Intramolecular and intermolecular enzymatic modulation of ion channels in excised membrane patches.

A calcium-activated potassium channel in posterior pituitary nerve terminals was modulated by phosphorylation and dephosphorylation. Nearly every patch of membrane containing this channel also contained both membrane bound protein phosphatase and membrane-bound protein kinase. By examining the statistical and kinetic nature of phosphorylation and dephosphorylation in excised patches, it was possible to evaluate two contrasting models for these enzymatic reactions. One of these models treated catalysis as an intermolecular process in which the enzyme and substrate are separate molecular species that diffuse and encounter one another during collisions. The second model treated catalysis as an intramolecular process in which the enzyme and substrate reside within a stable macromolecular complex. The study began with a Poisson analysis of the distribution of channel number in patches, and of the number of protein phosphatase-free and protein kinase-free patches. Subsequent kinetic analysis of dephosphorylation yielded an estimate of the mean number of protein phosphatase molecules per patch that was similar to the value obtained from Poisson analysis. Because these two estimates were independent predictions based on the intermolecular model, their agreement supported this model. Analysis of channel number in protein phosphatase-free patches and of the rarity of patches showing partial but incomplete rundown provided additional support for the intermolecular model over the intramolecular model. Furthermore, dephosphorylation exhibited monotonic kinetics with a rate well below the diffusion limit. Thus, several different lines of analysis support the intermolecular model for dephosphorylation, in which the protein phosphatase must encounter its substrate to effect catalysis. In contrast to the monotonic kinetics of dephosphorylation, the phosphorylation reaction exhibited sigmoidal kinetics, with a rate that depended on membrane potential. Voltage dependence is an unlikely property for a kinetic step involving encounters resulting from diffusion. Furthermore, the velocity of the phosphorylation reaction exceeded the diffusion limit, and this observation is inconsistent with the intermolecular model. Thus, both intermolecular and intramolecular enzymatic mechanisms operate in the modulation of the calcium-activated potassium channel of the posterior pituitary. These studies provide a functional characterization of the interactions between enzyme and substrate in intact patches of cell membrane.     

Formation of second messengers in response to activation of ion channels in excitable cells

1. Depolarization of excitable cells of the central nervous system results in the formation of the second messengers cyclic AMP, cyclic GMP, inositol phosphates, and diacylglycerides. 2. Depolarization-evoked accumulation of cyclic AMP in brain preparations can be accounted for mainly by the release of adenosine, which subsequently interacts with stimulatory adenosine receptor linked to adenylate cyclase. 3. Depolarization-evoked formation of cyclic GMP in brain preparations is linked to activation of voltage-dependent calcium channels, presumably leading to activation of guanylate cyclase by calcium ions. 4. In brain slices depolarization-evoked stimulation of phosphoinositide breakdown and subsequent formation of inositol phosphates and diacylglycerides are linked to activation of voltage-dependent calcium channels, which are sensitive to dihydropyridines, presumably leading to activation of phospholipase(s) C by calcium ions. 5. In the synaptoneurosome preparation depolarization-evoked stimulation of phosphoinositide breakdown does not involve activation of dihydropyridine-sensitive calcium channels and, instead, appears to be regulated primarily by the intracellular concentration of sodium ions. Thus, agents that induce increases in intracellular sodium--such as toxins that open or delay inactivation of voltage-dependent sodium channels; ouabain, an inhibitor of Na+/K+ ATPase that transports sodium outward and a sodium ionophore--all stimulate phosphoinositide breakdown. Mechanistically, increases in intracellular sodium either might directly affect phospholipase(s) C or might lead to influx of calcium ions through Na+/Ca2+ transporters. 6. Depolarization-evoked stimulation of cyclic AMP formation and phosphoinositide breakdown can exhibit potentiative interactions with responses to receptor agonists, thereby providing mechanisms for modulation of receptor responses by neuronal activity. 7. Since all these second messengers can induce phosphorylation of ion channels through the activation of specific kinases, it is proposed that depolarization-evoked formation of second messengers represents a putative feedback mechanism to regulate ion fluxes in excitable cells.     

Second messengers and ion channels in acetylcholine-induced chloride secretion in hen trachea

1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl⁻ equal to ~ 60–90 μA/cm².2. Radio-ligand-displacement for IP₃, cAMP and cGMP and ion channel selective drugs in voltage clamp setups were employed to characterize second messengers and Cl⁻, K⁺ and Ca²⁺ channels involved in the ACh response.3. ACh induced a significant rise in IP, in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 μM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold.4. Around 35% of ACh Cl⁻ secretion depends on Ca²⁺ mobilization from internal stores and about 65% on Ca²⁺ influx over basolateral membrane. The activated Ca²⁺ channel is insensitive to class I, II, III and IV Ca²⁺ antagonists.5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase.6. Two K⁺ channels are involved in ACh secretion, one sensitive to Ba²⁺ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA.7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl⁻ channel is NPPB-sensitive.     

Modal gating behavior of cardiac sodium channels in cell-free membrane patches.

Single voltage-activated Na+ channel currents were obtained from membrane patches of isolated ventricular cells of guinea pig hearts. The currents were compared when measured from cell-attached patches and from the same patch but at least 20 minutes after manual excision. The averaged currents showed a distinctly delayed decay in the excised patches due to the appearance of long lasting openings or bursts of openings. In contrast to control patches, the open time distribution in excised patches requires at least two exponentials. A short mean open time was voltage independent for cell-attached patches (0.38 ms +/- 0.07 ms between -60 and -20 mV, 6 cell-attached patches; and 0.41 +/- 0.1 ms, 7 excised patches). The long mean open time found in excised patches was clearly voltage dependent and increased from 0.48 +/- 0.14 ms (-80 mV) to 2.87 +/- 0.35 ms (-20 mV, regression coefficient +0.88, 7 patches). Sweeps with long openings appeared in clusters. The clustering of records with long openings, short openings, or without openings (nulls) was quantified by a runs analysis which showed a highly significant nonrandom ordering. The results show that in excised patches inactivation is temporally hibernating.     

Resonance phenomena in membranes containing ion channels with inactivation

A mathematical model of the ion transport across a membrane containing channel with inactivation has been analysed. Under certain conditions, such a membrane has been shown to behave as a selfoscillating circuit of a very high quality, its own frequency ranging for a variety of natural channels between 10(-1)-10(3) cycles. When exposed to an alternating electric field with a frequency approximating f0, the membrane displays resonance changes in its potential and channel conductivity. The average (over a period of forced oscillations) values of the potential and conductivity also show a resonance type of dependence on the frequency of the external field.     

Porin channels in Escherichia coli: studies with beta-lactams in intact cells

Wild-type Escherichia coli K-12 produces two porins, OmpF (protein 1a) and OmpC (protein 1b). In mutants deficient in both of these "normal" porins, secondary mutants that produce a "new" porin, protein PhoE (protein E), are selected for. We determined the properties of the channels produced by each of these porins by measuring the rates of diffusion of various cephalosporins through the outer membrane in strains producing only one porin species. We found that all porin channels retarded the diffusion of more hydrophobic cephalosporins and that with monoanionic cephalosporins a 10-fold increase in the octanol-water partition coefficient of the solute produced a 5- to 6-fold decrease in the rate of penetration. Electrical charges of the solutes had different effects on different channels. Thus, with the normal porins (i.e., OmpF and OmpC proteins) additional negative charge drastically reduced the penetration rate through the channels, whereas additional positive charge significantly accelerated the penetration. In contrast, diffusion through the PhoE channel was unaffected by the presence of an additional negative charge. We hypothesize that the relative exclusion of hydrophobic and negatively charged solutes by normal porin channels is of ecological advantage to E. coli, which must exclude hydrophobic and anionic bile salts in its natural habitat. The properties of the PhoE porin are also consistent with the recent finding (M. Argast and W. Boos, J. Bacteriol. 143:142-150, 1980; J. Tommassen and B. Lugtenberg, J. Bacteriol. 143:151-157, 1980) that its biosynthesis is derepressed by phosphate starvation; the channel may thus act as an emergency pore primarily for the uptake of phosphate and phosphorylated compounds.     

Spike latency and jitter of neuronal membrane patches with stochastic Hodgkin-Huxley channels

Ion channel stochasticity can influence the voltage dynamics of neuronal membrane, with stronger effects for smaller patches of membrane because of the correspondingly smaller number of channels. We examine this question with respect to first spike statistics in response to a periodic input of membrane patches including stochastic Hodgkin-Huxley channels, comparing these responses to spontaneous firing. Without noise, firing threshold of the model depends on frequency—a sinusoidal stimulus is subthreshold for low and high frequencies and suprathreshold for intermediate frequencies. When channel noise is added, a stimulus in the lower range of subthreshold frequencies can influence spike output, while high subthreshold frequencies remain subthreshold. Both input frequency and channel noise strength influence spike timing. Specifically, spike latency and jitter have distinct minima as a function of input frequency, showing a resonance like behavior. With either no input, or low frequency subthreshold input, or input in the low or high suprathreshold frequency range, channel noise reduces latency and jitter, with the strongest impact for the lowest input frequencies. In contrast, for an intermediate range of suprathreshold frequencies, where an optimal input gives a minimum latency, the noise effect reverses, and spike latency and jitter increase with channel noise. Thus, a resonant minimum of the spike response as a function of frequency becomes more pronounced with less noise. Spike latency and jitter also depend on the initial phase of the input, resulting in minimal latencies at an optimal phase, and depend on the membrane time constant, with a longer time constant broadening frequency tuning for minimal latency and jitter. Taken together, these results suggest how stochasticity of ion channels may influence spike timing and thus coding for neurons with functionally localized concentrations of channels, such as in “hot spots” of dendrites, spines or axons.     

Activation of mechanosensitive ion channels in plasma membrane of K562 cells

Patch clamp method in cell-attached configuration was used to search for mechanogated ion channels in plasma membrane of human myeloid leukemia K562 cells. A reversible activation of transmembrane currents in response to negative pressure applied to membrane patch was observed. Four types of mechanosensitive channels were identified in K562 cells: two main types were characterized with conductance values of 16 and 25 pS; while two others, showing higher conductance values (about 35 and 50 pS), were rarely met. In terms of gating, all channels described here could be assigned to the stretch-activated type. No inactivation of mechanosensitive channels at the sustained stimulation was observed. The activation of mechanosensitive channels in K562 cells was not dependent upon the presence of bivalent cations in the extracellular solution.     

Signal transduction and ion channels in guard cells.

Our understanding of the signalling mechanisms involved in the process of stomatal closure is reviewed. Work has concentrated on the mechanisms by which abscisic acid (ABA) induces changes in specific ion channels at both the plasmalemma and the tonoplast, leading to efflux of both K+ and anions at both membranes, requiring four essential changes. For each we need to identify the specific channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identified following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca2+, although stomata can close without any measurable global increase in cytoplasmic Ca2+. There is also evidence for the importance of several protein phosphatases and protein kinases in the regulation of channel activity. At the plasmalemma, loss of K+ requires depolarization of the membrane potential into the range at which the outward K+ channel is open. ABA-induced activation of a non-specific cation channel, permeable to Ca2+, may contribute to the necessary depolarization, together with ABA-induced activation of S-type anion channels in the plasmalemma, which are then responsible for the necessary anion efflux. The anion channels are activated by Ca2+ and by phosphorylation, but the precise mechanism of their activation by ABA is not yet clear. ABA also up-regulates the outward K+ current at any given membrane potential; this activation is Ca(2+)-independent and is attributed to the increase in cytoplasmic pH, perhaps through the marked pH-sensitivity of protein phosphatase type 2C. Our understanding of mechanisms at the tonoplast is much less complete. A total of two channels, both Ca(2+)-activated, have been identified which are capable of K+ efflux; these are the voltage-independent VK channel specific to K+, and the slow vacuolar (SV) channel which opens only at non-physiological tonoplast potentials (cytoplasm positive). The SV channel is permeable to K+ and Ca2+, and although it has been argued that it could be responsible for Ca(2+)-induced Ca2+ release, it now seems likely that it opens only under conditions where Ca2+ will flow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate efflux of Cl- from vacuole to cytoplasm, no vacuolar anion channel has yet been identified. There is clear evidence that ABA activates release of Ca2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca2+ are uncertain. The tonoplast and another membrane, probably ER, have IP3-sensitive Ca2+ release channels, and the tonoplast has also cADPR-activated Ca2+ channels. Their relative contributions to ABA-induced release of Ca2+ from internal stores remain to be established. There is some evidence for activation of phospholipase C by ABA, by an unknown mechanism; plant phospholipase C may be activated by Ca2+ rather than by the G-proteins used in many animal cell signalling systems. A further ABA-induced channel modulation is the inhibition of the inward K+ channel, which is not essential for closing but will prevent opening. It is suggested that this is mediated through the Ca(2+)-activated protein phosphatase, calcineurin. The question of Ca(2+)-independent stomatal closure remains controversial. At the plasmalemma the stimulation of K+ efflux is Ca(2+)-independent and, at least in Arabidopsis, activation of anion efflux by ABA may also be Ca(2+)-independent. But there are no indications of Ca(2+)-independent mechanisms for K+ efflux at the tonoplast, and the appropriate anion channel at the tonoplast is still to be found. There is also evidence that ABA interferes with a control system in the guard cell, resetting its set-point to lower contents, suggesting that stretch-activated channels also feature in the regulation of guard cell ion channels, perhaps through interactions with cytoskeletal proteins. (ABSTRACT TRUN     

Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin.

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.     

Functional ion channels in stem cells

Bioelectrical signals generated by ion channels play crucial roles in excitation genesis and impulse conduction in excitable cells as well as in cell proliferation,migration and apoptosis in proliferative cells.Recent studies have demonstrated that multiple ion channels are heterogeneously present in different stem cells;however,patterns and phenotypes of ion channels are species-and/or origin-dependent.This editorial review focuses on the recent findings related to the expression of functional ion channels and the roles of these channels in regulation of cell proliferation in stem cells.Additional effort is required in the future to clarify the ion channel expression in different types of stem cells;special attention should be paid to the relationship between ion channels and stem cell proliferation,migration and differentiation.     

Influences of Lovastatin on membrane ion flow and intracellular signaling in breast cancer cells

Lovastatin, an inhibitor of cellular cholesterol synthesis, has an apparent anti-cancer property, but the detailed mechanisms of its anti-cancer effects remain poorly understood. We investigated the molecular mechanism of Lovastatin anti-tumor function through the study of its effect on membrane ion flow, gap junctional intercellular communication (GJIC), and the pathways of related signals in MCF-7 mammary cancer cells. After treatment for 24–72 h with 4, 8 or 16 μmol/L Lovastatin, cellular proliferation was examined via the MTT assay, and changes in membrane potential and cellular [Ca²⁺]_i were monitored using confocal laser microscopy. In addition, the expression of plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA was analyzed via RT-PCR, the GJIC function was examined using the scrape-loading dye transfer (SLDT) technique, and MAPK phosphorylation levels were tested with the kinase activity assay. The results showed that Lovastatin treatment significantly inhibited the growth of MCF-7 breast cancer cells. It also increased the negative value of the membrane potential, leading to the hyperpolarization of cells. Moreover, Lovastatin treatment continuously enhanced [Ca²⁺]_i, although the levels of PMCA1 mRNA were unchanged. GJIC was also upregulated in MCF-7 cells, with transfer of LY Fluorescence reaching 4 to 5 rows of cells from the scraped line after treatment with 16 μmol/L Lovastatin for 72 h. Finally, downregulation of ERK1 and p38^MAPK phosphorylation were found in Lovastatin-treated MCF-7 cells. It could be deduced that Lovastatin can induce changes in cellular hyperpolarization and intracellular Ca²⁺ distributions, and increase GJIC function. These effects may result in changes in the downstream signal cascade, inhibiting the growth of MCF-7 cells.     

Mechanical transduction by membrane ion channels: a mini review

There are ion channels in the cell membrane that are sensitive to stress in the membrane cytoskeleton. Some channels turn on with stress, others turn off. In specialized receptors such as those involved in hearing, touch, etc. the role of the channels is clear. However, virtually all cells have these channels, and we don't yet know the physiological role of the channels although it is reasonable to suppose that they are involved in the control of cell size, either acutely as in volume regulation, or trophically as in the control of cell division.     

Thrombin stimulates L-type calcium channels of guinea pig cardiomyocytes in cell-attached patches but not after intracellular dialysis.

The action of the blood clotting enzyme thrombin on single channel and whole cell Ca(2+)-currents was studied in isolated mammalian cardiac myocytes. Thrombin, at a concentration of 10(-8) mol/l, increased the Ca(2+)-channel activity in cell-attached patches. The mean open probability of the channel was enhanced, while the number of sweeps without openings, which reflects the availability of the channel, was significantly reduced. Neither the single channel conductance nor the activation curve were affected by thrombin. Thrombin was added to the bath solution, and its effect is therefore indirect and probably mediated via a second messenger. However, thrombin did not affect whole-cell Ca(2+)-currents, whereas a beta-adrenergic stimulation in the same cell increased the Ca(2+)-current. It is concluded that thrombin affects an intracellular mechanism for Ca2+ channel current regulation, which is still unknown and which is rapidly lost during conventional whole-cell Ca2+ current measurements.     

On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells.

Diphtheria toxin (DT) in acidic media forms ion-conducting channels across the plasma membrane and inhibits protein synthesis of both highly and poorly DT-sensitive cell lines. This results in loss of cell potassium and in entry of both sodium and protons with a concomitant rapid lowering of membrane potential. The pH dependency of the permeability changes is similar to that of the inhibition of cell protein synthesis. DT-induced ion channels close when the pH of the external medium is returned to neutrality and cells recover their normal monovalent cation content. Similar permeability changes were induced by two DT mutants defective either in enzymatic activity or in cell binding, but not with a mutant defective in membrane translocation. The implication of these findings for the mechanism of DT membrane translocation is discussed.     

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.     

Nondestructive quantum dot-based intracellular serotonin imaging in intact cells

In recent years, quantum dots (Qdot), with their unique physical, chemical, and optical properties, have been used extensively as probes to visualize several cell membrane receptors and extracellular biomolecules. However, Qdot-based intracellular imaging has always been associated with vital lacunas. High affinity between quantum dots may induce serious aggregation in the cytoplasm; as a result, quantum dot aggregates are usually misinterpreted as quantum dot-probed intracellular molecules. Moreover, due to the more viscous nature of the cytoplasm versus the extracellular aqueous media, aggregation issues become more severe during intracellular studies. In this work, we suggest direct nondestructive serotonin imaging in an intact cell using the quantum dot-based immunoassay with a rapid tunable multicolor imaging system based on the acousto-optic tunable filter. Any false-positive intracellular serotonin molecules that appeared due to the aggregation of quantum dots could be completely discriminated from the real intracellular serotonin granules through multicolor cellular imaging. The developed method is quick and has wide applicability in targeting various intracellular proteins, coenzymes, and micronutrients.     

A molecular model for ion selectivity in membrane channels

In this article, the three-dimensional motion of an ion within a molecular channel is discussed for the first time; escape rates from binding sites are calculated using the transition state method. For a given ligand configuration and a particular pore radius the rates depend upon ion size and mass. It is found that the activation energies depend strongly on the ion size, i.e., they increase with decreasing ion radius. In contrast to the rates obtained from the mass dependence alone, the rates depending on both mass and size of the alkali ions yield the completely inverted sequence, namely the Eisenman sequence I.     

Elevated intracellular Ca2+ reveals a functional membrane nucleotide pool in intact human red blood cells

Elevated intracellular calcium generates rapid, profound, and irreversible changes in the nucleotide metabolism of human red blood cells (RBCs), triggered by the adenosine triphosphatase (ATPase) activity of the powerful plasma membrane calcium pump (PMCA). In the absence of glycolytic substrates, Ca(2+)-induced nucleotide changes are thought to be determined by the interaction between PMCA ATPase, adenylate kinase, and AMP-deaminase enzymes, but the extent to which this three-enzyme system can account for the Ca(2+)-induced effects has not been investigated in detail before. Such a study requires the formulation of a model incorporating the known kinetics of the three-enzyme system and a direct comparison between its predictions and precise measurements of the Ca(2+)-induced nucleotide changes, a precision not available from earlier studies. Using state-of-the-art high-performance liquid chromatography, we measured the changes in the RBC contents of ATP, ADP, AMP, and IMP during the first 35 min after ionophore-induced pump-saturating Ca(2+) loads in the absence of glycolytic substrates. Comparison between measured and model-predicted changes revealed that for good fits it was necessary to assume mean ATPase V(max) values much higher than those ever measured by PMCA-mediated Ca(2+) extrusion. These results suggest that the local nucleotide concentrations generated by ATPase activity at the inner membrane surface differed substantially from those measured in bulk cell extracts, supporting previous evidence for the existence of a submembrane microdomain with a distinct nucleotide metabolism.     

Stretch-activated ion channels in cultured mesangial cells

Membrane stretch, delivered by negative pressures in cell-attached patch pipettes, activated single-channel ionic currents in cultured mesangial cells. Channel opening probabilities were directly related to degree of suction, with threshold for activation being 5-10 mm Hg. The stretch-activated channels were permeable to Na+, K+, as well as Cl-, having conductances averaging 62 +/- 17 pS. These channels may represent a cellular mechano-reflex in mesangial cells.