Ion Substitution Experiments Research Articles

Swelling-induced changes in electrophysiological properties of cultured astrocytes and oligodendrocytes. II. Whole-cell currents

Using whole cell patch-clamp recordings we have found that swelling cultured cerebrocortial astrocytes or mouse spinal cord oligodendrocytes by perfusing them with hypotonic medium induced inward currents at the normal resting potential of −60 mV. The currents in the oligodendrocytes were always less than for astrocytes. We examined the reversal potentials of these responses by rapidly jumping the holding potential to different values and measuring the currents. We found that the hypotonic medium-induced conductance increase was always preceded by a conductance decrease in the case of oligodendrocytes, but only sometimes preceded by a conductance decrease in cultured astrocytes. The reversal potential of the conductance increase for astrocytes was around −40 mV, while the conductance decrease had a more negative reversal potential of −60 mV or less. For oligodendrocytes the reversal potential for the conductance increase was around −50 mV while the conductance decrease had a reversal potential of −90 mV or less. This suggests that K + conductance decreased in the initial phase, while the conductance increase was due to additional channel openings. Ion substitution experiments in the case of the astrocytes showed that the reversal potential was shifted to a more positive value when medium K + was increased, but was unaffected when Na + was substituted by N-methyl- d-glucamine or Cl − by d-glucuronate, when corrected for liquid junction potential changes. Thus, the channels opened in these cells are likely to include non-specific cation channels. It is of interest that the two cells show a difference in their responses, and in the case of astrocytes these are likely to be involved in the regulatory volume decrease processes documented in these cells.

Chloride channels in human platelets: Evidence for activation by internal calcium

Whole-cell patch-clamp recordings were made from freshly isolated human platelets. The pipette contained a high concentration of divalent cations, which permitted easy disruption of cell-attached membrane patches by suction. Single-channel currents were measured when the pipette contained isotonic BaCl2 or MgCl2 saline; over 30 sec -5 min an increasing number of channels appeared until conductance steps through individual channels could no longer be distinguished. The current-voltage relationship was curvilinear; chord conductance at -35 mV was 25 pS increasing to 45 to 52 pS at +45 mV. Ion substitution experiments showed the current to be primarily carried by Cl-. Erev was shifted 30 mV/10-fold change in external Cl- (replaced by gluconate), was similar with BaCl2 or MgCl2 in the pipette and was not significantly shifted by replacing external Na+ with K+. Addition of 1 mM BAPTA to the MgCl2 pipette saline prevented activation of Cl- currents; with isotonic CaCl2 internal saline, current appeared immediately upon patch rupture, suggesting that the Cl- channels are dependent on internal Ca2+. 5-nitro-2-(3-phenylpropylamino)-benzoate, reported to block a Cl- conductance in studies of rat epithelial cells, caused a potent flickery block and may be a useful tool with which to investigate the physiological role of Cl- currents in human platelets.

Model of ion transport regulation in chloride-secreting airway epithelial cells. Integrated description of electrical, chemical, and fluorescence measurements

An electrokinetic model was developed to calculate the time course of electrical parameters, ion fluxes, and intracellular ion activities for experiments performed in airway epithelial cells. Model variables included cell [Na], [K], [Cl], volume, and membrane potentials. The model contained apical membrane Cl, Na, and K conductances, basolateral membrane K conductance, Na/K/2 Cl and Na/Cl symport, and 3 Na/2 K ATPase, and a paracellular conductance. Transporter permeabilities and ion saturabilities were determined from reported ion flux data and membrane potentials in intact canine trachea. Without additional assumptions, the model predicted accurately the measured short-circuit current (Isc), cellular conductances, voltage-divider ratios, open-circuit potentials, and the time course of cell ion composition in ion substitution experiments. The model was used to examine quantitatively: (a) the effect of transport inhibitors on Isc and membrane potentials, (b) the dual role of apical Cl and basolateral K conductance in cell secretion, (c) whether the basolateral symporter requires K, and (d) the regulation of apical Cl conductance by cAMP and Ca-dependent signaling pathways. Model predictions gave improved understanding of the interrelations among transporting systems and in many cases gave surprising predictions that were not obvious without a detailed model. The model developed here has direct application to secretory or absorptive epithelial cells in the kidney thick ascending limb, cornea, sweat duct, and intestine in normal and pathophysiological states such as cystic fibrosis and cholera.

Extracellular tumor necrosis factor induces a decreased K+ conductance in an identified neuron of Aplysia kurodai

Recombinant human tumor necrosis factor (rhTNF) was pressure-applied onto the the soma of identified neuron R12 in the Aplysia abdominal ganglion. rhTNF induced a slow inward current (ITNF, 80-100 s in duration, 5-10 nA in amplitude) associated with a conductance decrease. ITNF begins 1-2 s after applying rhTNF and peaks in 5-6 s. ITNF was decreased by hyperpolarization and had a reversal potential of approximately -87 mV (close to the K+ equilibrium potential). Ion substitution and pharmacological experiments suggest that ITNF is due to a decreased K+ conductance and that TNF, a product of macrophages, may form an important link in communications between nervous and immune systems.

Potassium, chloride and non‐selective cation conductances opened by noradrenaline in rabbit ear artery cells.

1. The action of noradrenaline on cells isolated from the rabbit ear artery was studied with the whole-cell configuration of the patch clamp technique. In normal potassium-containing solutions at a holding potential of -50 mV noradrenaline elicited either outward, inward or mixed outward and inward currents. These responses were blocked by the alpha-adrenoceptor antagonist, phentolamine (10(-6) M). 2. The outward current occurred as a consequence of an increase in membrane conductance and the reversal potential was close to the potassium equilibrium potential (EK). It was possible to record outward currents without external calcium but not when the concentration of EGTA in the pipette was increased to 10 mM or when potassium was absent from the pipette solution. It is concluded that the outward current evoked by alpha-adrenoceptor stimulation is produced by a calcium mediated increase in potassium conductance. 3. The ionic basis of the inward current was investigated in potassium-free external and pipette solutions. When sodium chloride was the major constituent of the external and pipette solutions the reversal potential (Er) of the noradrenaline-induced current was 0.63 mV, close to ENa and ECl. 4. When most of the external sodium chloride was replaced by sucrose Er was intermediate between ENa and ECl but had shifted significantly towards ENa. Further ion substitution experiments suggest that noradrenaline increased the membrane conductance to both anions and cations. 5. When the current was carried predominantly by anions, depolarizing steps (from -50 mV) produced outward current relaxations with a time constant of about 40 ms. Bath-applied caffeine also produced a membrane current which rectified in the outward direction. 6. When the response to noradrenaline was mediated mainly by cations, the relationship between the membrane current and clamp potential was non-linear and the amplitude of the currents at potentials positive to -50 mV became disproportionately smaller. In addition on repolarization to -50 mV the instantaneous current was followed by an inward relaxation. 7. In high external barium solution noradrenaline evoked a membrane current with a reversal potential much more positive than ENa or ECl. This current was recorded in the presence of 10(-5) M-nifedipine and diltiazem and in 10(-4) M-cadmium which suggests that the voltage-dependent calcium channel is not implicated in the generation of the non-selective cation current to noradrenaline.(ABSTRACT TRUNCATED AT 400 WORDS)

High potassium selective permeability and extracellular ion regulation in the glial perineurium (blood-brain barrier) of the crayfish

Selective ion permeability, ion transport properties, and electrical resistance of the perineurial barrier, as they relate to interstitial ion regulation, where studied and characterized electrophysiologically in ion substitution experiments. In high external [K +] a transient spike-like voltage was generated across the perineurial barrier which fell over 1–2 min to a slowly decaying voltage. The glial perineurium had at least a 10 times greater permeability to K + than Cl −, and was effectively impermeant to Na +. The potential, in high external [K +], was determined by the K + and Cl − gradients and their relative permeabilities across the sheath. For other cations the selectivity sequence of the perineurial barrier, as determined from electrophysiological measurements, was K + ⩾ Rb + « NH + 4 > Cs + > Li + > Na + corresponding most closely to the Eisenman sequence IV. The perineurium had a resistance of 260 ± 23 Ω cm 2 in crayfish physiological solution. In high [K +] 0 the resistance fell by over half during the transient spike potential and then recovered towards resting levels as the voltage decayed. In the intact nerve cord interstitial [K +] rose to only 10–20 mM during a 2-min exposure to 100 mM K + 0. K influx and efflux were related to the change in barrier permeability and an increased selectivity to K + which, in these studies, was determined primarily by its electrochemical gradient across the perineurial barrier. The results suggest that the crayfish perineurium is a leaky epithelium capable of a high degree of ion regulation. Trans-perineurial barrier potential and conductance in high external [K +] are primarily functions of passive processes of the perineurial glial cell membranes and of the paracellular conductance channels driven by the electrochemical gradient for the K +. Accordingly, the mass transport of [K +] showed the same quantitative relationship in both directions.

An open trial of ondansetron (GR38032F) in the treatment of acute schizophrenia

Whole cell and patch clamp techniques were used to investigate the properties of 5-HT3 receptors of a murine neuroblastoma cell line (N1E-115) and adult rabbit nodose ganglion neurones. In addition, some preliminary results from guinea-pig nodose ganglion neurones are presented. In such cells, voltage-clamped at −60mV, 5-HT (10μM) induced an inward current associated with a conductance increase. The results of ion substitution experiments suggest that the 5-HT activated ion channel is permeable to both Na+ and K+ ions with a permeability ratio (PNa/PK) of 0.94 and 0.92 for rabbit nodose ganglion cells and N1E-115 cells respectively. On outside out membrane patches excised from rabbit nodose ganglion neurones, 5-HT (1 μM) activated clearly discernible single channel currents with a conductance of 16.6 ± 0.7 pS (n = 4). In contrast, fluctuation analysis of 5-HT induced whole cell currents suggests that the single channel conductance of N1E-115 cells is only 0.3 pS, a value some 50 fold lower.The 5-HT-induced whole cell currents recorded from all three preparations were antagonised by the selective 5-HT3 receptor antagonist ondansetron (GR38032F) and by the less selective agents metoclopromide, cocaine and (+)-tubocurarine. However, these preparations demonstrate a differential sensitivity to some antagonists. In particular, (+)-tubocurarine was a potent antagonist in N1E-115 cells (IC50 = 0.85 nM) but was approximately 200 fold (IC50 = 156 nM) and 1200 fold (IC50 = 10 μM) less potent in rabbit and guinea-pig nodose ganglion neurones respectively. Additionally, a novel effect of ketamine (10 μM) to potentiate the 5-HT-induced current of rabbit nodose ganglion neurones is described.

Electrophysiological characteristics of the Necturus proximal duodenal mucosa: effects of ion substitutions.

Voltage-sensitive glass micro-electrodes were used to determine the electrical characteristics of Necturus proximal duodenal epithelium. Some comparative experiments with amiloride were performed with gastric antrum. The apical and the basolateral cell membrane potential differences in duodenum averaged -32 mV and -34 mV (cell negative) respectively. The transepithelial potential difference was -2 mV (lumen negative). The EMF across the apical cell membrane was -29 mV and that across the basolateral cell membrane -39 mV. The transepithelial resistance (Rt) of 63 omega cm2 and the paracellular pathway resistance (Rs) of 80 omega cm2 are of magnitudes similar to that previously reported for more distal amphibian small intestine. The apical and basolateral cell membrane resistances, however, were lower than those reported for distal small intestine. Ion permeabilities for Na+, K+ and Cl- across the apical cell membrane were calculated from ion substitution experiments. The permeability sequence across the apical cell membrane was PK:PCl:PNa 3.02:1.31:1.00. Luminal amiloride (10(-4)M) was without significant effect, further indicating a low duodenal membrane conductance for Na+. The low conductances for K+, Na+ and Cl- suggest that the major ion transport modes across the apical duodenal cell membrane are electroneutral in nature. In contrast, amiloride caused a marked increase in the transmembrane potentials in the antrum.

Intracellularly injected inositol hexakisphosphate induces a biphasic current in identified neurons of Aplysia

Inositol hexakisphosphate (IP 6) was pressure-injected into the somata of the identified neurons (R9-R12) in the abdominal ganglion of Aplysia. Intracellular injection of IP 6 into a neuron voltage-clamped at −45 mV reproducibly induced a biphasic membrane current consisting of an inward current ( I i(IP 6)) followed by a slow outward current ( I o(IP 6)). Conductance is increased at the peaks of these currents. The extrapolated reversal potential of I i(IP 6) was −34 mV, whereas I o(IP 6) was increased by depolarization and decreased by hyperpolarization up to −75 mV, where it disappeared. Ion substitution and pharmacological experiments suggest that microinjection of IP 6 into the Aplysia neurons activates two or more separate types of ion channels.

Diabetes mellitus and the sodium electrochemical gradient across the brush border membrane of rat intestinal enterocytes.

The effects of chronic diabetes mellitus of 4-5 weeks duration on the potential difference across the brush border membrane of rat small intestine (Vm) and on Na+-dependent uptake of D-glucose by jejunal brush border vesicles have been studied. Diabetes increased Vm in the jejunum from a mean value of -47.2 mV in control tissue to -57.4 mV in diabetic tissue (P less than 0.001) but was without effect on ileal Vm. Measurements of Vm during ion-substitution experiments revealed that the conductance of Na+ of the jejunal brush border was reduced by diabetes, whilst K+ and Cl- permeabilities were unaltered. Uptake studies using brush border vesicles and an Na+-electrochemical gradient showed that diabetes caused a 56% increase in the initial rate of uptake of D-glucose but was without effect on the peak to equilibrium ratio. Taken together, data from these two studies suggest that the lower Na+ permeability of the brush border in diabetes enhances the electrical and chemical driving force for active Na+-dependent uptake of glucose by reducing glucose-independent movement of Na+ across this membrane. Finally, the possible humoral factors involved in this response to diabetes are discussed.

Intracellulary injected inositol 1,3,4,5,6-pentakisphosphate induces a slow inward current in identified neurons of Aplysia kurodai

Micropressure injection of inositol 1,3,4,5,6-pentakisphosphate (IP 5) into identified neurons (R9–R12) of Aplysia induced a slow inward current associated with a conductance increase. The IP 5-induced inward current was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of the current was −11 mV. The IP 5-induced inward current was sensitive to changes in [Na +] 0, [Ca 2+] 0 and [K +] 0 but not to changes in [Cl −] 0, and was resistant to tetrodotoxin. The current was not completely abolished by Ca 2+ channel blocking cations, Co 2+ and Mn 2+. Ion substitution and pharmacological experiments suggest that microinjection of IP 5 can activate a non-specific cation conductance in Aplysia neurons.

Single chloride channels in endosomal vesicle preparations from rat kidney cortex

Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl- channels with a single-channel conductance of 73 +/- 2 pS in symmetrical 140 mM KCl solution (n = 25). The current-voltage relationship was linear in the range of -60 to +80 mV, but channel kinetic properties depended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10-15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2-6 msec. Clamp voltages more negative than -50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl- = Br- = I- greater than SO4(2-) approximately F-. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, 1.0 mM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1 mM) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl(-)-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes.

A patch‐clamp study of effects of dihydroavermectin on Ascaris muscle

1. Effects of bath-application of the anti-parasitic agent 22,23-dihydroavermectin B1a (DHAVM 20 fM to 0.2 microM) on outside-out and inside-out patches from collagenase-treated muscle membranes of Ascaris suum were examined. 2. DHAVM was tested as a gamma-aminobutyric acid (GABA) agonist by application to outside-out patches. DHAVM failed to open characteristic GABA channels even when high concentrations (20 nM to 0.2 microM) were used. These high concentrations of DHAVM also failed to potentiate opening of channels activated by application of 3-4 microM GABA; instead they acted to depress GABA-activated channel currents by reducing mean conductances and P open. 3. In outside-out patches, low concentrations of DHAVM (1 pM to 100 pM) produced opening of 0-13 channels, the number depending on the patch. The progressive opening of channels gave rise to a 'Staircase' effect. The conductance of these channels was 9-15 pS and open times were long (greater than 100 mS). Ion substitution experiments showed these channels to be permeable to Cl. The channels were not blocked by 20 microM picrotoxin. There was a long delay (greater than 15 s) between DHAVM application and channel opening; this delay and lipophilic nature of DHAVM suggested a site of action in the lipid phase of the membrane. 4. The effect of bath-application of low concentrations of DHAVM on inside-out patches was investigated under low-Ca conditions (to avoid Ca-activated Cl channels). DHAVM (20 fM to 1 nM) did not produce opening of Cl channels but produced opening after a long delay (mean 2.5 minutes) of noisy cation-selective channels which had conductances of 5-30 pS. 5. The actions of DHAVM are discussed.

Chloride channels in cultured glomus cells of the rat carotid body

As part of our investigations on the chemosensory mechanisms in the rat carotid body, we are studying the physiology of the parenchymal glomus cells by the patch-clamp technique. Here we characterize a large-conductance chloride channel (approximately 296 pS) with random open and closed kinetics in inside-out patches of cultured glomus cells. The open-state probability (Po; mean = 0.61) was hardly affected by membrane potential (-50 to +50 mV) and cytoplasmic calcium (0-1 mM). Similarly, the channel did not appear to be regulated by cytoplasmic nucleotides (1 mM) or pH (6.5-8). Ion-substitution experiments yielded the following selectivity sequence: chloride greater than bicarbonate greater than sulfate greater than glutamate approximately sodium. Single-channel currents were reversibly reduced or blocked by anthracene-9-carboxylic acid (5-10 mM) but were unaffected by stilbene derivatives (0.5-1 mM), by furosemide (1 mM), and by 5-nitro-2-(3-phenyl-propylamino)benzoic acid (0.01 mM). Because these cultured glomus cells have been shown to express carbonic anhydrase, it is inferred that the chloride channels may play an important role in the physiology of glomus cells by aiding in the regulation of pHi and the resting potential via bicarbonate and chloride permeability.

Interaction of the B subunit of cholera toxin with endogenous ganglioside GM1 causes changes in membrane potential of rat thymocytes.

The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca2+-sensitive K+ channel. Reduction of extracellular Ca2+ to less than 1 microM by the addition of [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K+ channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na+ was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na+/K+ ATPase, Na+/H+ antiporter and Na+/K+/Cl- cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K+ by triggering the opening of Ca2+-sensitive K+ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.

Aspartate neurotoxicity on cultured cortical neurons.

L-aspartate neurotoxicity was quantitatively characterized in murine cortical cell cultures. Five-minute exposure to 30 microM-3 mM L-aspartate resulted in concentration-dependent (ED50 about 190 microM) neuronal destruction over the next 10 hr; glia were not injured. D-aspartate and L-aspartate were roughly equipotent neurotoxins. Ion substitution experiments suggested that L-aspartate neurotoxicity is comprised of both acute, sodium-dependent "excitotoxicity" and delayed, calcium-dependent degeneration, with the latter predominant under conditions of brief exposure. Aspartate neurotoxicity could be attenuated by D-2-amino-5-phosphonovalerate (D-APV), dextrorphan, ketamine, and kynurenate, but not by L-glutamate diethyl ester or gamma-D-glutamylaminomethyl sulfonate, consistent with principal involvement of N-methyl-D-aspartate receptors. D-APV and dextrorphan produced different effects on the aspartate concentration-toxicity relation; the former drug was consistent with a competitive and the latter with a noncompetitive mechanism of antagonism.

Characteristics of GABA‐activated chloride channels in mammalian dorsal root ganglion neurones.

1. The properties of gamma-aminobutyric acid (GABA)-activated chloride channels in dorsal root ganglion (DRG) neurones obtained from rats and cats were examined using the single-electrode voltage clamp in conjunction with suction-electrode techniques. 2. GABA-evoked currents showed voltage-sensitive kinetics. Time constants (tau D) were measured from voltage-jump relaxations and tau D became briefer with membrane hyperpolarization. tau D was 33 ms at -120 mV with 60 microM-GABA and changed e-fold for 188 mV. tau D decreased as GABA concentration was increased - the extrapolated tau D at 'zero' GABA concentration was approximately equal to 50 ms at -120 mV. 3. The steady-state current in GABA was curvilinear, rectifying at negative potentials. The instantaneous current was linear with symmetrical chloride concentrations (140 mM) on both sides of the cell membrane. 4. Muscimol was a more effective agonist than GABA, while piperidine-4-sulphonic acid and ethylenediamine monocarbamate were only weakly effective agonists. Taurine and glycine had no detectable agonist activity. 5. Ion substitution experiments revealed the permeability sequence I- greater than Br- greater than Cl- greater than F- greater than propionate (1.88 greater than 1.21 greater than 1.0 approximately equal to 0.1 approximately equal to 0.1). 6. The presence of iodide and bromide ions externally caused an increase in chloride efflux at membrane potentials more negative than -40 mV, and caused a prolongation of voltage-jump relaxations. Relaxations in fluoride and propionate solutions were faster than those seen in chloride.

Inositol trisphosphate and activators of protein kinase C modulate membrane currents in tail motor neurons of Aplysia.

1. We have investigated how activation of the inositol lipid second messenger pathway may contribute to modulation of membrane currents in tail motor neurons of Aplysia. Specifically, we examined the effects of injected inositol 1,4,5-trisphosphate (IP3) and analogues of diacylglycerol (DAG), both of which are products of the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2). 2. Injection of IP3 produced an outward current associated with an apparent increase in membrane conductance. Ion substitution experiments, the sensitivity of the response to low concentrations of TEA and its attenuation by intracellular injections of EGTA suggest that the current produced by injection of IP3 is a calcium-activated K+ current (IK,Ca). 3. The response to IP3 was mimicked by intracellular injection of Ca2+. Injection of Ca2+ produced an outward current that was associated with an apparent increase in input conductance of the membrane. The same manipulations that affected the response to IP3 (see above) also affected the response to injections of Ca2+. 4. Injections of activators of protein kinase C (PKC) produced a relatively slow inward current. The inward current has not been fully analyzed, but it does not appear to be due to the actions of any single conventional ion channel. 5. Activators of PKC attenuated responses to subsequent injections of IP3 indicating that one component of PIP2 hydrolysis can attenuate the other. 6. The results suggest that hydrolysis of inositol phospholipids is a mechanism for regulation of membrane properties in tail motor neurons of Aplysia.

A nonselective cation channel activated by membrane deformation in oocytes of the ascidianBoltenia villosa

Cell-attached patch clamp recordings from unfertilized oocytes of the ascidian Boltenia villosa reveal an ion channel which is activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette, but not in the absence of suction or during voltage steps. The estimated density of these stretch-activated channels is about 1.5/microns2, a figure equal to or greater than the density of known voltage-dependent channels in the oocyte. Ion substitution experiments done with combined whole-cell and attached patch recording, so absolute potentials are known, indicate that the channel passes Na+, Ca2+ and K+, but not Cl-. The channel has at least two open and two closed states, with the rate constant that leaves the longer-lived closed state being the primary site of stretch sensitivity. External Ca2+ concentration affects channel kinetics: at low calcium levels, long openings predominate, whereas at high calcium virtually all openings are to the short-lived open state. In multiple channel patches, the response to a step change in suction is highly phasic, with channel open probability decreasing over several hundred milliseconds to a nonzero steady-state level after an initial rapid increase. This channel may play a role in the physiological response of cells of the early embryo to the membrane strains associated with morphogenetic events.

Components of resting membrane electrogenesis in Lepidopteran skeletal muscle

The partial contributions of K +, Na +, Cl −, Mg 2+ and an electrogenic, metabolic pump to resting membrane electrogenesis in skeletal muscles of Chilo partellus (Lepidoptera) have been quantified. The contributions of the ions were evaluated by ionic substitution experiments, whilst the role of the pump was investigated by cooling and application of ouabain. In response to changes in extracellular K +, Na + and Cl − activities, membrane potentials changed in rapid, graded, sustained and reversible steps. For a 10-fold change in the extracellular activity, the maximal changes in the membrane potentials were 31 mV (K +), 2 mV (Na +) and 17 mV (Cl −). In constrast, changing the extracellular Mg 2+ concentration had no immediate effect on resting potentials. Cooling preparations by 14°C from room temperature resulted in rapid membrane depolarizations of some 12 mV. However, application of 1 mM ouabain in Ringers containing varying concentrations of K + had no effect on the membrane potential. It followed, therefore, that a metabolic mechanism was also involved in electrogenesis, but this was unlikely to be an ouabain-sensitive (Na +K +) ATPase. The contributions of K +, Na +, Cl − and the metabolic pump were sufficient to account quantitatively for the prevailing resting membrane potential (mean, −46 mV) with an error margin of some 7%.