Ouabain-resistant hyperpolarization induced by insulin in aggregates of embryonic heart cells.

Spheroidal aggregates formed from trypsin-dissociated 14-day embryonic chicken hearts after 48 hr of rotation on a gyratory shaker. Intracellularly recorded resting membrane potentials of aggregates bathed in 1.3 mM K+ balanced salt solution had a mean (+/- SD) of 64 +/- 4 mV. After a stable potential was achieved, addition of 1-100 nM sodium bovine insulin caused a slow hyperpolarization of up to 19 mV after 4-5 min, followed, in some cases, by a further, more rapid, shift to a potential near EK. Equivalent hyperpolarizations were observed when insulin was added in the presence of 10 mM ouabain, indicating that enhanced Na+,K+ pump activity was not responsible for the change in membrane potential. The concentration of insulin that produced half-maximal hyperpolarization (2 nM) corresponded to the association constant of a high-affinity insulin receptor, suggesting that binding to this class of receptors led to the change in membrane potential. Steady-state current-voltage curves from current clamp experiments suggested that insulin produced an increase in slope conductance at potentials near rest by inducing an outward current with an apparent potential negative to -90 mV.     

Triiodothyronine affects the electrical properties of GH3 cells

Electrophysiological experiments show that T3 has a direct effect on the cell membrane of GH3 cells, a transformed line from the rat pituitary. Slope conductance versus membrane potential, resting membrane resistance, potential, capacity and action potentials were measured in this study. Using a current clamp technique, the effects of tetrodotoxin, tetraethylammonium, apamin, and nifedipine were measured and compared with those directly evoked by T3. T3 increased the slope conductance: 1. at around -60 mV, as did tetrodotoxin (Na+ channel blocker); 2. at about -40 mV, as did nifedipine (Ca2+ channel blocker), but decreased this conductance strongly between -20 and -30 mV, as did both nifedipine and apamin (Ca2(+)-sensitive K+ channel blocker). Action potentials were inhibited by T3 and by nifedipine. Action potentials in these cells are primarily related to Ca2+ ions. It seems that T3 inhibits the Ca2+ current and, as a consequence, the Ca2(+)-sensitive K+ current.     

Evidence for electrogenic Na+ pumping in human atrial myocardium

The resting potential of "sodium-loaded' cardiac cells can transiently hyperpolarize to levels negative to the steady state resting potential [3,5,6]. Hyperpolarization is associated with the coupled efflux of Na+ and influx of K+ driven by an active transport process and may result from an increased K+ equilibrium potential (EK), an outward pump current or both. Using conventional microelectrode techniques, we found that Na+-loaded human atrial myocardium can also transiently hyperpolarize. Na+ loading was induced by cooling to 2 degrees to 3 degrees C. Upon rewarming to 37 degrees C in a 20 mM K+ solution, the resting potential transiently hyperpolarized to levels at least 11 mV negative to the calculated EK and 29 +/- 2 mV (mean +/- S.E.) negative to the steady state level (- 33 +/- 2 mV) recorded some 15-20 minutes later. An increase in K+ conductance induced by acetylcholine exposure [2,7,10] during the transient hyperpolarization caused a depolarization, indicating that the resting potential was indeed negative to EK. These findings cannot be explained by either conductance changes or electroneutral Na+ pumping and concomitant extracellular K+ depletion. We conclude that the Na+-loaded human atrium can generate net pump current.     

An inward rectifier and a voltage-dependent K+ current in single, cultured pericytes from bovine heart

OBJECTIVE: The purpose of this study was to describe passive electrical properties and major membrane currents in coronary pericytes. METHODS: 78 single, cultured bovine pericytes were studied with the patch-clamp technique in the whole-cell mode. RESULTS: The membrane potential of the cells was -48.9+/-9.6 mV (mean+/-S.D.) with 5 mM and -23.2+/-2.2 mV with 60 mM extracellular K+. The membrane capacitance was 150.2+/-123.2 pF. The current-voltage relation of the pericytes was dominated by an inward current at hyperpolarized potentials and an outward current at depolarized potentials. Increasing extracellular K+ from 5 to 60 mM led to an increase of the inward current and to a shift of this current to more depolarized potentials. The inward current was very sensitive to extracellular barium (50 microM). The maximum slope conductance of the cells at hyperpolarized potentials was 2.9+/-2.8 nS. Inward rectification of whole-cell currents was steep (slope factor = 6.8 mV). With elevated external K+ the outward current reversed near the potassium equilibrium potential. Onset of the outward current was sigmoid and inactivation of this current was monoexponential, slow (time constant = 12.8 s) and incomplete. Voltage-dependence of outward current steady-state activation was steep (slope factor = 4.6 mV). The outward current was very sensitive to 4-aminopyridine (dissociation constant = 0.1 mM). The maximum slope conductance at depolarized potentials was 16.6+/-15.6 nS. CONCLUSION: We report for the first time, patch-clamp recordings from coronary pericytes. An inward rectifier and a voltage-dependent K+ current were identified and characterized. Regulation of these currents may influence coronary blood flow.     

Voltage-dependent activation in purified reconstituted sodium channels from rabbit T-tubular membranes.

We have examined the voltage-dependent gating of batrachotoxin-modified sodium channels purified from rabbit T-tubular membranes in two ways. First, purified channels were reconstituted into planar bilayers and single-channel properties were measured. Batrachotoxin-activated channels showed steep voltage-dependent activation with half-maximal opening probabilities at potentials between -95 and -116 mV. The single-channel conductance (500 mM Na+ cis, 200 mM Na+ trans) averaged 20 pS and was independent of membrane potential. Channels usually inserted with their extracellular faces on the trans side of the bilayer; addition of tetrodotoxin to the cis side had no effect, whereas addition to the trans side blocked greater than 95% of channel openings at -77 mV. A second approach was used to establish that this voltage dependence was a characteristic of the entire population of purified channels and not just those few channels observed in planar bilayers. Channels reconstituted into egg phosphatidylcholine vesicles were functionally oriented by inclusion of internal saxitoxin; vesicle membrane potentials were then generated by K+ gradients in the presence of valinomycin. After batrachotoxin activation, Vm was altered by shifts of K+o. All of the specific 22Na+ influx activated by batrachotoxin and blocked by saxitoxin was found to be voltage sensitive, activating between predicted membrane potentials of -100 and -50 mV. The single-channel properties of the purified T-tubular sodium channel correspond closely to those seen with native sodium channels from rat sarcolemma. The voltage-dependent activation of the batrachotoxin-modified reconstituted channel is the same as that seen with native channels in situ or in bilayers after exposure to this toxin. Most importantly, this voltage-dependent gating is a property of all of the purified channels capable of specific pharmacological activation.     

Mechanism of high-affinity potassium uptake in roots of Arabidopsis thaliana.

Potassium is a major nutrient in higher plants, where it plays a role in turgor regulation, charge balance, leaf movement, and protein synthesis. Terrestrial plants are able to sustain growth at micromolar external K+ concentrations, at which K+ uptake across the plasma membrane of root cells must be energized despite the presence of a highly negative membrane potential. However, the mechanism of energization has long remained obscure. Therefore, whole-cell mode patch clamping has been applied to root protoplasts from Arabidopsis thaliana to characterize membrane currents resulting from the application of micromolar K+. Analysis of whole cell current/voltage relationships in the presence and absence of micromolar K+ enabled direct testing of K+ transport for possible energization by cytoplasmic ATP and the respective trans-membrane gradients of Na+, Ca2+, and H+. Subtracted current/voltage relations for K(+)-dependent membrane currents are independent of ATP and reverse at potentials that imply H(+)-coupled K+ transport with a ratio of 1 H+:K+. Furthermore, the reversal potential of the K+ current shifts negative as external H+ activity is decreased. K(+)-dependent currents saturate in the micromolar concentration range with an apparent Km of 30 microM, a value in close agreement with previously reported Km values for high-affinity K+ uptake. We conclude that our results are consistent with the view that high-affinity K+ uptake in higher plants is mediated by a H+:K+ symport mechanism, competent in driving K+ accumulation to equilibrium ratios in excess of 10(6)-fold.     

Transmembrane electrical potential difference regulates Na+/HCO3- cotransport and intracellular pH in hepatocytes.

We have examined the hypothesis that a regulatory interplay between pH-regulated plasma membrane K+ conductance (gK+) and electrogenic Na+/HCO3- cotransport contributes importantly to regulation of intracellular pH (pHi) in hepatocytes. In individual cells, membrane depolarization produced by transient exposure to 50 mM K+ caused a reversible increase in pHi in the presence, but not absence, of HCO3-, consistent with voltage-dependent HCO3- influx. In the absence of HCO3-, intracellular alkalinization and acidification produced by NH4Cl exposure and withdrawal produced membrane hyperpolarization and depolarization, respectively, as expected for pHi-induced changes in gK+. By contrast, in the presence of HCO3-, NH4Cl exposure and withdrawal produced a decrease in apparent buffering capacity and changes in membrane potential difference consistent with compensatory regulation of electrogenic Na+/HCO3- cotransport. Moreover, the rate of pHi and potential difference recovery was several-fold greater in the presence as compared with the absence of HCO3-. Finally, continuous exposure to 10% CO2 in the presence of HCO3- produced intracellular acidification, and the rate of pHi recovery from intracellular acidosis was inhibited by Ba2+, which blocks pHi-induced changes in gK+, and by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, which inhibits Na+/HCO3- cotransport. These findings suggest that in hepatocytes, changes in transmembrane electrical potential difference, mediated by pH-sensitive gK+, play a central role in regulation of pHi through effects on electrogenic Na+/HCO3- cotransport.     

Relation of Na+, K(+)-ATPase to delayed motor nerve conduction velocity: effect of aldose reductase inhibitor, ADN-138, on Na+, K(+)-ATPase activity

The role of sorbitol, myo-inositol, and Na+, K(+)-adenosine triphosphatase (ATPase) activity on motor nerve conduction velocity (MNCV) in streptozotocin (STZ)-diabetic rats was studied. Reduction of MNCV and Na+, K(+)-ATPase in caudal nerves appeared after 3 weeks of diabetes, and at this time treatment with aldose reductase inhibitor (ARI), ADN-138 and 1% myo-inositol supplement was begun. One percent myo-inositol supplement for 3 weeks resulted in a significant increase in myo-inositol levels in diabetic nerves, but left MNCV and sorbitol levels unchanged. In contrast, treatment with ADN-138 for 3 weeks reduced sorbitol levels in diabetic nerves and resulted in significant increases in MNCV and Na+, K(+)-ATPase in the nerves. Since ADN-138 did not restore myo-inositol levels, the increase in Na+, K(+)-ATPase levels by ADN-138 treatment was independent of myo-inositol levels. Also, nerve Na+ levels in ADN-138-treated rats were reduced and the ratio of K+ to Na+ was raised, while 1% myo-inositol supplement did not affect them. These results suggest that treatment with ADN-138 elevates MNCV through a series of processes: ARI----reduction of sorbitol level----increase in Na+, K(+)-ATPase activity----correction of K+, Na+ imbalance----increase in MNCV.     

Electrical properties of cultured human adrenocorticotropin-secreting adenoma cells: effects of high K+, corticotropin-releasing factor, and angiotensin II

ACTH-secreting pituitary adenoma cells were cultured from specimens obtained by transphenoidal hypophysectomy in five patients with Cushing's disease. The majority of adenoma cells (90%) stained specifically with antiserum against human ACTH. The electrophysiological properties and response to hormones of these cells were studied with intracellular recording techniques under current clamp and voltage clamp conditions. Most (80%) of the cells fired action potentials that were Ca2+-dependent inasmuch as they were blocked by Co2+ (5 mM) and by removal of Ca2+ from the medium, but were unaffected by tetrodotoxin (0.3 mM) and by Na+ removal. The cells responded to factors known to stimulate ACTH release, including high K+, CRF, and angiotensin II (AII). High K+ (50 mM) induced a membrane depolarization in association with an increase in conductance. CRF (100 nM) produced a depolarization, a decrease in conductance, an increase in spike firing, and an increase in spike duration. Although AII was inactive in ordinary recordings, in cells loaded with lithium (Li+) to promote the phospholipid-dependent second messenger system, the peptide produced an increase in spike firing and spike duration with no change in membrane potential. The combination of CRF and AII (CRF + AII; 100 nM each) in Li+-loaded cells caused a greater excitatory effect than either peptide alone. Under voltage clamp, the response either to CRF or to CRF + AII could be attributed, at least in part, to the inhibition of a slow, voltage-dependent K+ current that is persistently active at resting potential. These results indicate that modulation of action potential firing may be an early step in the regulation of ACTH release from pituitary cells by known secretagogues. Since action potentials in these cells are associated with Ca2+ entry, the resulting changes in intracellular Ca2+ levels could mediate the effects of the hormones on secretion.     

Inward-rectifying K+ channels in guard cells provide a mechanism for low-affinity K+ uptake.

The molecular mechanisms by which higher plant cells take up K+ across the plasma membrane (plasmalemma) remain unknown. Physiological transport studies in a large number of higher plant cell types, including guard cells, have suggested that at least two distinct types of K(+)-uptake mechanisms exist, permitting low-affinity and high-affinity K+ accumulation, respectively. Recent patch clamp studies have revealed the presence of inward-conducting (inward-rectifying) K+ channels in the plasma membrane of higher plant cells. Research on guard cells has suggested that these K+ channels provide a major pathway for proton pump-driven K+ uptake during stomatal opening. In the present study the contribution of inward-rectifying K+ channels to higher plant cell K+ uptake was investigated by examining kinetic properties of guard cell K+ channels in Vicia faba in response to changes in the extracellular K+ concentration. Increasing the extracellular K+ concentration in the range from 0.3 mM to 11.25 mM led to enhancement of inward K+ currents and changes in current-voltage characteristics of K+ channels. The increase in K+ conductance as a function of the extracellular K+ concentration revealed a K(+)-equilibrium dissociation constant (Km) of approximately 3.5 mM, which suggests that inward-rectifying K+ channels can function as a molecular mechanism for low-affinity K+ uptake. Lowering the extracellular K+ concentration in the range from 11 mM to 1 mM induced negative shifts in the activation potential of K+ channels, such that these channels function as a K+ sensor, permitting only K+ uptake. At low extracellular K+ concentrations of 0.3 mM K+, inward-rectifying K+ channels induce hyperpolarization. Results from the present study suggest that inward-rectifying K+ channels constitute an essential molecular mechanism for plant nutrition and growth control by providing a K(+)-sensing and voltage-dependent pathway for low-affinity K+ uptake into higher plant cells and additionally by contributing to plasma membrane potential regulation.     

Single-channel recording of inwardly rectifying potassium currents in developing myocardium

Properties of the inwardly rectifying K+ channel, which contributes to the maintenance of the resting membrane potential, were studied in neonatal rabbit ventricular myocytes using the patch-clamp technique. Inward rectification was evident in single-channel current-voltage (I-V) relations at potentials positive to the potassium equilibrium potential (Ek = 0 mV with [K+]o = [K+]i = 150 mM, [Mg2+]i = 2 mM). The single-channel conductance was 3.2 +/- 0.1 pS in physiological (5.4 mM) [K+]o. The zero-current potential shifted 48.4 +/- 2.4 mV for a ten-fold change in [K+]o in neonatal cells. External Ba2+ blocked the current in a dose-dependent manner. The voltage dependence, open-state probability and channel density of this channel were compared between neonatal and adult ventricular myocytes isolated by similar techniques. The open-state probability of the channel was approximately the same in neonatal (0.39 +/- 0.06, n = 13) as in adult cells (0.4 +/- 0.05, n = 11). However, in symmetrical transmembrane K+ concentration [( K+]o = [K+]i = 150 mM), the single channel conductance was significantly smaller in neonatal (25 +/- 0.3 pS, n = 25) as compared with adult cells (31 +/- 0.4 pS, n = 12). In addition, the relationship between resting membrane potential and [K+]o was measured in neonatal and adult myocytes. The resting membrane potential in the neonate was less dependent on [K+]o than in the adult. These results are consistent with an age-related change in resting membrane K+ permeability which may result from a developmental change in the single-channel conductance properties of the inwardly rectifying K+ channel.     

Novel arrhythmogenic mechanism revealed by a long-QT syndrome mutation in the cardiac Na(+) channel

Variant 3 of the congenital long-QT syndrome (LQTS-3) is caused by mutations in the gene encoding the alpha subunit of the cardiac Na(+) channel. In the present study, we report a novel LQTS-3 mutation, E1295K (EK), and describe its functional consequences when expressed in HEK293 cells. The clinical phenotype of the proband indicated QT interval prolongation in the absence of T-wave morphological abnormalities and a steep QT/R-R relationship, consistent with an LQTS-3 lesion. However, biophysical analysis of mutant channels indicates that the EK mutation changes channel activity in a manner that is distinct from previously investigated LQTS-3 mutations. The EK mutation causes significant positive shifts in the half-maximal voltage (V(1/2)) of steady-state inactivation and activation (+5.2 and +3.4 mV, respectively). These gating changes shift the window of voltages over which Na(+) channels do not completely inactivate without altering the magnitude of these currents. The change in voltage dependence of window currents suggests that this alteration in the voltage dependence of Na(+) channel gating may cause marked changes in action potential duration because of the unique voltage-dependent rectifying properties of cardiac K(+) channels that underlie the plateau and terminal repolarization phases of the action potential. Na(+) channel window current is likely to have a greater effect on net membrane current at more positive potentials (EK channels) where total K(+) channel conductance is low than at more negative potentials (wild-type channels), where total K(+) channel conductance is high. These findings suggest a fundamentally distinct mechanism of arrhythmogenesis for congenital LQTS-3.     

Erythrocyte membrane lipids and cationic transport systems in men.

OBJECTIVE: The relationship between erythrocyte membrane and plasma lipids and various transmembrane erythrocyte cationic fluxes was examined in 53 normal men. DESIGN: Different measurements of erythrocyte transport systems were obtained: Na(+)-Li+ countertransport activity; Na+, K+ cotransport activity; Na+, K(+)-ATPase pump activity and the ground membrane permeability for Na+ and K+ as well as the intra-erythrocyte Na+, K+ and Mg2+ concentrations. Plasma cholesterol, triglycerides, phospholipids, free fatty acids, low- and high-density lipoprotein cholesterol levels and the erythrocyte membrane contents of cholesterol, phospholipids and free fatty acids were obtained from fasting subjects. RESULTS: In single regression analysis the erythrocyte Na(+)-Li+ countertransport and Na+, K+ cotransport activities were negatively related to the erythrocyte membrane cholesterol, phospholipids and free fatty acids contents. The Na+, K(+)-ATPase pump activity as assessed by the ouabain-sensitive Na+ efflux was also inversely related to the membrane cholesterol and phospholipids contents. In multiple regression analysis the red blood cell Na(+)-Li+ countertransport activity was independently and negatively related to the membrane cholesterol and free fatty acids contents. CONCLUSION: Our data show that an elevated level of erythrocyte membrane lipids in normal men is accompanied by lower Na(+)-Li+ countertransport, Na+, K+ cotransport and Na+, K(+)-ATPase pump activities.     

Muscarinic control of pancreatic B cell function involves sodium-dependent depolarization and calcium influx

Mouse pancreatic islets were used to investigate the mechanisms and functional significance of the B cell membrane depolarization by acetylcholine (ACh). At low glucose (3mM), ACh (20 microM) increased 22Na+ influx, and slightly depolarized the B cell membrane but did not induce electrical activity or stimulate 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and barely affected basal insulin release. At a stimulatory concentration of glucose (10 mM), ACh stimulated 22Na+ influx, depolarized the B cell membrane, increased glucose-induced electrical activity, and stimulated 45Ca2+ influx. ACh also accelerated 86Rb+ and 45Ca2+ efflux and strongly potentiated insulin release. Omission of extracellular Ca2+ did not impair ACh stimulation of 22Na+ influx or 86Rb+ efflux, slightly modified the acceleration of 45Ca2+ efflux, and almost completely suppressed the increase in insulin release. Na+ omission (with N-methyl-D-glucamine as substitute) prevented the B cell membrane depolarization and the stimulation of 45Ca2+ influx, largely inhibited the acceleration of 86Rb+ efflux and insulin release, and suppressed the late phase of 45Ca2+ efflux otherwise produced by ACh. On the other hand, ACh stimulation of 3H efflux from islets prelabeled with myo-[2-3H]inositol was not affected by Na+ omission. All effects of ACh were blocked by atropine and unaffected by nicotinic antagonists. It is concluded that activation of muscarinic receptors depolarized the B cell membrane by increasing its permeability to Na+. When the membrane is already depolarized by glucose, this further depolarization augments Ca2+ influx and, hence, potentiates insulin release.     

Sodium channels induced by depolarization of the Xenopus laevis oocyte.

An electrically gated Na+ channel can be made to appear in the membrane of the Xenopus laevis oocyte by simple depolarization. This membrane normally responds passively to imposed transmembrane currents with resting potentials around -60 mV, but when it is held depolarized to more than about +30 mV it becomes possible to obtain long-lasting regenerative depolarizations up to +80 mV; these depolarizations can last as long as 20 min. This potential is due to an "induction" of a Na+-dependent channel that is electrically gated open and closed. Its threshold for opening is about -20 mV and it is selective for Na+ over Cs+ and choline+ but is blocked by relatively small quantities of Li+. When a long voltage clamp step to a positive potential under ENa (+70 to +90 mV) is applied, an inward current is observed for many minutes, implying that this channel does not have an inactivation mechanism. The inward Na+ current is blocked by 0.50 mM tetrodotoxin. When the membrane is held at or near resting potential, the excitability will disappear with time, but it can be made to reappear by again depolarizing the membrane.     

Presynaptic membrane potential affects transmitter release in an identified neuron in Aplysia by modulating the Ca2+ and K+ currents

We have examined the relationships between the modulation of transmitter release and of specific ionic currents by membrane potential in the cholinergic interneuron L10 of the abdominal ganglion of Aplysia californica. The presynaptic cell body was voltage-clamped under various pharmacological conditions and transmitter release from the terminals was assayed simultaneously by recording the synaptic potentials in the postsynaptic cell. When cell L10 was voltage-clamped from a holding potential of -60 mV in the presence of tetrodotoxin, graded transmitter release was evoked by depolarizing command pulses in the membrane voltage range (-35 mV to + 10 mV) in which the Ca(2+) current was also increasing. Depolarizing the holding potential of L10 results in increased transmitter output. Two ionic mechanisms contribute to this form of plasticity. First, depolarization inactivates some K(+) channels so that depolarizing command pulses recruit a smaller K(+) current. In unclamped cells the decreased K(+) conductance causes spike-broadening and increased influx of Ca(2+) during each spike. Second, small depolarizations around resting potential (-55 mV to -35 mV) activate a steady-state Ca(2+) current that also contributes to the modulation of transmitter release, because, even with most presynaptic K(+) currents blocked pharmacologically, depolarizing the holding potential still increases transmitter release. In contrast to the steady-state Ca(2+) current, the transient inward Ca(2+) current evoked by depolarizing clamp steps is relatively unchanged from various holding potentials.     

Enkephalin inhibits release of substance P from sensory neurons in culture and decreases action potential duration.

Sensory neurons grown in dispersed cell culture in the absence of non-neuronal cell types contain immunoreactive substance P that is chemically similar to synthetic substance P. When depolarized in high-K+ media (30-120 mM), the neurons release this peptide by a Ca2+-dependent mechanism. An enkephalin analogue, [D-Ala2]enkephalin amide, at 10 micron inhibits the K+-evoked release of substance P. At the same or lower concentrations, [D-Ala2]enkephalin amide and enkephalin decrease the duration of the Ca2+ action potential evoked and recorded in dorsal root ganglion cell bodies without affecting the resting membrane potential or resting membrane conductance. This modulation of voltage-sensitive channels may account for the inhibition of substance P release.     

Na+, K(+)-adenosine triphosphatase regulation in hypertrophied vascular smooth muscle cells.

Vascular smooth muscle cell hypertrophy is a normal compensatory state that may play a pathogenic role in hypertension. Angiotensin II stimulates a hypertrophic response in cultured vascular smooth muscle cells. As part of the growth response, angiotensin II rapidly activates the Na(+)-H+ exchanger, increasing Na+ influx. Because Na+, K(+)-ATPase is the major cellular mechanism for regulating intracellular Na+, we studied the effects of angiotensin II-induced hypertrophy on Na+, K(+)-ATPase expression and activity. Angiotensin II caused rapid increases in both steady-state Na+, K(+)-ATPase activity (ouabain-sensitive 86Rb uptake) and intracellular [Na+]. Angiotensin II also caused a sustained increase in Na+, K(+)-ATPase at 24 hours with a 73% increase in maximal 86Rb uptake per milligram protein and a fourfold increase in Na+, K(+)-ATPase alpha-1 messenger RNA levels. Thus, angiotensin II hypertrophy was associated with rapid increases in Na+, K(+)-ATPase activity due to increased Na+ entry and sustained increases due to a specific increase in Na+, K(+)-ATPase expression. These data demonstrate dynamic regulation of Na+, K(+)-ATPase at the functional and molecular level and suggest that similar compensatory mechanisms should be present in vivo. Alterations in such compensatory pathways may be fundamental to the pathogenesis of hypertension.     

Voltage-dependent Ca2+ influx into right-side-out plasma membrane vesicles isolated from wheat roots: characterization of a putative Ca2+ channel.

We report on the identification of a voltage-dependent Ca2+ transport system that mediates Ca2+ influx across the plasma membrane (PM) of wheat (Triticum aestivum) root cells. The experimental approach involved the imposition of transmembrane electrical potentials (via K+ diffusion potentials) in populations of purified, right-side-out PM vesicles isolated from wheat roots. Using 45Ca2+ to quantify Ca2+ influx into the PM vesicles, the voltage-dependent characteristics of Ca2+ transport were found to be similar to those exhibited by L-type voltage-gated Ca2+ channels in animal cells. The putative PM Ca2+ channel opened upon depolarization of the membrane potential, and Ca2+ flux increased to a maximum upon further depolarization and then decreased back to zero upon further successive depolarizations. This channel was found to be selective for Ca2+ over Mg2+, Sr2+, K+, and Na+; was blocked by very low concentrations of La3+; was unaffected by high concentrations of the K+ channel blocker tetraethylammonium; and exhibited Michaelis-Menten-type transport kinetics. Based on these transport properties, we argue that this transport system is a PM Ca2+ channel. We suggest that the use of radiotracer flux analysis of voltage-clamped PM vesicles derived from plant roots is a straightforward approach for the characterization of certain voltage-gated ion channels functioning in cellular membranes of higher plant cells.     

Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle.

Addition of membrane vesicles prepared from transverse tubule (T-tubule) membranes of rabbit skeletal muscle to the aqueous phase of a planar lipid bilayer induces a stepwise increase in conductance. This conductance is both voltage and Ca2+ dependent. At 1 mM Ca2+, the steady-state conductance is maximal at voltages higher than +20 mV and decreases for more negative voltages. (Voltages refer to the side to which the vesicles are added, cis) Decreasing the Ca2+ concentration reversibly shifts the conductance-voltage curve toward the right along the voltage axis. Furthermore, Ca2+ can activate the conductance only if added to the cis compartment. Neither Mg2+, Ba2+, nor Cd2+ can activate the conductance induced by T-tubule vesicles. Addition of 5 mM tetraethylammonium ion to the trans, but not the cis, side abolishes the T-tubule-induced conductance. The Ca2+-dependent conductance appears as a consequence of ionic channel formation. Single-channel activity appears in bursts followed by periods of time in which the channel remains "silent". The conductance of the open channel averages 226 pS in 0.1 M KC1 and is voltage and Ca2+ independent. However, the fraction of time that the channel remains in the open state is voltage and Ca2+ dependent in a manner that parallels the voltage and Ca2+ dependence of the multichannel membrane. The channel is 6.6 times more permeable to K+ than to Na+ and is impermeable to C1-.