Chloride cotransport in the membrane of earthworm body wall muscles

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

Membrane potential in human myeloid leukemia cell line ML-1: responsiveness of granulocytic and monocytic differentiated cells.

The membrane potential responsiveness of human myeloid leukemia cells (ML-1 line) was studied with the voltage sensitive fluorescent dye diS-C3-(5). The experimental procedure used in this study enabled us to assess the magnitude of the membrane potential change in cells treated with ouabain, 12-0-tetradecanoylphorbol-13-acetate (TPA) and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), relative to the membrane potential in the untreated control. Inhibition of the Na, K-ATPase by ouabain was followed by a (20 +/- 4) mV depolarization. In undifferentiated homogeneous cell population TPA caused a (19.4 +/- 4.4) mV depolarization while FMLP had virtually no effect. Cells in which granulocytic or monocytic differentiation was induced by retinoic acid or 1,25-dihydroxyvitamin D3 exhibited under the effect of TPA a (57.8 +/- 7.1) mV and (34.8 +/- 10.9) mV depolarization, respectively. A very small transient depolarization was also observed up on treating of the cells with FMLP. The changes in the membrane potential responsiveness in the induced cells are obviously connected with the cell differentiation.     

Membrane potential of rat calvaria bone cells: dependence on temperature

The membrane potentials of bone cells derived from calvaria of new born rats was shown to be strongly dependent on temperature. When we lowered the temperature from 36 degrees C to 26 degrees C, cells with spontaneous resting membrane potentials (MP) of -80 to -50 mV depolarized (mean amplitude 8 mV; n = 33), and the membrane resistance increased by approximately 80% (n = 20). The temperature response depended on the actual MP, the reversal potential being in the range of -80 to -90 mV. With the application of ouabain (0.1-1 mmol/liter; n = 12), cells depolarized. Simultaneously, the reversal potential of the temperature response was shifted towards more positive values and approached the actual MP level of the cells. Consequently, the depolarization amplitudes induced by lowering temperature were reduced at spontaneous MP levels. The rise of the membrane resistance during cooling was unaffected. When the extracellular chloride concentration was reduced from 133 to 9 mmol/liter, temperature-dependent depolarizations persisted at spontaneous MP values (n = 5). The findings indicate that the marked effects of temperature changes on the MP of bone-derived cells are mainly determined by changes of the potassium conductance.     

Changes in resting membrane potential induced by active sensitization in the guinea-pig vas deferens

Smooth muscles hyperresponsiveness is a common feature in anaphylaxis and allergic diseases. The aim of the present work was to investigate whether the enhanced reactivity of sensitized guinea-pig vas deferens was associated with changes in the resting membrane potential (Er) of the smooth muscle cells. Active sensitization was performed by subcutaneous injection of egg albumen. Er was measured in vitro in isolated vas deferens with conventional KCl-filled microelectrodes. Quantification of [3H]ouabain binding sites, measurements of 86Rb efflux, and measurements of Na and K contents were also performed. In normal physiological solution, at 35 degrees C, Er was a mean of -54.1+/-0.3 mV (mean +/- SEM) in control vas deferens. Sensitization resulted in depolarizing Er by about 7 mV. In control and sensitized preparations, the 3H-ouabain binding site concentration, the efflux of 86Rb, and the K content were similar. In guinea-pig vas deferens, active sensitization induced a partial depolarization of the resting membrane potential of the smooth muscle cells, which did not result from a downregulation of Na+ -K+ pump sites.     

Modulation of membrane potential by an acetylcholine-activated potassium current in trout atrial myocytes

Application of the current-clamp technique in rainbow trout atrial myocytes has yielded resting membrane potentials that are incompatible with normal atrial function. To investigate this paradox, we recorded the whole membrane current (I(m)) and compared membrane potentials recorded in isolated cardiac myocytes and multicellular preparations. Atrial tissue and ventricular myocytes had stable resting potentials of -87 +/- 2 mV and -83.9 +/- 0.4 mV, respectively. In contrast, 50 out of 59 atrial myocytes had unstable depolarized membrane potentials that were sensitive to the holding current. We hypothesized that this is at least partly due to a small slope conductance of I(m) around the resting membrane potential in atrial myocytes. In accordance with this hypothesis, the slope conductance of I(m) was about sevenfold smaller in atrial than in ventricular myocytes. Interestingly, ACh increased I(m) at -120 mV from 4.3 pA/pF to 27 pA/pF with an EC(50) of 45 nM in atrial myocytes. Moreover, 3 nM ACh increased the slope conductance of I(m) fourfold, shifted its reversal potential from -78 +/- 3 to -84 +/- 3 mV, and stabilized the resting membrane potential at -92 +/- 4 mV. ACh also shortened the action potential in both atrial myocytes and tissue, and this effect was antagonized by atropine. When applied alone, atropine prolonged the action potential in atrial tissue but had no effect on membrane potential, action potential, or I(m) in isolated atrial myocytes. This suggests that ACh-mediated activation of an inwardly rectifying K(+) current can modulate the membrane potential in the trout atrial myocytes and stabilize the resting membrane potential.     

Effects of hyperthermia on the membrane potential and Na+ transport of V79 fibroblasts

The effects of hyperthermia (41-43 degrees C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41 degrees C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37 degrees C. The hyperpolarization was inhibited 50% by 1 mM ouabain or 0.25 mM amiloride, an inhibitor of Na+:H+ exchange. Shifting temperature to 41 degrees C increased ouabain-sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43 degrees C for 60 min, the membrane potential of log phase cells depolarized (20-35 mV). Parallel studies demonstrated enhanced Na+ uptake at 41 degrees C only in the presence of ouabain. At 43 degrees C, Na+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43 degrees C, 0.25 mM amiloride inhibited heat-stimulated Na+ uptake. Na+ efflux was enhanced at 41 degrees C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41 degrees C is activation of Na+:H+ exchange with the resultant increase in cytosolic [Na+] activating the electrogenic Na+ pump. At temperatures greater than or equal to 43 degrees C, the Na+ pump is inhibited.     

Comparative study of the effects of antibodies to S-100 protein and ouabain on neurons of Helix pomatia

Ouabain, used in 5.10(-4) M concentration, elicits 12 +/- 5 mV (15 experiments) depolarization of membrane of snail Helix neurons. In 80% of experiments depolarization is not accompanied by changes in membrane conductance, in 20% of experiments the decrease of the membrane conductance is observed. Application of the antibodies to S-100 protein (their concentration in the micropipette being 0.05 mg/ml) induces similar effects. The effects of ouabain and antibodies to S-100 protein are not additive and the main difference in their action lies in the ability of the cell to recover the resting potential of the membrane in the solution containing ouabain.     

The fluorescent cationic dye rhodamine 6G as a probe for membrane potential in bovine aortic endothelial cells.

The membrane potential of cultured bovine aortic endothelial cells was assessed by a fluorescent probe as an alternative to direct methods. We used the fluorescent cationic dye rhodamine 6G, a lipophilic probe with high permeability in cell membranes. A linear relationship was obtained between fluorescence intensity (F.I.) and membrane potential (Em) as a function of the extracellular Na(+) concentration in the presence of the ionophore gramicidin. From the equation derived from the linear relationship F.I. = -0.004 Em + 0. 03 (P < 0.001), the fluorescence measurements could be converted to membrane potential. The resting plasma membrane potential obtained was -65 +/- 7 mV. Nigericin (27 microM), ouabain (1 mM), and bradykinin (20 nM) induced a decrease in F.I. (depolarization), while ATP (25-100 microM) induced an increase in F.I. (hyperpolarization). Mitochondrial membrane potential inhibitors myxothiazol (3 microM) and oligomycin (4 microM) did not influence F. I. measured in the cultured bovine aortic endothelial cells. The results indicate that rhodamine 6G can be used as a sensitive and specific dye in studies of substances that affect the membrane potential of endothelial cells.     

A packed Cytodex microbead array for three-dimensional cell-based biosensing

A packed Cytodex 3 microbead array was fabricated as a simple three-dimensional (3-D) cell-based biosensing format. Resting membrane potentials and voltage-gated calcium channel (VGCC) function of SH-SY5Y human neuroblastoma cells cultured on the microbead array versus collagen-coated flat (2-D) substrates were evaluated by confocal microscopy with a potentiometric dye, tetramethylrhodamine methyl ester, and a calcium fluorescent indicator, Calcium Green-1. SH-SY5Y cells, differentiated with 1mM dibutyryl cAMP and 2.5 microM 5-bromodeoxyuridine, showed significant resting membrane potential establishment on the topographical scaffolds in a period of 13 days into differentiation, in contrast to the previously reported insignificant resting membrane potential establishment of the same cells within collagen hydrogels. On days 2, 8 and 13 into differentiation, cells on collagen-coated flat substrates developed resting membrane potentials of -6.0+/-19.5 mV (n=198), -30.5+/-19.9 mV (n=191) and -21.7+/-18.9 mV (n=308), in contrast to values for cells on 3-D scaffolds of -25.8+/-14.7 mV (n=112), -37.6+/-13.1 mV (n=120) and -28.7+/-12.2 mV (n=158), respectively. The development of VGCC function, as measured by percentage of cells responsive to 50 mM high K(+) depolarization, was significantly slower for cells on 3-D scaffolds (20.0% on day 13 into differentiation) than for cells on 2-D substrates (30.7% on day 8 into differentiation). The exaggerated 2-D cell calcium dynamics, in comparison with those of 3-D cells, is consistent with previous 2-D/3-D comparative studies. This study established the rationale and feasibility of the microbead array format for 3-D cell-based biosensing.     

Chronic carbon monoxide enhanced IbTx-sensitive currents in rat resistance pulmonary artery smooth muscle cells

Exogenous carbon monoxide (CO) can induce pulmonary vasodilation by acting directly on pulmonary artery (PA) smooth muscle cells. We investigated the contribution of K+ channels to the regulation of resistance PA resting membrane potential on control (PAC) rats and rats exposed to CO for 3 wk at 530 parts/million, labeled as PACO rats. Whole cell patch-clamp experiments revealed that the resting membrane potential of PACO cells was more negative than that of PAC cells. This was associated with a decrease of membrane resistance in PACO cells. Additional analysis showed that outward current density in PACO cells was higher (50% at +60 mV) than in PAC cells. This was linked to an increase of iberiotoxin (IbTx)-sensitive current. Chronic CO hyperpolarized membrane of pressurized PA from -46.9 +/- 1.2 to -56.4 +/- 2.6 mV. Additionally, IbTx significantly depolarized membrane of smooth muscle cells from PACO arteries but not from PAC arteries. The present study provides initial evidence of an increase of Ca2+-activated K+ current in smooth muscle cells from PA of rats exposed to chronic CO.     

The diffusion and electrogenic components of the membrane potential of hypoxic myocardium

The diffusion and electrogenic components of the resting potential of hypoxic ventricular muscle were separated by inhibition of the sodium pump with 10(-4) M ouabain. The response to varying external K concentrations (Ko) was studied. Arterially perfused rabbit hearts were submitted to 60 min hypoxia in Krebs solution containing 5 mM K throughout or to different external K concentrations during the last 20 min of hypoxia. For K concentrations between 1.5 and 10 mM, hypoxia did not change the resting potential except for a slight hyperpolarization in 7.5 mM K. The diffusion component of the resting potential did not differ from the resting potential at Ko less than 5 mM. An electrogenic potential of -3 to -6 mV was detectable at Ko values between 5 and 10 mM. The internal K concentration, Ki, was estimated from extrapolations to zero potential of the relation resting potential vs. Ko in normoxic and hypoxic hearts. These experiments revealed a decline of Ki of 16 mM with hypoxia. The variation of the diffusion potential with external K was fitted by a PNa:PK ratio five times lower than in normoxia. It has been concluded that an increase in K permeability and the persistence of electrogenic Na extrusion during hypoxia of rather short duration prevent membrane depolarization despite the myocardial K loss.     

Effects of chronic nicotine exposure on electrogenic activity of the Na+, K(+)-ATPase and contractility in the rat diaphragm muscle

Rats were chronically treated with nicotine via subcutaneous injections up to a dose 6 mg/kg/day during 2-3 weeks. After this period, resting membrane potential and action potentials of muscle fibres as well as isometric twitch and tetanic (20 s(-1) and 50(-1)) contractions of isolated rat diaphragm were studied. To estimate electrogenic contribution of the alpha2 isoform of the Na+, K(+)-ATPase ouabain in concentration 1 microM was used. Chronic nicotine exposure induced depolarization of resting membrane potential of 2.2 +/- 0.6 mV (p < 0.01). In rats chronically exposed to nicotine, electrogenic contribution of the Na+, K(+)-ATPase alpha2 isoform was twofold lesser than in control animals (3.7 +/- 0.6 mV and 6.4 +/- 0.6 mV, respectively, p < 0.01). Chronic nicotine exposure did not affect force of twitch and tetanic contractions in response to direct or indirect stimulation. A decrease in the twitch contraction time as well as in the rise time of tetanic contractions was observed. Fatigue dynamics was unchanged. The results suggest that chronic nicotine exposure leads to decrease of the Na+, K(+)-ATPase alpha2 isoform electrogenic activity, and as a consequence to damage of the rat diaphragm muscle electogenesis.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

The effect of altered transmembrane ion gradients on membrane potential and aggregation of human platelets in blood plasma

The resting membrane potential of platelets suspended in blood plasma buffer mixture measured using tritiated triphenylmethylphosphonium was 48 ± 13 mV. Increases in extracellular potassium concentration caused depolarization of the platelet membrane. Ouabain (1.0 μM) produced a depolarization of 13 ± 5 mV. Depolarization with elevated extracellular potassium or ouabain potentiated the aggregating effects of adenosine diphosphate even in the presence of methysergide suggesting that the effects are not mediated through ion linked serotonin transport. These observations are consistent with the suggestion that transmembrane ion gradients and membrane potential can regulate platelet sensitivity to the aggregating agent adenosine diphosphate.     

Participation of electrogenic Na+-K+-ATPase in the membrane potential of earthworm body wall muscles

The effect of Na+-K+-ATPase inhibitor ouabain on the resting membrane potential (Vm) was studied by glass microelectrodes in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris and compared with frog sartorius muscle. In earthworm muscle, Vm was -49 mV (inside negative) in a reference external solution with 4 mmol/l K+. The electrogenic participation of Na+-K+-ATPase was absent in solutions with very low concentrations of 0.01 mmol/l K+, higher in 4 and 8 mmol/l K+ (4-5 mV) and maximal (13 mV) in solutions containing 12 mmol/l K+ where Vm was -46 mV in the absence and -33 mV in the presence of 1 x 10(4) M ouabain. The electrogenic participation of Na+-K+-ATPase was much smaller in m. sartorius of the frog Rana temporaria bathed in 8 and 12 mmol/l K+. The results indicate that the Na+-K+-ATPase is an important electrogenic factor in earthworm longitudinal muscle fibres and that its contribution to Vm depends directly on the concentration of K+ in the bathing solution.     

Hyperpolarizing potentials induced by Ca-mediated K-conductance increase in hamster submandibular ganglion cells.

The mechanisms of three types of hyperpolarizing electrogenesis in hamster submandibular ganglion cells were analyzed with intracellular microelectrodes. These included (1) spike-induced hyperpolarizing afterpotential (S-HAP), (2) spontaneous transient hyperpolarizing potential (HP), and (3) the hyperpolarizing (H) phase of postsynaptic potential (PSP). Most of these hyperpolarizing potentials were due to conductance increases and reversed polarity at membrane potential (Em) between -70 and -85 mV, which was close to the K-equilibrium potential. The average resting potential of ganglion cells was -53 mV. Action potential overshoot increased slightly in high [Ca2+]0 and decreased in low [Ca2+]0. In most neurons action potentials were completely suppressed by 10(-7)-M tetrodotoxin (TTX). The S-HAP has an initial component due to delayed rectification and a late component. The late component is enhanced by increasing [Ca2+]0, or by applying Ca-ionophore (A23187), TEA, caffeine, or dibutyryl cyclic (DBc-) AMP; it is suppressed by decreasing [Ca2+]0, or by applying Mn2+. Perfusion with Cl--free saline reduced membrane potential slightly but did not modify the S-HAP. Depolarizing pulses also induced hyperpolarizing afterpotential (D-HAP), similar to the S-HAP. Spontaneous transient HPs occurred in some neurons at irregular intervals. HPs were insensitive to TTX but were suppressed by Mn2+. Caffeine induced low frequency rhythmic HPs in many neurons, often alternating with periods of repetitive spiking. The PSP was a monophasic depolarizing (D-) potential in some neurons, but in others the D-phase was followed by a small H-phase. Perfusion with A23187, caffeine or DBc-AMP increased the H-phase of the PSP. Perfusion with K+-free saline or treatment with 10(-5)M ouabain did not abolish the H-phase of PSPs. These membrane potential-dependent phenomena appear to be induced mainly by Ca-mediated K-conductance increases. This mechanism contributes to the regulation of low-frequency repetitive firing in submandibular ganglion cells.     

Coupling of Li+ distribution to the plasma membrane potential of rat cortical synaptosomes

The effect of the plasma membrane potential delta psi p on the transport rate and steady state distribution of Li+ was assessed in rat cortical synaptosomes. Up to 15 mM Li+ failed to saturate Li+ influx into polarized synaptosomes in a Na+-based medium with 3 mM external K+. Veratridine increased and tetrodotoxin, ouabain, or high external K+ decreased the rate of Li+ influx. At steady state, Li+ was concentrated about 3-fold in resting synaptosomes at 0.3 to 1 mM Li+ externally. Subsequent depolarization of the plasma membrane by veratridine or high external K+ induced an immediate release of Li+. When graded depolarizations were imposed onto the plasma membrane by varying concentrations of ouabain, veratridine, or external K+, steady state distribution of Li+ was linearly related with K+ distribution or electrochemical activity coefficients. It was concluded that uptake rate and steady state distribution of Li+ depend significantly on delta psi p. However, Li+ gradients were lower than predicted from delta psi p, suggesting that (secondary) active transport systems counteracted passive equilibration by uphill extrusion of Li+. The electrochemical potential difference delta mu Li+ maintained at a delta psi p of -72 mV was calculated to 4.2 kJ/mol of Li+. At physiological external K+, Li+ was not actively transported by the sodium pump. The ouabain sensitivity resulted from the coupling of Li+ uptake to the pump-dependent K+ diffusion potential. In low K+ and K+-free media, however, active transport of Li+ by the sodium pump contributed to total uptake. In the absence of K+, Li+ substituted for K+ in generating a delta psi p of -64 mV maximally, as calculated from TPMP+ distribution at 40 mM external Li+. Since Li+ gradients were far too low to account for a diffusion potential, it was assumed that Li+ gave rise to an electrogenic pump potential.     

Role of membrane potential in vasomotion of isolated pressurized rat arteries

Vasomotion, the phenomenon of vessel diameter oscillation, regulates blood flow and resistance. The main parameters implicated in vasomotion are particularly the membrane potential and the cytosolic free calcium in smooth muscle cells. In this study, these parameters were measured in rat perfused-pressurized mesenteric artery segments. The application of norepinephrine (NE) caused rhythmic diameter contractions and membrane potential oscillations (amplitude; 5.3 +/- 0.3 mV, frequency; 0.09 +/- 0.01 Hz). Verapamil (1 microM) abolished this vasomotion. During vasomotion, 10(-5) M ouabain (Na(+)-K(+) ATPase inhibitor) decreased the amplitude of the electrical oscillations but not their frequency (amplitude; 3.7 +/- 0.3 mV, frequency; 0.08 +/- 0.002 Hz). Although a high concentration of ouabain (10(-3) M) (which exhibits non-specific effects) abolished both electrical membrane potential oscillations and vasomotion, we conclude that the Na+-K+ ATPase could not be implicated in the generation of the membrane potential oscillations. We conclude that in rat perfused-pressurized mesenteric artery, the slow wave membrane type of potential oscillation by rhythmically gating voltage-dependent calcium channels, is responsible for the oscillation of intracellular calcium and thus vasomotion.     

Lymphocyte membrane potential assessed with fluorescent probes

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3'-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an inophore which adds a highly K+-selective permeability membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl-, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential-the "valinomycin null point" - and under the conditions used the resting potential was approx.-60 mV. B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells. In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around -45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel. Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.