Basolateral membrane potential of a tight epithelium: Ionic diffusion and electrogenic pumps

Summary The contribution of specific ions to the conductance and potential of the basolateral membrane of the rabbit urinary bladder has been studied with both conventional and ion-specific microelectrode techniques. In addition, the possibility of an electrogenic active transport process located at the basolateral membrane was studied using the polyene antibiotic nystatin. The effect of ion-specific microelectrode impalement damage on intracellular ion activities was examined and a criterion set for acceptance or rejection of intracellular activity measurements. Using this criterion, we found (K⁺)=72mm and (Cl^–)=15.8mm. Cl^– but not K⁺ was in electrochemical equilibrium across the basolateral membrane. The selective permeability of the basolateral membrane was measured using microelectrodes, and the data analyzed using the Goldman, Hodgkin-Katz equation. The sodium to potassium permeability ratio (P _Na/P _K) was 0.044, and the chloride to potassium permeability ratio (P _Cl/P _K) was 1.17. Since K⁺ was not in electrochemical equilibrium, intracellular (K⁺) is maintained by active metabolic processes, and the basolateral membrane potential is a diffusion potential with K⁺ and Cl^– the most permeable ions. After depolarizing the basolateral membrane with high serosal potassium bathing solutions and eliminating the apical membrane as a rate limiting step for ion movement using the polyene antibiotic nystatin, we found that the addition of equal aliquots of NaCl to both solutions caused the basolateral membrane potential to hyperpolarize by up to 20 mV (cell interior negative). This popential was reduced by 80% within 3 min of the addition of ouabain to the serosal solution. This hyperpolarization most probably represents a ouabain sensitive active transport process sensitive to intracellular Na⁺. An equivalent electrical circuit for Na⁺ transport across rabbit urinary bladder is derived, tested, and compared to previous results. This circuit is also used to predict the effects that microelectrode impalement damage will have on individual membrane potentials as well as time-dependent phenomena; e.g., effect of amiloride on apical and basolateral membrane potentials.     

细胞静息膜电位的产生和维持

根据中学生物学教学及教材中涉及到有关细胞膜电位的内容,特别请我刊编委、北京师范大学生命科学学院左明雪教授撰写了相关知识,将在2006年4、5、6期连续刊登。     

Proton-induced endocytosis is dependent on cell membrane fluidity,lipid-phase order and the membrane resting potential

Recently it has been shown that decreasing the extracellular pH of cells stimulates the formation of inward membrane invaginations and vesicles, accompanied by an enhanced uptake of macromolecules. This type of endocytosis was coined as proton-induced uptake (PIU). Though the initial induction of inward membrane curvature was rationalized in terms of proton-based increase of charge asymmetry across the membrane, the dependence of the phenomenon on plasma membrane characteristics is still unknown. The present study shows that depolarization of the membrane resting potential elevates PIU by 25%, while hyperpolarization attenuates it by 25%. Comparison of uptake in suspended and adherent cells implicates that the resting-potential affects PIU through remodeling the actin-cytoskeleton. The pH at the external interface of the cell membrane rather than the pH gradient across it determines the extent of PIU. PIU increases linearly upon temperature increase in the range of 4–36 °C, in correlation with the membrane fluidity. The plasma membrane fluidity and the lipid phase order are modulated by enriching the cell's membrane with cholesterol, tergitol, dimethylsulfoxide, 6-ketocholestanol and phloretin and by cholesterol depletion. These treatments are shown to alter the extent of PIU and are better correlated with membrane fluidity than with the lipid phase order. We suggest that the lipid phase order and fluidity influence PIU by regulating the lipid order gradient across the perimeter of the lipid-condensed microdomains (rafts) and alter the characteristic tension line that separates the higher ordered lipid-domains from the lesser ordered ones.     

Electro-permeabilization of cell membranes: effect of the resting membrane potential

Electric field induced permeabilization of cell membranes is an important technique for gene transfection and cell hybridization. Mechanistic studies of this process revealed that the uptake of fluorescent indicator by plant protoplasts occurs predominantly on the hemisphere facing the positive electrode, while in erythrocyte ghosts the probes exit through the hemisphere facing the negative electrode. To reconcile these observations symmetrical pore formation and a mechanism of molecular exchange by electroosmosis has been proposed. In light of these controversial observations, we conducted a systematic study of electroporation of NIH3T3 cells with varying electric field strength, waveform and frequency. Our data revealed that (i) symmetrical permeabilization of the cell membrane occurs only with bipolar a.c. fields. (ii) When a critical membrane breakdown potential, Vc, is applied using either an unipolar a.c. fields or a single d.c. square pulse, the cell membrane becomes permeabilized only at the hemisphere facing the positive electrode. (iii) When the pulse-induced membrane potential, Vm, is approximately equal to or larger than the intrinsic membrane potential (i.e. using d.c. or unipolar a.c. field), asymmetric permeabilization was observed with the hemisphere facing the positive electrode being most permeable. (iv) The rate of fluorescent indicator uptake is dependent on the concentration of the indicator. These results indicate that electro-permeabilization of cell membranes is affected by its resting potential and that electroosmosis is not the dominant mechanism for the cellular uptake of foreign molecules in electroporation.     

Generation of resting membrane potential

This brief review is intended to serve as a refresher on the ideas associated with teaching students the physiological basis of the resting membrane potential. The presentation is targeted toward first-year medical students, first-year graduate students, or senior undergraduates. The emphasis is on general concepts associated with generation of the electrical potential difference that exists across the plasma membrane of every animal cell. The intention is to provide students a general view of the quantitative relationship that exists between 1) transmembrane gradients for K(+) and Na(+) and 2) the relative channel-mediated permeability of the membrane to these ions.     

Glycine modulates membrane potential,cell volume,and phagocytosis in murine microglia

Phagocytes form engulfment pseudopodia at the contact area with their target particle by a process resembling cell volume (CV) regulatory mechanisms. We evaluated whether the osmoregulatory active neutral amino acid glycine, which contributes to CV regulation via activation of sodium-dependent neutral amino acid transporters (SNATs) improves phagocytosis in isotonic and hypertonic conditions in the murine microglial cell line BV-2 and primary microglial cells (pMG). In BV-2 cells and pMG, RT-PCR analysis revealed expression of SNATs (Slc38a1, Slc38a2), but not of GlyRs (Glra1–4). In BV-2 cells, glycine (5 mM) led to a rapid Na⁺-dependent depolarization of membrane potential (V _mem). Furthermore, glycine increased CV by about 9 %. Visualizing of phagocytosis of polystyrene microspheres by scanning electron microscopy revealed that glycine (1 mM) increased the number of BV-2 cells containing at least one microsphere by about 13 %. Glycine-dependent increase in phagocytosis was suppressed by the SNAT inhibitor α-(methylamino)isobutyric acid (MeAIB), by replacing extracellular Na⁺ with choline, and under hypertonic conditions, but not by the GlyR antagonist strychnine or the GlyR agonist taurine. Interestingly, hypertonicity-induced suppression of phagocytosis was rescued by glycine. These findings demonstrate that glycine increases phagocytosis in iso- and hypertonic conditions by activation of SNATs.     

细胞静息膜电位的测量

实验表明,大细胞表面积与体积的比率是相当大的,以至于离子梯度的下降是极为缓慢的。即使代谢类毒物阻断了依赖ATP的能量代谢,静息膜电位也能维持相当长的时间,这表明依赖ATP的泵不是维持膜电位的直接能源。例如,应用地高辛(cardiac glycol— side)来抑制枪鸟贼巨轴突膜上的Na—K泵,细胞膜电     

Steady-state diffusion and the cell resting potential

The steady-state diffusion of ions through separate, selective channels is described according to irreversible thermodynamics. Ion fluxes thus obtained are the same as those in the parallel conductance model. The equivalent electric circuit set up to describe the system has its electromotive forces expressed by the chemical potentials of the diffusing ions. The expression obtained for the potential differs from the Goldman-Hodgkin-Katz formula, and is reputed to be more accurate. In order for the passive diffusion flows to remain steady, active transport mechanisms must pump the ions up their electrochemical potentials. Such pumps have been incorporated into the equivalent circuit. They supply energy lost in the dissipation caused by preexisting passive forces without affecting the potential, which can thus hardly be called passive diffusion potential. Ion pumps can also create an electric potential in excess of that by passive forces, especially when secondary active transport is involved. The same equivalent circuit is, however, able to describe the whole range of seemingly different situations – from passive diffusion of an electrolyte to active extrusion of anions from the living cell. It has been applied to explain the measured plasma membrane potential of cells, especially those whose potential does not behave as the potassium electrode. Received: 6 July 1998 / Revised version: 7 November 1998 / Accepted: 10 November 1998     

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.     

Contribution of an electrogenic pump to the resting membrane polarization in a crustacean heart.

1. In the neurogenic heart of the isopod crustacean Porcellio dilatatus, external K+ removal depolarized the membrane (K0 effect) whereas subsequent restoration of K+ resulted in a rapid hyperpolarization (K1 effect). 2. The amplitude of the K1 effect depended on the duration of the prior K+ deprivation and on the subsequent K+ concentration. 3. The membrane resistance slightly increased during the K0 effect; during the K1 effect, it only returned to its control value. 4. Ouabain, cooling and replacement of external Na+ by Li+ also produced depolarization. 5. The K1 effect was suppressed by ouabain and markedly depressed by lowering the temperature to 4-6 degrees C. It was abolished if Li+ replaced Na+ during the prior privation of K+; moreover Li+ was unable to act as a substitute for external K+ in generating the K1 effect if used at equivalent concentration, but enhanced the effect at high concentration. 6. The findings are consistent with the presence of an electrogenic sodium pump in the myocardium of Porcellio contributing to the resting membrane potential. 7. Changes in the spontaneous rhythm observed during K0 and K1 are further suggestive of the presence of an electrogenic Na+ pump in the pacemaker neurons of the cardiac ganglion. Another explanation is also proposed. 8. The magnitude of the spontaneous contractions of the heart was increased during the K0 effect and markedly decreased during the K1 effect. An indirect effect of the changes in internal Na+ concentration on the contractile processes is suggested.     

Kinetic model of the effects of electrogenic enzymes on the membrane potential

Electrogenic enzymes contribute to the electrical field existing across biological membranes by using a source of free energy to generate an ionic current. The model introduced here permits one to evaluate this contribution. Since the model incorporates the electrogenic enzyme in the form of a sequential kinetic diagram, it permits one to study the kinetic effects of the concentration of the enzyme, the substrates and the different ligands on the membrane potential. Ionic electrodiffusion is expressed in terms of a chemical reaction; ionic permeabilities are thus treated as voltage-dependent rate constants. We use the condition of global electroneutrality to obtain an expression for the electrical potential difference across the membrane; such expression constitutes an extension of the Goldman-Hodgkin-Katz equation. The enzyme-related terms appear in the equation as functions of the rate constants and the diverse concentrations. The model is used to analyze the case of a cell membrane traversed by Na+ and K+ by simple diffusion, and by electrogenic transport mediated by a Na+-K+ ATPase. The enzyme reaction is represented by the six-step scheme proposed by Chapman et al. (1983, J. membr. Biol. 74, 139-153). The main results of the numerical calculations are that, within a certain interval, the membrane potential difference depends linearly on the enzyme density and hyperbolically on the ATP concentration. A similar behavior has been experimentally observed for the electrogenic proton pump of Neurospora crassa. Thus, the model here can be useful in the explanation and prediction of effects of electrogenic enzymes on the membrane potential.