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The membrane potential of guinea-pig spermatozoa was investigated by using the fluorescent dye, diS-C3-(5). The potential did not vary when K replaced Na in the suspending medium, and in a physiological salt solution the resting potential was found to be about +13 mV.
The transmembrane potential of female rat liver cells in situ was-52.4 mV. This was decreased to -42.9 mV by ethacrynic acid (4 mg/100 g), but not by ouabain (1 mg). DL-Ethionine (25-100 mg) caused a decrease in the membrane potential and tissue ATP content. A high dose ethionine (100 mg) increased tissue Na content and decreased K content. By applying adenine (25 mg) to the animals treated with ethionine (100 mg), the membrane potential, ATP content and K content were increased and the Na content was decreased. The repolarized membrane potential in the animals treated with adenine following the ethionine was again depolarized by the administration of ouabain, but not by the administration of ethacrynic acid. These results suggest that two kinds of active ion transport mechanisms, ethacrynic acid-sensitive and ouabain-sensitive mechanisms, may be involved in maintenance of the membrane potential of rat liver cells.
When slices of ovine luteal tissue were perfused with medium containing luteinizing hormone (LH), the output of progesterone was increased significantly (P less than 0.01) in eleven of twelve experiments. However, addition of LH to the medium did not influence the luteal cell membrane potential. The addition of 47 mM potassium to the medium resulted in increased progesterone output (P less than 0.01) and depolarization of the luteal cell membrane within 2 min. Progesterone output decreased to approximate pretreatment levels within 2 min of the return to normal potassium levels in the perfusion medium. High levels of potassium further increased the output of progesterone from tissue stimulated with LH. Perfusion of the slices with sodium-free medium also resulted in increased (P less than 0.01) progesterone output within 2 min, which returned to pretreatment levels within 2 min after normal sodium levels were restored to the medium. Perfusion of the slices with sodium-free medium did not influence the membrane potential. Perfusion of the tissue with LH, 47 mM potassium, or sodium-free medium had no effect on progesterone output if the medium was calcium-free and/or contained 2 mM EGTA. These data suggested that the calcium ion plays an important role in mediating the steroidogenic response of ovine luteal tissue to LH. A second series of experiments was designed to ascertain if luteal cells were coupled electrically. Sixty-six pairs of luteal cells separated by 150-300 mum were penetrated with electrodes and the membrane potential of both cells was studied. One cell of each pair was hyperpolarized by passage of 0.4 nA current into the cell, but in no case was there an effect on the membrane potential of the other penetrated cell. Likewise, when five cells were injected iontophoretically with Procion Yellow there was no evidence of diffusion of the dye to adjacent cells. There was no evidence obtained in this study which suggested that ovine luteal cells were coupled electrically.
The effect of 3 microM norepinephrine (NE) on intracellular sodium activity (aiNa) and resting membrane potential was studied by continuous intracellular recordings with a conventional and an ion-selective microelectrode. The electrodes were impaled simultaneously in small (diameter, 0.3 mm) superfused trabeculae of the beating guinea pig ventricle at 37 degrees C. In the absence of NE, changes of the beating rate produced an increase of aiNa by 1.5 +/- 0.17 mM (from 0 to 1 Hz) and 1.9 +/- 0.47 mM (from 0 to 2 Hz). In the presence of NE, there was a very small significant increase of aiNa during constant stimulation (1 Hz) and at at [K+]o of 4.7 and 11.5 mM. After 7 minutes of exposure, aiNa increased by 0.5 +/- 0.19 mM (mean +/- SEM, n = 4) at [K+]o of 4.7 mM and by 0.5 +/- 0.22 (n = 6) at [K+]o of 11.5 mM. Resting membrane potential became more positive by 1 mV at both levels of [K+]o. The effect of NE became also clearly manifest from the configurational changes of action potentials (profound increase in plateau height and duration). Stimulation of the Na(+)-K+ pump by NE became manifest from the changes of resting membrane potential and aiNa after abrupt cessation of stimulation. The magnitude and the rate of the decrease in aiNa and the initial rate of hyperpolarization were significantly greater in the presence of NE than in its absence.(ABSTRACT TRUNCATED AT 250 WORDS)
Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis.
Many models of cortical dynamics have focused on the high-firing regime, in which neurons are driven near their maximal rate. Here we consider the responses of neurons in auditory cortex under typical low-firing rate conditions, when stimuli have not been optimized to drive neurons maximally. We used whole-cell patch-clamp recording in vivo to measure subthreshold membrane potential fluctuations in rat primary auditory cortex in both the anesthetized and awake preparations. By analyzing the subthreshold membrane potential dynamics on single trials, we made inferences about the underlying population activity. We found that, during both spontaneous and evoked responses, membrane potential was highly non-Gaussian, with dynamics consisting of occasional large excursions (sometimes tens of millivolts), much larger than the small fluctuations predicted by most random walk models that predict a Gaussian distribution of membrane potential. Thus, presynaptic inputs under these conditions are organized into quiescent periods punctuated by brief highly synchronous volleys, or "bumps." These bumps were typically so brief that they could not be well characterized as "up states" or "down states." We estimate that hundreds, perhaps thousands, of presynaptic neurons participate in the largest volleys. These dynamics suggest a computational scheme in which spike timing is controlled by concerted firing among input neurons rather than by small fluctuations in a sea of background activity.
ITP hydrolysis catalysed by the ATPase of submitochondrial particles from both bovine heart and rat liver is shown to be linked to the generation of a membrane potential, and therefore also to proton translocation. The magnitude of the membrane potential is similar to that observed during ATP hydrolysis at equivalent concentrations of phosphate and nucleoside tri- and diphosphates. An explanation is suggested for why in other reports ITP was found to be a poor substrate for supporting energy-linked reactions that are driven by the membrane potential.
Despite the marked influence on neuronal physiology of the low-voltage activated T-type Ca(2+) currents, little is known about the intracellular pathways and neurotransmitters involved in their regulations. Here, we report that in thalamocortical neurons a phosphorylation mechanism induces an increase both in the current amplitude (1.5 +/- 0.27-fold in the ventrobasal nucleus) and its inactivation kinetics. Dialysis of the neuron with an ATP-free solution suppresses the T-current potentiation, whereas it becomes irreversible in the presence of ATPgammaS. Phosphorylation occurs when the channels are inactivated and is slowly removed when they recover from inactivation and remain in closed states (time constants of the induction and removal of the potentiation: 579 +/- 143 msec and 4.9 +/- 1.1 sec, respectively, at 25 degrees C). The resulting apparent voltage sensitivity of this regulation follows the voltage dependence of the current steady-state inactivation. Thus, the current is paradoxically inhibited when the preceding hyperpolarization is lengthened, and maximal currents are generated after transient hyperpolarizations with a duration (0.7-1.5 sec) that is defined by the balance between the kinetics of the dephosphorylation and deinactivation. In addition, the phosphorylation will facilitate the generation of T current at resting membrane potential. This potentiation, which is specific to sensory thalamocortical neurons, would markedly influence the electroresponsiveness of these neurons and represent the first evidence of a regulation of native Cav3.1 channels.
K+ current which take part in the controlling of membrane potential in myocytes isolated from rat resistance mesenteric arteries have been investigated using conventional patch clamp method. The mean resting potential of myocytes was--37 mV. Charybdotoxin (200 nM)--selective blocker of large conductance Ca(2+)-activated K+ (KCa) channels--inhibited transmembrane outward K+ current by 60%. 1 mM of tetraethylammonium inhibited outward K+ current same as 200 nM of charybdotoxin, also it inhibited spontaneous spike-like hyperpolarizations and did not affect the membrane potential. Transmembrane current had a 4 aminopyridine (4-AP) sensitive component of delayed rectifier current (KV). Addition of 5 mM of 4-AP evoked membrane depolarization with mean significance of 12.0 +/- 1.5 mV in 5 from 7 single myocytes which had resting potential in the range of -50 ... -35 mV. The obtained results suggest that large conductance KCa channels do not determine the resting potential, but may serve as a negative feedback mechanism at the considerable membrane depolarization. In contrast, 4-AP sensitive KV current take part in the controlling of the resting membrane potential of single myocytes from rat resistance mesenteric arteries.
The effects of low concentrations of extracellular ATP on cytosolic Ca(2+), membrane potential, and transcription of IL-6 were studied in monocyte-derived human macrophages. During inflammation or infection many cells secrete ATP. We show here that application of 10 microM ATP or 10 microM UTP induces oscillations in cytosolic Ca(2+) with a frequency of approximately 12 min(-1) and oscillations in membrane potential. RT-PCR analysis showed expression of P2Y(1), P2Y(2), P2Y(11), P2X(1), P2X(4), and P2X(7) receptors, large-conductance (KCNMA1 and KCNMB1-4), and intermediate-conductance (KCNN4) Ca(2+)-activated K(+) channels. The Ca(2+)oscillations were unchanged after removal of extracellular Ca(2+), indicating that they were mainly due to movements of Ca(2+) between intracellular compartments. Comparison of the effects of different nucleotides suggests that the Ca(2+) oscillations were elicited by activation of P2Y(2) receptors coupled to phospholipase C. Patch-clamp experiments showed that ATP induced a transient depolarization, probably mediated by activation of P2X(4) receptors, followed by membrane potential oscillations due to opening of Ca(2+)-activated K(+) channels. We also found that 10 microM ATP gamma S increased transcription of IL-6 approximately 40-fold within 2 h. This effect was abolished by blockade of P2Y receptors with 100 microM suramin. Our results suggest that ATP released from inflamed, damaged, or metabolically impaired cells represents a "danger signal" that plays a major role in activating the innate immune system.
1. The resting membrane potential of Ascaris muscle fibres, which is normally relatively insensitive to ion changes in the medium, has been measured under a wide variety of conditions.2. The results have been interpreted in terms of a form of the constant field equation containing additional terms for the contribution of ions and charged groups other than potassium, sodium and chloride.3. Normally the contribution of the additional terms is large and tends to outweigh the contributions of potassium, sodium and chloride.4. The contribution of the additional terms is considerably reduced in the absence of sodium and in the presence of gamma-amino butyric acid and acetylcholine.5. It is suggested that the additional terms may represent the contribution of an electrogenic active transport mechanism to the factors determining the membrane potential.
We have used the cyanine dye fluorescence technique to measure the membrane potential of human erythrocytes as a function of temperature. With erythrocytes starved of glucose, there is an abrupt decrease in membrane potential centered at 38 degrees which is reversible up to 41 degrees, and irreversible at higher temperatures. With erythrocytes supplemented with glucose, the thermally induced transition is centered at 41 degrees and is reversible up to the highest temperature measured, 45 degrees. These results extend previous spectroscopic studies with erythrocyte membranes which demonstrated a thermally induced transition in protein tertiary or quaternary structure that is irreversible above 42 degrees.
1. The effects of Na, Ca and membrane potential on desensitization of postjunctional glutamate receptors on locust muscle were investigated. 2. The kinetics of desensitization were measured ionophoretically. 3. Replacement of Na by equimolar concentrations of the permeant cations Li, NH4 and guanidine and the impermeant cation choline accelerated desensitization onset, increased the steady-state leve of desensitization and reduced the rate of recovery from desensitization. 4. Desensitization onset rates and steady-state levels of desensitization were not significantly altered either by changing the extracellular Ca concentration or by changing the membrane potential of voltage clamped muscle fibres.
BACKGROUND: Fluorescence detection of cell membrane potentials is an important technique in neurobiology, cell physiology and pharmaceutical screening, but traditional one-fluorophore indicators either respond too slowly or have limited sensitivity. Recently, we introduced two-component sensors based on the transfer of fluorescence resonance energy from fluorescent lectins bound on one side of the plasma membrane to highly fluorescent oxonol acceptors that electrophorese from one face of the membrane to the other in response to membrane potential. RESULTS: We have found that fluorescent lectins can often be advantageously replaced in such sensors by fluorescently labeled phospholipids. A coumarinlabeled phosphatidylethanolamine donor and a bis(1,3-dihexyl-2-thiobarbiturate)trimethineoxonol acceptor gave the largest sensitivity of fluorescence ratio (>50% per 100 mV) ever reported. The response was also speeded several-fold by lengthening the mobile dye to the pentamethineoxonol analog, the <0.4 ms time constant of which was shorter than action potential durations. Photodynamic damage due to singlet oxygen was reduced by administering a natural carotenoid, astaxanthin. CONCLUSIONS: Voltage-sensitive fluorescence resonance energy transfer already gives record-setting performance on single cells and will continue to be rationally improvable.
The transmembrane chemical gradient for Na+ that most living cells maintain represents an energy source which is not sufficient to account for observed cellular sugar or amino acid gradients. This short review summarizes the literature pointing to the above conclusion, and described experimental evidence from a variety of model systems which indicates that the membrane potential can provide an additional driving force. Ordinarily, solute leak pathways compromise the full gradient forming capability of the Na+-dependent concentration systems and obscure the extent to which membrane potentials are important. When these "leaks" are experimentally controlled, solute gradients are established that identify the membrane potential as a quantitatively very important energy input. Furthermore, if the electrochemical gradient for Na+ is the sole source of energy, an extremely high efficiency of energy transduction must occur during gradient-coupled transport. An experimental approach is described here which can provide clues to the mechanistic role for membrane potentials in intestinal Na+-dependent sugar transport.
Effects of membrane potential and cytosolic free Ca2+ concentrations ([Ca2+]i) on acetycholine (ACh)-induced inositol phosphate production were investigated in insulin secreting betaTC3 cells. ACh (10 microM) caused a rapid inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and increase in [Ca2+]i reaching a maximum within 5 s. The rise in Ins(1,4,5)P3 production was reduced by 79 +/- 5% when [Ca2+]i was kept low in cells loaded with the Ca2+ chelator BAPTA. The ACh-evoked Ins(1,4,5)P3 production also depended on the membrane potential as it was reduced by 31 +/- 6% in cells hyperpolarized by diazoxide, an opener of ATP-sensitive K+ channels. The Ca2+ ionophore ionomycin caused a rapid increase in [Ca2+]i and in the cellular Ins(1,4,5)P3 content. We conclude that stimulation-induced changes in membrane potential and [Ca2+]i play an important role in controlling Ins(1,4,5)P3 production in insulin-secreting betaTC3 cells.
Water and electrolyte contents, cell pH, membrane potential and 125I- uptake were determined in cultured follicular cells of turtle thyroid. The Na+, K+ and Cl- concentrations in the cultured thyroid cells were 59.2, 119.0 and 50.9 mmol/l cell water respectively. Treatment with TSH (10 mu./ml for 24 h) increased the K+ and Cl- and decreased the Na+ concentrations in cells. The water and protein contents of these cells were 81.6 and 8.7 g/100 g cells respectively. The cell pH was 6.91. With glass micro-electrodes, the resting membrane potential of thyroid cells cultured in Medium 199 averaged 33.9 +/- 0.63 mV which is slightly higher than 29.8 +/- 1.6 mV as calculated from the data on the uptakes of [14C]methyltriphenylphosphonium and 3H2O by the cells. The potential varied linearly with the log of external K+ concentration (between 15 and 120 mmol/l) with a slope of about 24 mV per tenfold change in K+ concentration. Both TSH and cyclic AMP depolarized the cell membrane. Calculations based on the values for the electrolyte concentrations in cells and in culture medium indicated that Na+, K+ and Cl- were not distributed according to their electrochemical gradients across the cell membrane. Na+ was actively transported out of the cells and K+ and Cl- into the cells. Follicular cells of turtle thyroid cultured in the medium without addition of TSH formed a monolayer. Their iodide-concentrating ability was low and they did not respond to TSH with an increase in iodide uptake. In contrast, cells cultured in medium containing TSH tended to aggregate and organize to form follicles. They had higher ability to concentrate iodide and respond to TSH.
Mechanotransduction is required for a wide variety of biological functions. The aim of this study was to determine the effect of activation of a mechanosensitive Ca(2+) channel, present in human jejunal circular smooth muscle cells, on whole cell currents and on membrane potential. Currents were recorded using patch-clamp techniques, and perfusion of the bath (10 ml/min, 30 s) was used to mechanoactivate the L-type Ca(2+) channel. Perfusion resulted in activation of L-type Ca(2+) channels and an increase in outward current from 664 +/- 57 to 773 +/- 72 pA at +60 mV. Membrane potential hyperpolarized from -42 +/- 4 to -50 +/- 5 mV. In the presence of nifedipine (10 microM), there was no increase in outward current or change in membrane potential with perfusion. In the presence of charybdotoxin or iberiotoxin, perfusion of the bath did not increase outward current or change membrane potential. A model is proposed in which mechanoactivation of an L-type Ca(2+) channel current in human jejunal circular smooth muscle cells results in increased Ca(2+) entry and cell contraction. Ca(2+) entry activates large-conductance Ca(2+)-activated K(+) channels, resulting in membrane hyperpolarization and relaxation.