2+ release occurred after the steady state had been reached, whereas caffeine-pretreated vesicles did not spontaneously release Ca2+. Addition of IP3 at steady state [Ca2+] induced similar Ca2+ release followed by Ca2+ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca2+ was released from all Ca2+ pools including the IsCaP. Following Ca2+ reuptake after IP3 had been added, a second addition of IP3 to control vesicles induced further but smaller Ca2+ release, and a third addition resulted in a steady Ca2+ efflux by which all Ca2+ that had been taken up was released. This steady Ca2+ release started at a Ca2+ concentration between 5.5-8 x 10(-7) mol/liter and could also be induced by the IP3 analogue inositol 1,4,5-trisphosphorothioate (IPS3) or by addition of Ca2+ itself. Ruthenium red (10(-5) mol/liter) inhibited both caffeine-induced as well as Ca2(+)-induced but not IP3-induced Ca2+ release. Heparin (100 micrograms/ml) inhibited IP3- but not caffeine-induced Ca2+ release. The data indicate the presence of at least three separate Ca2+ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca2+ uptake these Ca2+ pools appear to be separate.(ABSTRACT TRUNCATED AT 400 WORDS)

Muscarinic responses of gastric parietal cells.

Abstract: Isolated rabbit gastric glands were used to study the nature of the muscarinic cholinergic responses of parietal cells. Carbachol (CCh, 100 microM) stimulation of acid secretion, as measured by the accumulation of aminopyrine, was inhibited by the M1 antagonist, pirenzepine, with an IC50 of 13 microM; by the M2 antagonist, 11,2-(diethylamino)methyl-1 piperidinyl acetyl-5,11-dihydro-6H-pyrido 2,3-b 1,4 benzodiazepin-6-one (AF-DX 116), with an IC50 of 110 microM; and by the M1/M3 antagonist, diphenyl-acetoxy-4-methylpiperidinemethiodide (4-DAMP), with an IC50 of 35 nM. The three antagonists displayed equivalent IC50 values for the inhibition of carbachol-stimulated production of 14CO2 from radiolabeled glucose, which is a measure of the turnover of the H,K-ATPase, the final step of acid secretion. Intracellular calcium levels were measured in gastric glands loaded with FURA 2. Carbachol was shown to both release calcium from an intracellular pool and to promote calcium entry across the plasma membrane. The calcium entry was inhibitable by 20 microM La3+. The relative potency of the three muscarinic antagonists for inhibition of calcium entry was essentially the same as for inhibition of acid secretion or pump related glucose oxidation. Image analysis of the glands showed the effects of carbachol, and of the antagonists, on intracellular calcium were occurring largely in the parietal cell. The rise in cell calcium due to release of calcium from intracellular stores was inhibited by 4-DAMP with an IC50 of 1.7 nM, suggesting that the release pathway was regulated by a low affinity M3 muscarinic receptor or state; Ca entry and acid secretion are regulated by a high affinity M3 muscarinic receptor or state, inhibited by higher 4-DAMP concentrations (greater than 30 nM), suggesting that it is the steady-state elevation of Ca that is related to parietal cell function rather than the [Ca]i transient. Displacement of 3H N-methyl scopolamine (NMS) binding to purified parietal cells by CCh showed the presence of two affinities for CCh, but only a single affinity for 4-DAMP and lower affinity for pirenzepine and AFDX 116, providing further evidence for the parietal cell location of the [Ca]i response. Elevation of steady-state [Ca]i levels with either ionomycin or arachidonic acid did not replicate M3 stimulation of acid secretion or glucose oxidation, hence elevation of [Ca]i is necessary but not sufficient for acid secretion.

Patch clamping VDAC in liposomes containing whole mitochondrial membranes.

Abstract: Whole mitochondrial membranes isolated from Neurospora crassa were reconstituted into liposomes and patch clamped. Clear activity characteristic of the mitochondrial channel VDAC was found, namely: open state conductance of 650 pS (in 150 mM KCl, 1 mM CaCl2, 20 mM HEPES, pH 7.2), voltage-dependent closure at both positive and negative potentials, change in conductance upon channel closure of about 450 pS in response to negative and positive potentials, and increased voltage dependence in the presence of Konig's polyanion. This is the first clear demonstration of VDAC single channels using the patch-clamp technique, even though others used this method before to study whole mitochondrial membranes and liposomes containing mitochondrial proteins. We also found one other channel with a conductance change of about 120 pS.

Tumor necrosis factor inhibits K + current expression in cultured oligodendrocytes

Abstract: The effects of tumor necrosis factor-α (TNF-α), a cytokine secreted by activated macrophages, on the electrical membrane properties of cultured adult ovine oligodendrocytes (OLGs) were investigated using the whole-cell voltage-clamp technique. Treatment with recombinant human TNF-α (rhTNF) for 24 to 72 hr produces (i) process retraction in some but not all OLGs, (ii) a reduction in the resting membrane potential with no significant change in membrane capacitance or input resistance over control cells and (iii) a decrease in the expression of both the inwardly rectifying and outward K+ current. The magnitude of the membrane potential change as well as K+ current inhibition was larger in cells with retracted processes. The electrophysiological effects of rhTNF were attenuated when rhTNF was neutralized with a polyclonal anti-rhTNF antibody. The binding of rhTNF to its receptor has been reported to increase GTP binding, to increase GTPase activity of a pertussis-sensitive G protein, and to produce an elevation in intracellular cAMP in other cell types. However, pretreatment of OLGs with activated pertussis toxin failed to attenuate or mimic the effects of rhTNF. Chronic exposure of OLGs to the membrane permeant analogue of cAMP, 8-bromo-cAMP, resulted primarily in an inhibition of the inwardly rectifying K+ current, an effect which was less than that produced by rhTNF alone and without any of the associated rhTNF-induced morphological changes. This indicates that the effects of rhTNF cannot be entirely accounted for by an elevation in intracellular cAMP. Cycloheximide (CHX), an inhibitor of protein synthesis, mimicked the effects of rhTNF; however, the effects of rhTNF and CHX were not additive. The finding that both ionic current expression and membrane potential were reduced in cells treated with rhTNF that appeared morphologically normal suggests that abnormal ion channel expression in OLGs precedes and may contribute to eventual myelin swelling and damage.