Showing papers in "The Journal of Membrane Biology in 1995"

Lipids in biological membrane fusion.

Abstract: The results reviewed suggest that membrane fusion in diverse biological fusion reactions involves formation of some specific intermediates: stalks and pores. Energy of these intermediates and, consequently, the rate and extent of fusion depend on the propensity of the corresponding monolayers of membranes to bend in the required directions. Proteins and peptides can control the bending energy of membrane monolayers in a number of ways. Monolayer lipid composition may be altered by different phospholipases [50, 85, 90], flipases and translocases [4, 50]. Proteins and peptides can change monolayer spontaneous curvature or hydrophobic void energy by direct interaction with membrane lipids [20, 32, 111]. Proteins may also provide some barriers for lipid diffusion in the plane of the monolayer [83, 141]. If diffusion of lipids at some specific membrane sites (e.g., in the vicinity of fusion protein) is somehow hindered, the energy of the bent fusion intermediates would reflect the elastic properties of these particular sites rather than the spontaneous curvature of the whole monolayers. Proteins may deform membranes while bringing them locally into close contact. The alteration of the geometric (external) curvature will certainly change the elastic energy of the initial state and, thus affect the energetic barriers of the formation of the intermediates [143]. In addition, the area and the energy of the stalk can be reduced by preliminary bending of the contacting membranes [111]. The possible effects of proteins and polymers on local elastic properties and local shapes of the membranes have been recently analyzed [22, 39, 45, 63]. These studies may provide a good basis for future development of theoretical models of protein-mediated fusion. Various models for biological fusion have been presented as hypothetical sequences of intermediate conformations of proteins, with membrane lipids just covering the empty spaces between the proteins. Although the results discussed above do not allow us to draw an allexplaining cartoon of the fusion mechanism, they do indicate which properties of membrane lipid bilayers (if modified by fusion proteins) would get these bilayers to fuse. In addition, these data suggest a specific geometry to bent fusion intermediates (stalks and pores) and imply a contribution by lipids to the energy of these intermediates. We think that the synthesis of rapidly developing structural information on fusion proteins with the analysis of the physics of membrane rearrangement may soon yield a real understanding of the fascinating and fundamental phenomenon of membrane fusion.

The AQP2 water channel: Effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats

Abstract: Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats.

A Method for Determining the Unitary Functional Capacity of Cloned Channels and Transporters Expressed in Xenopus Laevis Oocytes

Abstract: The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212±48 particles/μm2, mean, sd) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/μm2) only on the P face. The utility and generality of the method were demonstrated by estimating the “gating charge” per particle of the Na+/ glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like Intramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a “gating charge” of ∼3.5 electronic charges for SGLT1 and ∼9 for the mutant Shaker K+ channel from the ratio of Q max to density of particles measured on the same oocytes. The “gating charges” were 3-fold larger than the “effective valences” calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (p f s) of 1.4 × 10−14 cm3/ sec for CHIP and of 1.5 × 10−16 cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (P f ) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit ∼100-fold less effective than CHIP.

Excitation-calcium release uncoupling in aged single human skeletal muscle fibers

Abstract: The biological mechanisms underlying decline in muscle power and fatigue with age are not completely understood. The contribution of alterations in the excitation-calcium release coupling in single muscle fibers was explored in this work. Single muscle fibers were voltage-clamped using the double Vaseline gap technique. The samples were obtained by needle biopsy of the vastus lateralis (quadriceps) from 9 young (25–35 years; 25.9 ± 9.1; 5 female and 4 male) and 11 old subjects (65–75 years; 70.5 ± 2.3; 6 f, 5 m). Data were obtained from 36 and 39 fibers from young and old subjects, respectively. Subjects included in this study had similar physical activity. Denervated and slow-twitch muscle fibers were excluded from this study. A significant reduction of maximum charge movement (Qmax) and DHP-sensitive Ca current were recorded in muscle fibers from the 65–75 group. Qmax values were 7.6 ± 0.9 and 3.2 ± 0.3 nC/μF for young and old muscle fibers, respectively (P < 0.01). No evidences of charge inactivation or interconversion (charge 1 to charge 2) were found. The peak Ca current was (−)4.7 ± 0.08 and (−)2.15 ± 0.11 μA/μF for young and old fibers, respectively (P < 0.01). The peak calcium transient studied with mag-fura-2 (400 μm) was 6.3 ± 0.4 μm and 4.2 ± 0.3 μm for young and old muscle fibers, respectively. Caffeine (0.5 mm) induced potentiation of the peak calcium transient in both groups. The decrease in the voltage-/ Ca-dependent Ca release ratio in old fibers (0.18 ± 0.02) compared to young fibers (0.47 ± 0.03) (P < 0.01), was recorded in the absence of sarcoplasmic reticulum calcium depletion. These data support a significant reduction of the amount of Ca available for triggering mechanical responses in aged skeletal muscle and, the reduction of Ca release is due to DHPR-ryanodine receptor uncoupling in fast-twitch fibers. These alterations can account, at least partially for the skeletal muscle function impairment associated with aging.

Cytoplasmic Ca2+ inhibits the ryanodine receptor from cardiac muscle.

Abstract: Ca2+-dependent inhibition of native and isolated ryanodine receptor (RyR) calcium release channels from sheep heart and rabbit skeletal muscle was investigated using the lipid bilayer technique. We found that cytoplasmic Ca2+ inhibited cardiac RyRs with an average Km = 15 mm, skeletal RyRs with Km = 0.7 mm and with Hill coefficients of 2 in both isoforms. This is consistent with measurements of Ca2+ release from the sarcoplasmic reticulum (SR) in skinned fibers and with [3H]-ryanodine binding to SR vesicles, but is contrary to previous bilayer studies which were unable to demonstrate Ca2+-inhibition in cardiac RyRs (Chu, Fill, Stefani & Entman (1993) J. Membrane Biol. 135, 49–59). Ryanodine prevented Ca2+ from inhibiting either cardiac or skeletal RyRs. Ca2+-inhibition in cardiac RyRs appeared to be the most fragile characteristic of channel function, being irreversibly disrupted by 500 mm Cs+, but not by 500 mm K+, in the cis bath or by solublization with the detergent CHAPS. These treatments had no effect on channel regulation by AMP-PNP, caffeine, ryanodine, ruthenium red, or Ca2+-activation. Ca2+-inhibition in skeletal RyRs was retained in the presence of 500 mm Cs+. Our results provide an explanation for previous findings in which cardiac RyRs in bilayers with 250 mm Cs+ in the solutions fail to demonstrate Ca2+-inhibition, while Ca2+-inhibition of Ca2+ release is observed in vesicle studies where K+ is the major cation. A comparison of open and closed probability distributions from individual RyRs suggested that the same gating mechanism mediates Ca2+-inhibition in skeletal RyRs and cardiac RyRs, with different Ca2+ affinities for inhibition. We conclude that differences in the Ca2+-inhibition in cardiac and skeletal channels depends on their Ca2+ binding properties.

Transcriptional control of sodium transport in tight epithelial by adrenal steroids.

Abstract: The model for the adrenal steroid action on Na transport in tight epithelia as depicted in Fig. 3A and B dissociates two phases: an early phase during which the pre-existing Na transport machinery is activated and a late phase during which the transport capacity of the machinery is increased. These two sequential phases have been distinguished based on differences in functional aspects of the induced transport, on selective effects of agents interfering with transcriptional regulation and on a correlation of the late response phase with an increase in transport protein synthesis and expression [26, 45, 46, 98, 99, 124]. These observations suggest that a bimodal stimulation of Na transport could involve two different gene networks which are directly (in the physiological meaning) and independently stimulated by the action of the hormone-receptor complex and the following “molecular” cascades (see section Molecular and Physiological Cascades). The relatively clear temporal dissociation of the responses found in experimental situations is probably the consequence of inherent properties of the two networks. Indeed, to generate rapid functional changes, the genes involved in the early response must encode products which have relatively short half-lifes at the mRNA and protein levels. In contrast, the constitutive elements of the Na transport machinery that are increased during the late phase of adrenal steroid action have, as shown for the Na,K-ATPase [82], relatively long half-lifes. Consequently, even though changes in transcription may take place early in the course of the hormonal treatment, they impact on protein synthesis and pools only slowly and after a substantial lag period.

Structure and function of amiloride-sensitive Na+ channels

Abstract: A new molecular biological epoch in amiloride-sensitive Na+ channel physiology has begun. With the application of these new techniques, undoubtedly a plethora of new information and new questions will be forthcoming. First and foremost, however, is the question of how many discrete amiloride-sensitive Na+ channels exist. This question is important not only for elucidating structure-function relationships, but also for developing strategies for pharmacological or, ultimately, genetic intervention in such diseases as obstructive nephropathy, Liddle's syndrome, or salt-sensitive hypertension where amiloride-sensitive Na+ channel dysfunction has been implicated [17, 62]. Epithelia Na+ channels purified from kidney are multimeric. However, it is not yet clear which subunits are regulatory and which participate directly as a part of the Na+ conducting core and what is the nature of the gate. The combination of electrophysiologic techniques such as patch clamp and the ability to study reconstituted channels in planar lipid bilayers along with molecular biology techniques to potentially manipulate the individual subunits should provide the answers to questions that have puzzled physiologists for decades. It seems clear that the robust versatility of the channel in responding to a wide range of differing and potentially synergistic regulatory inputs must be a function of its multimeric structure and relation to the cytoskeleton. Multiple mechanisms of regulation imply multiple regulatory sites. This hypothesis has been validated by the demonstration that enzymatic carboxyl methylation and phosphorylation have both individual and synergistic effects on the purified channel in planar lipid bilayers. Of the multiple mechanisms proposed for channel regulation, evidence is now available to support the ideas that channels may be activated (or inactivated) by direct modifications including phosphorylation and carboxyl methylation, by activation or association of regulatory proteins such as G proteins, and by recruitment from subapical membrane domains. The observation that channel gating is achieved primarily through regulation of open probability without alterations in conductance may simplify future understanding of the molecular events involved in gating once the regulatory sites have been identified. As more Na+ channels or Na+ channel subunits are cloned from different epithelia, it will become possible to piece together the puzzle of epithelial Na+ channels. It is interesting to observe that renal Na+ channel proteins contain a subunit which falls into the 70 kD range. This size protein is in the range reported for the aldosterone-induced proteins [12, 46, 153].(ABSTRACT TRUNCATED AT 400 WORDS)

Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept.

Abstract: The ubiquity of cholesterol in cell membranes and changes in its concentration during development, aging and in various diseases suggest that it plays an important role in modulating cell function. We examined this possibility by monitoring the effects of cholesterol on the activity of the calcium-activated potassium (BK) channel reconstituted into lipid bilayers from rat brain homogenates. Increasing the cholesterol concentration to 11% of total lipid weight resulted in a 70% reduction in channel mean open time and a reduction of the open probability of the channel by 80%. Channel conductance was reduced by 7%. Cholesterol is known to change the order state and the modulus of compressibility of bilayers. These physico-chemical changes may be translated into an overall increase in the structural stress in the bilayer, and this force may be transmitted to proteins residing therein. By examining the characteristics of the BK channel as a function of temperature, in the presence and absence of cholesterol, we were able to estimate the activation energy based on Arrhenius plots of channel kinetics. Cholesterol reduced the activation energy of the BK channel by 50% for the open to closed transition. This result is consistent with an increased stress energy in the bilayer and favors the channel moving into the closed state. Taken together, these data are consistent with a model in which cholesterol induces structural stress which enhances the transition from the open to the closed state of the channel. We suggest that this is an important mechanism for regulating the activity of membrane-integral proteins and therefore membrane function, and that the concept of structural stress may be relevant to understanding the modulation of ion channel activity in cell membranes.

The inositol 1,4,5-trisphosphate (InsP3) receptor

Abstract: In this review, we have described the functional properties and regulation of the InsP3R. Not all aspects of InsP3R function and regulation were covered, the main focus was on the most recent and physiologically important data. Information about the structure, heterogeneity, functional properties, and regulation of the InsP3R is useful for understanding the spatiotemporal aspects of Ca signaling. The combination of biochemical, biophysical and molecular biological techniques has revealed the intricacies of the InsP3R over the past decade. However, questions about the functional differences between various isoforms and splice variants of the InsP3R, the structural determinants responsible for regulation of InsP3R by Ca and ATP, the functional effects of InsP3R phosphorylation and many others remain to be elucidated. Future investigations can be expected to provide answers to these important questions.

Transduction of chemostimuli by the type I carotid body cell.

Abstract: The postulated mechanisms for hypoxic and acidic chemotransduction by type I cells that we have described here are summarized in the diagrams of Fig. 4. Most if not all of these require more complete evaluation and, as we have described, there are obvious points of contention that need to be resolved. Nevertheless, it is apparent that studies of isolated type I cell preparations carried out over the last six years have provided significant advancements in our understanding of chemotransduction in the type I cell. Only when the functioning of these cells has been fully described can we hope to understand the mechanisms underlying the responses of the intact organ to chemostimuli.

Membrane fatty acid composition of tissues is related to body mass of mammals

Abstract: Phospholipids were extracted from tissues (heart, skeletal muscle, kidney cortex, liver and brain) of mammals representing a 9,000-fold range in body mass (mouse, rat, rabbit, sheep and cattle) and their fatty acid composition was determined. In heart, skeletal muscle and kidney cortex, there were significant allometric decreases in the Unsaturation Index (UI; average number of double bonds per 100 fatty acid molecules) with increasing body mass. There were significant inverse allometric relationships between body mass and the proportion of docosahexaenoic acid (22∶6ω3) in heart and skeletal muscle. In heart, skeletal muscle and kidney cortex, larger mammals also had shorter fatty acid chains in their phospholipids and a higher proportion of monounsaturates. In liver, smaller mammals had a higher UI than larger mammals (except the rabbit, which had the lowest UI and very low proportions of ω3 fatty acids). The brain of all mammals maintained a high UI with similar levels of polyunsaturated fatty acids, especially 22∶6ω3. Our results suggest that in heart, skeletal muscle and kidney cortex the activity of the elongases and desaturases are reduced in large mammals compared to small mammals. The allometric trends in membrane composition may be involved in modifying membrane permeability. It is proposed that the elevated degree of polyunsaturation in the membranes of several tissues from small mammals is related to their higher metabolic activity.

The biology of the P-glycoproteins

Abstract: The initial discovery of p-glycoprotein in the plasma membrane of MDR cancer cell lines was followed quickly by the cloning of its gene. Sequence analysis of cloned cDNAs revealed that p-glycoprotein was a member of the ABC family of membrane transporters. Subsequent biochemical characterization demonstrated the binding of chemotherapeutic drugs and ATP to p-glycoprotein. P-glycoprotein-mediated drag transport and drug-stimulated ATPase activity were documented in plasma membrane vesicles and in proteoliposomes containing the partially purified protein. P-glycoprotein was shown to be phosphorylated and the effect of this modification on the protein's biological function was explored. P-glycoproteins were found in many normal tissues and their overexpression was documented in numerous cancers. An important role for p-glycoprotein in intrinsic and acquired drug resistance in clinical oncology was established. Despite all that has been learned about p-glycoprotein over the last few years, additional studies will be necessary to address the many questions that have been left unanswered. Determination of p-glycoprotein structure and membrane topology should help elucidate the nature of chemotherapeutic drug binding sites and the mechanism whereby drug movement is coupled to ATP hydrolysis. Complete purification and functional reconstitution of p-glycoprotein into defined lipid vesicles will permit further characterization of drug transport and ATPase activity and give us the means by which p-glycoprotein's apparent dual function as a transporter and a channel can be clarified. Structural and functional studies on p-glycoprotein will also provide information needed to develop specified inhibitors that can be used clinically to overcome MDR in cancer patients. Further study of the mechanisms whereby p-glycoprotein expression is induced and regulated during malignant transformation is indicated. The development of biliary phospholipid deficiency in mdr2 knockout mice and xenobiotic hypersensitivity in mdr3 knockout mice have given us the first clues into the normal physiologic roles for the p-glycoproteins. The search for endogenous substrates for the p-glycoproteins will continue to be an area of active investigation. Continued investigation of p-glycoprotein's functions should result in better understanding of an important class of prokaryotic and eukaryotic membrane transporters. The potential of exploiting the knowledge garnered from these studies in the treatment of neoplastic, parasitic and inherited and acquired liver disease may be greater than we can now imagine.

The gating of the sheep skeletal sarcoplasmic reticulum Ca2+-release channel is regulated by luminal Ca2+

Abstract: The effects of changes in luminal [Ca2+] have been investigated in sheep skeletal sarcoplasmic reticulum (SR) Ca2+-release channels after activation of the channels by different ligands from the cytosolic side of the channel. Native heavy SR membrane vesicles were incorporated into planar phospholipid bilayers under voltage-clamp conditions. Experiments were carried out in symmetrical 250 mm Cs+. Lifetime analysis indicates that channels activated solely by cytosolic Ca2+ exhibit at least two open and five closed states. The open events are very brief and are close to the minimum resolvable duration. When channels are activated solely by cytosolic Ca2+, luminal Ca2+ does not appear to exert any regulatory effect. The P0 and duration of the open and closed lifetimes are unchanged. However, if channels are activated by ATP alone or by ATP plus cytosolic Ca2+, increases in luminal [Ca2+] produce marked increases in P0 and in the duration of the open lifetimes. Our results demonstrate that maximum activation of the skeletal SR Ca2+-release channel by ATP cannot be obtained in the absence of millimolar luminal [Ca2+].

Kinetics and specificity of the renal Na + / myo -inositol cotransporter expressed in Xenopus Oocytes

Abstract: The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V m ), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: K 0.5 MI , K 0.5 Na , and the Hill coefficient n. At 100 mM NaCl, K 0.5 MI was about 50 μm and was independent of V m . At 0.5 mm myo-inositol, K 0.5 Na ranged from 76 mm at V m =−50 mV to 40 mm at V m =−150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K I of 64 μm at V m =−50 mV and 130 μm at V m = −150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V m =−150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, α-methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V m =−150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, α-methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na+-dependent uptake of 3H-d-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na+ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording.

Abstract: Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 +/- 25 pS and 350 +/- 14 pS in 400 mM symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca2+ and glutamate-. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mechanosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.

NO3- transport across the plasma membrane of Arabidopsis thaliana root hairs: Kinetic control by pH and membrane voltage

Abstract: High-affinity nitrate transport was examined in intact root hair cells of Arabidopsis thaliana using electrophysiological recordings to characterise the response of the plasma membrane to NO3- challenge and to quantify transport activity. The NO3(-)-associated membrane current was determined using a three-electrode voltage clamp to bring membrane voltage under experimental control and to compensate for current dissipation along the longitudinal cell axis. Nitrate transport was evident in the roots of seedlings grown in the absence of a nitrogen source, but only 4-6 days postgermination. In 6-day-old seedlings, additions of 5-100 microM NO3- to the bathing medium resulted in membrane depolarizations of 8-43 mV, and membrane voltage (Vm) recovered on washing NO3- from the bath. Voltage clamp measurements carried out immediately before and following NO3- additions showed that the NO3(-)-evoked depolarizations were the consequence of an inward-directed current that appeared across the entire range of accessible voltages (-300 to +50 mV). Both membrane depolarizations and NO3(-)-evoked currents recorded at the free-running voltage displayed quasi-Michaelian kinetics, with apparent values for Km of 23 +/- 6 and 44 +/- 11 microM, respectively and, for the current, a maximum of 5.1 +/- 0.9 muA cm-2. The NO3- current showed a pronounced voltage sensitivity within the normal physiological range between -250 and -100 mV, as could be demonstrated under voltage clamp, and increasing the bathing pH from 6.1 to 7.4-8.0 reduced the current and the associated membrane depolarizations 3- to 8-fold. Analyses showed a well-defined interaction between the kinetic variables of membrane voltage, pHo and [NO3-]o. At a constant pHo of 6.1, depolarization from -250 to -150 mV resulted in an approximate 3-fold reduction in the maximum current but a 10% rise in the apparent affinity for NO3-. By contrast, the same depolarization effected an approximate 20% fall in the Km for transport as a function in [H+]o. These, and additional characteristics of the transport current implicate a carrier cycle in which NO3- binding is kinetically isolated from the rate-limiting step of membrane charge transit, and they indicate a charge-coupling stoichiometry of 2(H+) per NO3- anion transported across the membrane. The results concur with previous studies showing a high-affinity NO3- transport system in Arabidopsis that is inducible following a period of nitrogen-limiting growth, but they underline the importance of voltage as a kinetic factor controlling NO3- transport at the plant plasma membrane.

Calcium and inositol trisphosphate receptors.

Abstract: It is almost twenty years since the link between hormone effects on phosphoinositide and Ca 2+ metabolism was first proposed [55] and more than a decade since inositol 1,4,5-trisphosphate (IP3) was shown to release Ca 2+ from intracellular stores [7, 90]. More recently, there has been a rapid growth in our understanding of the means whereby IP 3 stimulates Ca 2+ mobilization and initiates more complex Ca 2+ signals [4, 76]. The receptors to which IP 3 binds, initially identified by structure-activity [ 12] and radioligand-binding [68, 88] studies, have been purified [92], functionally reconstituted into both lipid vesicles [23] and bilayers from which single channel events have been resolved [102], and the primary structures of IP 3 receptors have been deduced from cDNA cloning [28, 59]. These studies have revealed that IP 3 receptors are in many ways related to another family of intracellular Ca 2+ channels, ryanodine receptors. Both families of receptors form poorly selective cation channels with conductances severalfold larger than those of Ca 2+ channels in the plasma membrane, and each displays four similarly spaced subconductance states [86, 99, 102], an observation that may be related to their tetrameric structures. Although IP 3 and ryanodine receptors lack cation selectivity, Ca 2+ is the only cation with a significant electrochemical gradient across the membranes of the endoplasmic and sarcoplasmic reticula; both receptors therefore behave as intracellular Ca 2+ channels in their native setting. The two families of in-

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43.

Abstract: The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 μm monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.

Modulation of a volume-regulated chloride current by F-actin.

Abstract: We have examined whether F-actin integrity is involved in activation of a volume-regulated Cl− current (VRChlC) in B-lymphocytes. VRChlC activation was initiated in response to establishing a whole cell recording in the presence of a hyposmotic gradient. Parallel confocal microscopy experiments using Rhodamine Phalloidin (R-P) as a specific marker of F-actin showed that the submembrane actin ring is reversibly disrupted in response to an hyposmotic gradient. Disruptions of cortical F-actin integrity by 50 μm cytochalasin B (CB) does not trigger activation of VRChlC under isosmotic conditions or potentiate the rate of activation when the osmolarity of the extracellular solution was decreased by 75%. However, incubation with CB increased the rate of VRChlC activation in response to a 90% hyposmotic gradient. Phalloidin, a stabilizer of F-actin, decreases the rate of VRChlC activation in response to a 90% gradient, but has no effect in response to a 75% gradient. These observations suggest that disassembly of cortical F-actin is not critical for VRChlC activation in B-lymphocytes. The integrity of cortical F-actin, however, can exert a modulatory effect on the rate of VRChlC activation in the presence of a hyposmotic gradient.

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus.

Abstract: Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10−4 m and 10−4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na+/H+ exchanger, demonstrated by NH4Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na+/H+ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl−/HCO 3 − exchanger was also found in the chloride cells, inhibited by 10−4 m DIDS but not involved in the hyperosmotic response. Ca2+ concentration in the medium was critical for the stimulation of Cl− secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na+K+2Cl− cotransporter, the Na+/H+ exchanger and opening of Cl− channels.

Magnitude and modulation of pancreatic beta-cell gap junction electrical conductance in situ.

Abstract: The parallel gap junction electrical conductance between a β-cell and its nearest neighbors was measured by using an intracellular microelectrode to clamp the voltage of a β-cell within a bursting islet of Langerhans. The holding current records consisted of bursts of inward current due to the synchronized oscillations in membrane potential of the surrounding cells. The membrane potential record of the impaled cell, obtained in current clamp mode, was used to estimate the behavior of the surrounding cells during voltage clamp, and the coupling conductance was calculated by dividing the magnitude of the current bursts by that of the voltage bursts. The histogram of coupling conductance magnitude from 26 cells was bimodal with peaks at 2.5 and 3.5 nS, indicating heterogeneity in extent of electrical communication within the islet of Langerhans. Gap junction conductance reversibly decreased when the temperature was lowered from 37 to 30°C and when the extracellular calcium concentration was raised from 2.56 to 7.56 mm. The coupling conductance decreased slightly during the active phase of the burst. Activation of adenylate cyclase with forskolin (10 μm) resulted in an increase in cell-to-cell electrical coupling. We conclude that β-cell gap junction conductance can be measured in situ under near physiological conditions. Furthermore, the magnitude and physiological regulation of β-cell gap junction conductance suggest that intercellular electrical communication plays an important role in the function of the endocrine pancreas.

Evaluation of optimal voltage-sensitive dyes for optical monitoring of embryonic neural activity.

Abstract: To evaluate the suitability of a variety of fast voltage-sensitive dyes for optical recording of rapid transmembrane potential activity in the embryonic nervous system, we screened over twenty dyes, including several newly synthesized probes, in three different embryonic neural preparations: cervical vagus nerve bundle, nodose ganglion, and brainstem from 7-day old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recordingsystem. Signal size, signal-to-noise ratio, photobleaching, and phototoxicity were examined. Several promising new merocyanine-rhodanine dyes for embryonic nervous systems were found.

Voltage-dependent sodium currents recorded from dissociated rat taste cells

Abstract: Voltage-dependent sodium currents were analyzed in detail from dissociated mammalian taste receptor cells using the whole-cell patch clamp technique. Approximately 50–75% of all taste receptor cells expressed sodium currents. These currents activated close to −50 mV (holding potential = −80 mV) with maximal currents most often occurring at −10 mV. The distribution of maximal inward currents across all cells appeared to display two peaks, at −254 pA and −477 pA, possibly due to differences in sodium channel density. Inward currents were eliminated by replacing 90% of external sodium with N-methyl-D-glucamine. The currentvoltage relationship of the activated current, as measured by a tail current analysis, was linear, suggesting an ohmic nature of the open channel conductance. The relationship between the time to the peak activated current and the step potential was well fit by a double exponential curve (τ1 = 6.18, τ2 = 37.8 msec). Development of inactivation of the sodium current was dependent upon both voltage- and temporal-parameters. The voltage dependence of the time constant (τ) obtained from removal of inactivation, development of inactivation, and decay of the sodium current displayed a bell-shaped curve with a maximum of 55 msec at −70 mV. In addition to fast inactivation (half maximal at −50 mV), these currents also displayed a slow inactivation (half maximal at −65 mV). Voltage-dependent sodium currents were reversibly inhibited by nanomolar concentrations of tetrodotoxin (Kd = 10−8 m). There was no evidence of a TTX-insensitive sodium current. This description broadens our understanding of gustatory transduction mechanisms with a particular relevance to the physiological role of receptor cell action potentials.

Amino acid current through anion channels in cultured human glial cells.

Abstract: During volume regulation in hypotonic media, glial cells release a large portion of their amino acids These amino acid losses appear to be mediated by a diffusion type of transport and a swelling-activated chloride channel seems to be involved The objective of this project was to provide direct evidence that amino acids could diffuse through a Cl− channel Using a human glial cell line, Cl− currents activated in hypotonic media were measured in whole-cell patch clamp To measure the currents produced by amino acids, it was necessary to increase the pH of external solutions to basic values reaching 96 and 100 to raise the concentration of the anionic form of these amino acids Introducing external hypotonic media containing high concentrations of amino acids, like glycine, taurine, glutamine and glutamate, it was possible to measure their respective current-voltage curves with NMDG-Cl-filled pipettes From the reversal potentials, their permeability ratios with respect to chloride were determined It was found that the low molecular weight amino acids, like glycine, were most permeant, while the larger ones, like glutamine, had a lower permeability with respect to chloride The amino acids with two carboxyl groups, like glutamate, had a much lower permeability ratio The reversal potentials for some metabolites, like lactate and malate were also measured for comparison These results demonstrate that amino acids can diffuse through anion channels and that activation of these channels in pathological conditions could be at least partly responsible for the observed increase in external amino acids

Low conductance states of a single ion channel are not 'closed'.

Abstract: We have used a polymer-exclusion method to estimate the sizes of the high and low-conductance states of Staphylococcus aureus α-toxin channels across planar lipid bilayers. Despite a >10-fold difference in conductance between high and low-conductance states, the size differs by <2-fold. We conclude that factors other than the dimensions have a strong influence on the conductance of α-toxin channels. We also show that the high conductance state is destabilized by the presence of high molecular weight polymers outside the channel, compatible with the removal of channel water as the high conductance state “shrinks” to the low conductance state.

The ion channel behavior of the nuclear pore complex.

Abstract: Macromolecule-conducting pores have been recently recognized as a distinct class of ion channels. The poor role of macromolecules as electrical charge carriers can be used to detect their movement along electrolyte-filled pores. Because of their negligible contribution to electrical ion currents, translocating macromolecules reduce the net conductivity of the medium inside the pore, thus decreasing the measured pore ion conductance. In the extreme case, a large translocating macromolecule can interrupt ion flow along the pore lumen, reflected as a negligible pore conductance. Therefore, ion conductance serves as a measurement of macromolecular transport, with lesser values indicating greater macromolecular translocation (in size and/or number). Such is the principle of operation of the widely used Coulter counter, an instrument for counting and sizing particles. It has long been known that macromolecules translocate across the central channel of nuclear pore complexes (NPCs). Recently, large conductance ion channel activity (100–1000 pS) was recorded from the nuclear envelope (NE) of various preparations and it was suggested that NPCs may be the source of this activity. Despite its significance to understanding the regulation of transcription, replication, mRNA export, and thus gene expression of normal and pathological states, no report has appeared demonstrating that this channel activity corresponds to ion flow along the central channel of the NPC. Here we present such a demonstration in adult mouse cardiac myocyte nuclei. In agreement with concepts introduced for macromolecule-conducting channels, our patch clamp experiments showed that ion conductance is reduced, and thus that ion flow is restricted during translocation of macromolecules containing nuclear targeting signals. Ion flow was blocked by mAb414, a monoclonal antibody raised against a major NPC glycoprotein and known to localize on the NPC channel where it blocks macromolecular transport. These results also establish patch clamp as a useful technique for the measurement of macromolecular translocation along the large central channel of the NPC and provide a basis for the design of future investigations of nuclear signaling for control of gene activity, mRNA export for gene expression, as well as other processes subservient to NPC-mediated nucleocytoplasmic exchange.

Phospholipid surface density determines the partitioning and permeability of acetic acid in DMPC:cholesterol bilayers.

Abstract: Relationships between the permeability coefficient (PHA) and partition coefficient (Km/w) of acetic acid and the surface density of DMPC:cholesterol bilayers have been investigated Permeability coefficients were measured in large unilamellar vesicles by NMR line broadening Bilayer surface density, σ, was varied over a range of 05–09 by changing cholesterol concentration and temperature The temperature dependence of PHA for acetic acid exhibits Arrhenius behavior with an average apparent activation energy (E a ) of 22±3 kcal/mole over a cholesterol mole fraction range of 000–040 This value is much greater than the enthalpy change for acetic acid partitioning between bulk decane and water (ΔH° = 48±08 kcal/mole) and the calculated E a (= 80 kcal/mole) assuming a “bulk phase” permeability model which includes the enthalpy of transfer from water to decane and the temperature dependence of acetic acid's diffusion coefficient in decane These results suggest that dehydration, previously considered to be a dominant component, is a minor factor in determining E a Values of 1n PHA decrease linearly with the normalized phospholipid surface density with a slope of κ = -124±11 (r = 090) Correction of PHA for those temperature effects considered to be independent of lipid chain order (ie, enthalpy of transfer from water to decane and activation energy for diffusion in bulk hydrocarbon) yielded an improved correlation (κ = -117±05 (r = 096)) The temperature dependence of Km/w is substantially smaller than that for PHA and dependent on cholesterol composition Values of 1n Km/w decrease linearly with the surface density with a slope of κ = -46±03 (r = 095), which is 27-fold smaller than the slope of the plot of 1n PHAvs σ Thus, chain ordering is a major determinant for molecular partitioning into and transport across lipid bilayers, regardless of whether it is varied by lipid composition or temperature

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.

Abstract: We determined the extent of Na+-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na+-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na+-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, β-alanine, α-aminoisobutyric acid (AIB), α-methylaminoisobutyric acid (MeAIB), τ-amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (Isc) in voltage-clamped Caco-2 cell monolayers in Na+-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pHi and inward Isc. In conclusion, Caco-2 cells express a Na+-independent, H+-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.

Preparation, characterization, and structure of half gap junctional layers split with urea and EGTA.

Abstract: Gap junctions, collections of membrane channels responsible for intercellular communication, contain two paired hemichannels (also called connexons). We have investigated conditions for splitting the membrane pair using urea. We have developed a protocol which consistently splits the gap junction samples with 60–90% efficiency. Our results indicate that hydrophobic forces are important in holding the two connexons together but that Ca2+ ions are also important in the assembly of the membrane pair. Greater yields and better structural integrity of split junctions were obtained with a starting preparation of gap junctions which had been detergent treated. Image analysis of edge views of single connexon layers reveal an asymmetry in the appearance of the cytoplasmic and extracellular surface. Cryo-electron microscopy and image analysis of split junctions show that the packing and structural detail of membranes containing arrays of single connexons are the same as for intact junctions, and that the urea treatment causes no gross structural changes in the connexon assembly.