Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

The High Energy Demand of Neuronal Cells Caused by Passive Leak Currents is Not a Waste of Energy

It is estimated that maintenance of the resting potential of neurons consumes between 15 % (in gray matter) and 44 % (in fully myelinated white matter) of the brain’s total energy budget [1]. This poses the intriguing question why evolution has not strived to lower the permeability of passive ion channels to cut the high resting-state energy budget of the brain. Based on a conceptual mathematical model of neuronal ion currents and action potential (AP) firing we demonstrate that a neuron endowed with small leak currents and correspondingly low energy consumption by the Na⁺/K⁺-ATPase in the resting state may indeed recapitulate all features of normal AP firing. However, the activation and inactivation of such a “low-energy-cost neuron” turns out to be extremely sensitive to small fluctuation of Na⁺ currents associated with Na⁺-dependent secondary-active transport that is indispensable for the metabolic integrity of the cell and neurotransmitter recycling. We provide evidence that sufficiently large leak currents function as important stabilizers of the membrane potential and thus are required to allow robust AP firing. Our simulations suggest that the energy demand of the Na⁺/K⁺-ATPase needed to counterbalance passive leak currents cannot be significantly dropped below observed values.     

Solute transport at the plasmalemma and the early evolution of cells

The evolution of the plasmalemma and its porter systems is considered in relation to selective pressures on primitive cells. Initially the polar lipid bilayer acted to separate the genetic apparatus of the protocell from the rest of the world. The requirement for the supply of nutrients and removal of waste products resulted in the evolution of passive uniporters for a number of organic and inorganic solutes. There was also a requirement for primary active transport, whereby one or more solutes is transported across the membrane contrary to the direction predicted from passive driving forces, with an energy input from light, redox reactions, “high-energy phosphate” or some other metabolic process. Active transport is discussed in terms of cytoplasmic pH regulation, cytoplasmic volume regulation, Ca²⁺ exclusion/phosphate accumulation, and the accumulation of organic (heterotrophic) substrates.It is suggested that volume regulation in wall-less cells was initially achieved by Na⁺ exclusion with active Na⁺ extrusion as a later refinement; the same applies to the maintenance of the characteristically low free Ca²⁺ level in the cytoplasm. A requirement for active phosphate influx is also likely in view of the high concentrations of orthophosphate required for phosphorylation reactions relative to the likely external concentration of phosphate and the inside-negative potential difference. This p.d., which results inter alia from Na⁺ extrusion, makes the maintenance of intracellular pH via passive H⁺ fluxes very difficult in the face of continued intracellular production of H⁺ during fermentation. Hence an early role for primary active extrusion (uniport) of H⁺ is very likely. Such uniport is of universal occurrence in present-day cells. Besides its role in pH regulation and in energy-coupling, H⁺ transport energises secondary (H⁺-linked) transport of many other solutes. We suggest that transport of HCO₃^? might also have a pH-regulating role, but apparently HCO₃^? cannot substitute for H⁺ with respect to energy-coupling and secondary active transport.     

Modeling Plasmalemma Ion Transport of the Aquatic Plant Egeria densa

Fresh-water plants generate extraordinarily high electric potential differences at the plasma membrane. For a deeper understanding of the underlying transport processes a mathematical model of the electrogenic plasmalemma ion transport was developed based on experimental data mainly obtained from Egeria densa. The model uses a general nonlinear network approach and assumes coupling of the transporters via membrane potential. A proton pump, an outward-rectifying K⁺ channel, an inward-rectifying K⁺ channel, a Cl⁻ channel and a (2H-Cl)⁺ symporter are considered to be elements of the system. The model takes into consideration the effects of light, external pH and ionic content of the bath medium on ion transport. As a result it does not only satisfactorily describe the membrane potential as a function of these external physiological factors but also succeeds in simulating the effects of specific inhibitors as well as I-V-curves obtained with the patch-clamp technique in the whole cell mode. The quality of the model was checked by stability and sensitivity analyses. Received: 18 March 1996/Revised: 17 July 1996     

The functions of plant cation/proton antiporters

The cation/H⁺ exchange is a basic process in transmembrane transport. The acquisition of genome sequences has now established that plants possess genes encoding a large number of cation/proton antiporter 1 (CPA1) proteins, few of which have been characterized with respect to their contribution to ion homeostasis. The CPA1s comprise plasma membrane, vacuolar, and endosomal forms, and they have been identified as important for a salinity tolerance. They are, however, also involved in both the control of cellular pH and K⁺ homeostasis, and regulate processes over a wide range of physiological events, from vesicle trafficking to development.     

How erythrocyte volume is regulated, or what mathematical models can and cannot do for biology

Modern concepts of the red blood cell (RBC) volume regulation are considered. It is shown that the system of ion pumps and channels in the cell membrane ensures the physiological value of volume with a precision of about 10% even at 5- to 7-fold variations of passive membrane permeability for ions. Particular attention is paid to mathematical models for evaluation of the role of different molecular mechanisms in the RBC volume control. It is shown that many questions, for example, ‘why the Na⁺,K⁺-ATPase pumps the ions in opposite directions’ or ‘what is the physiological role of Ca²⁺-activated K⁺-channels’, cannot be answered without adequate mathematical models of such complex control systems as cell volume control.     

Action of local anaesthetics on passive and energy-linked ion translocation in the inner mitochondrial membrane

The effect of dibucaine on passive and respiration-driven ion translocation and oxidative phosphorylation in submitochondrial particles from beef-heart has been studied.Dibucaine inhibited the nigericin-mediated H⁺/K⁺ exchange diffusion and the electrogenic, valinomycin-mediated K⁺ translocation in submitochondrial particles.The local anaesthetic exerted a direct stimulatory effect on the respiration-driven proton uptake and on the passive proton-diffusion reactions. The increase of the respiration-linked proton turnover caused by dibucaine was accompanied by uncoupling of oxidative phosphorylation. It is concluded that spontaneous noncoupled as well as ionophoremediated K⁺ translocation in mitochondria occurs across phospholipid bilayer regions of the membrane whilst other components of the membrane would be specifically involved in active and passive proton translocation across the membrane.The results indicate that polar groups of membrane phospholipids play an important role in energy conservation and transfer in the mitochondrial membrane.     

Ion channel–transporter interactions

All living cells require membrane proteins that act as conduits for the regulated transport of ions, solutes and other small molecules across the cell membrane. Ion channels provide a pore that permits often rapid, highly selective and tightly regulated movement of ions down their electrochemical gradient. In contrast, active transporters can move moieties up their electrochemical gradient. The secondary active transporters (such as SLC superfamily solute transporters) achieve this by coupling uphill movement of the substrate to downhill movement of another ion, such as sodium. The primary active transporters (including H⁺/K⁺-ATPases and Na⁺/K⁺-ATPases) utilize ATP hydrolysis as an energy source to power uphill transport. It is well known that proteins in each of these classes work in concert with members of the other classes to ensure, for example, ion homeostasis, ion secretion and restoration of ion balance following action potentials. More recently, evidence is emerging of direct physical interaction between true ion channels, and some primary or secondary active transporters. Here, we review the first known members of this new class of macromolecular complexes that we term “chansporters”, explore their biological roles and discuss the pathophysiological consequences of their disruption. We compare functional and/or physical interactions between the ubiquitous KCNQ1 potassium channel and various active transporters, and examine other newly discovered chansporter complexes that suggest we may be seeing the tip of the iceberg in a newly emerging signaling modality.     

The Gárdos channel: a review of the Ca2+-activated K+ channel in human erythrocytes

Ca²⁺-dependent K⁺ efflux from human erythrocytes was first described in the 1950s. Subsequent studies revealed that a K⁺-specific membrane protein (the Gárdos channel) was responsible for this phenomenon (the Gárdos effect). In recent years several types of Ca-activated K⁺ channel have been identified and studied in a wide range of cells, with the erythrocyte Gárdos channel serving as both a model for a broader physiological perspective, and an intriguing component of erythrocyte function.The existence of this channel has raised a number of questions. For example, what is its role in the establishment and maintenance of ionic distribution across the red cell membrane? What role might it play in erythrocyte development? To what extent is it active in circulating erythrocytes? What are the cell-physiological implications of its dysfunction?This review summarises current knowledge of this membrane protein with respect to its function and structure, its physiological roles (some putative) and its contribution to various disease states, and it provides an introduction to adaptable NMR methods, which is our own area of technical expertise, for such ion transport analysis.     

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase

Summary A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K⁺ so that the membrane voltage approaches the Nernst potential for K⁺. With constant external K⁺ concentration, the time course of internal K⁺ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K⁺ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.     

The association between K+ and H+ distribution in skeletal muscles: implication to linked transport.

Data from the literature and results from a mathematical model of steady state fluid-electrolyte balance are used to support the observation that a relationship exists between the concentration gradients of K⁺ and H⁺ in the fluids of skeletal muscle over a range of acid-base disturbances. This relationship is shown to be consistent with the premise that the steady state electrochemical potential gradients for these ions remain constant under these conditions. Using a pump-leak model of ion transport, and the constant electric field assumption, it is also demonstrated that the steady state rates of active transport of K⁺ and H⁺ are related. These results suggest that the relations between both the steady state concentration gradients and the active transport rates for these ions are not necessarily the result of fixed biochemical mechanisms, but may come about simply from coupling through macroscopic thermodynamic processes.     

Monovalent ion and calcium ion fluxes in sarcoplasmic reticulum

Summary The ion permeability of sarcoplasmic reticulum vesicles from skeletal and heart muscle has been characterized by radioisotope flux, osmotic and membrane potential measurements, and by incorporating vesicles into planar phospholipid bilayers. The sarcoplasmic reticulum membrane is uniquely permeable to various biologically relevant monovalent ions. At least two and possibly three separate passive permeation systems for monovalent ions have been identified: 1) a K⁺, Na⁺ channel, 2) an anion channel, and 3) a H⁺ (OH^–) permeable pathway which may or may not be synonymous with the anion channel. A possible physiological function of these monovalent ion permeation systems is to permit rapid movement of K⁺, Na⁺, H⁺ and Cl across the membrane to counter electrogenic Ca²⁺ fluxes during Ca²⁺ release and uptake by sacroplasmic reticulum.     

Membrane voltage as a dynamic platform for spatiotemporal signaling,physiological, and developmental regulation

Membrane voltage arises from the transport of ions through ion-translocating ATPases, ion-coupled transport of solutes, and ion channels, and is an integral part of the bioenergetic “currency” of the membrane. The dynamics of membrane voltage—so-called action, systemic, and variation potentials—have also led to a recognition of their contributions to signal transduction, both within cells and across tissues. Here, we review the origins of our understanding of membrane voltage and its place as a central element in regulating transport and signal transmission. We stress the importance of understanding voltage as a common intermediate that acts both as a driving force for transport—an electrical “substrate”—and as a product of charge flux across the membrane, thereby interconnecting all charge-carrying transport across the membrane. The voltage interconnection is vital to signaling via second messengers that rely on ion flux, including cytosolic free Ca²⁺, H⁺, and the synthesis of reactive oxygen species generated by integral membrane, respiratory burst oxidases. These characteristics inform on the ways in which long-distance voltage signals and voltage oscillations give rise to unique gene expression patterns and influence physiological, developmental, and adaptive responses such as systemic acquired resistance to pathogens and to insect herbivory.

Membrane voltage serves as a platform coordinating ion flux to transmit and transduce biological signals.

Advances

• The biophysics of transport that determine membrane voltage are well-described with quantitative flux equations.
• In the models of the guard cell and the giant algae Chara and Nitella these charge-transporting processes accurately describe and predict physiological behavior, including the coupling of membrane voltage oscillations with ion flux, [Ca²⁺]_i, pH, their consequences for cellular osmotic adjustments, and their spatial propagation.
• Unlike neuronal and other animal tissues, action potentials in plants are mediated by a temporal sequence of ion flux through Ca²⁺ and Cl^- channels with voltage recovery driven by ion flux through K⁺ channels. The interplay of channel-mediated ion flux and changes in H⁺-ATPase activity are likely responsible for the slower propagation of variation and systemic potentials.
• In terrestrial plants, membrane voltage transients may propagate along vascular traces, both through the parenchymal cells lining the xylem and through the phloem. Propagation of such voltage transients is associated with glutamate receptor-like channels that may contribute to plasma membrane Ca²⁺ flux and [Ca²⁺]_i elevations.
• Changes in [Ca²⁺]_i, pH, and reactive oxygen species are key mediators that translate voltage signals into physiological, developmental, and adaptive responses in plant tissues.

     

Neuroprotection — rationale for pharmacological modulation of Na+-channels

Summary The primary factor detrimental to neurons in neurological disorders associated with deficient oxygen supply or mitochondrial dysfunction is insufficient ATP production relative to their requirement. As a large part of the energy consumed by brain cells is used for maintenance of the Na⁺ gradient across the cellular membrane, reduction of energy demand by down-modulation of voltage-gated Na⁺-channels is a rational strategy for neuroprotection. In addition, preservation of the inward Na⁺ gradient may be beneficial because it is an essential driving force for vital ion exchanges and transport mechanisms such as Ca²⁺ homeostasis and neurotransmitter uptake.     

Dynamics of cellular homeostasis: Recovery time for a perturbation from equilibrium

In the collecting ductin vivo, the principal cell encounters a wide range in luminal flow rate and luminal concentration of NaCl. As a consequence, there are substantial variations in the transcellular fluxes of Na⁺ and Cl⁻, conditions which would be expected to perturb cell volume and cytosolic concentrations. Several control mechanisms have been identified which can potentially blunt these perturbations, and these entail cellular regulation of the luminal membrane Na⁺ channel and peritubular membrane K⁺ and Cl⁻ channels. To illustrate the impact of these regulated channels, a mathematical model of the principal cell of the rat cortical collecting duct has been developed, in which ion channel permeabilities are either constant or regulated. In comparison to the model with fixed permeabilities, the model with regulated channels demonstrates enhanced cellular homeostasis following steady-state variation in luminal NaCl. However, in the transient response to a cytosolic perturbation, the difference in recovery time between the models is small. An approximate analysis is presented which casts these models as dynamical systems with constant coefficients. Despite the presence of regulated ion channels, concordance of each model with its linear approximation is verified for experimentally meaningful perturbations from the reference condition. Solution of a Lyapunov equation for each linear system yields a matrix whose application to a perturbation permits explicit estimation of the time to recovery. Comparison of these solution matrices for regulated and non-regulated cells confirms the similarity of the dynamic response of the two models. These calculations suggest that enhanced homeostasis by regulated channels may be protective, without necessarily hastening recovery from cellular perturbations.     

Interaction of dichlone with human erythrocytes. I. Changes in cell permeability

The uptake of the fungicide dichlone (2,3-dichloro-1,4-naphthoquinone) by human erythrocytes was extremely rapid, reaching a maximum within 5 min of treatment. Most of the dichlone taken up was present in the interior of the cell; only a small fraction of the pesticide (less than 5%) was bound to the cell membrane. Dichlone (3 · 10^?5M-10^?4M) induced a rapid loss of intracellular potassium from the erythrocytes; the leakage of K⁺ varied with the fungicide concentration as well as with cell concentration. Pretreatment of the cells with glutathione was able to reduce potassium loss. Cells exposed to dichlone showed increased osmotic fragility. Dichlone also inhibited Na⁺-K⁺ ATPase, which is associated with active ion transport. However, the leakage of potassium in dichlone-treated cells does not appear to be related to the interference with active ion transport. An extensive loss of potassium within a relatively short time after treatment suggests that dichlone produces its effect by increasing passive cation permeability, probably as a result of direct action on the membrane structure. Dichlone was able to induce hemolysis, but only at concentrations higher than those which resulted in K⁺ loss. The loss of hemoglobin appeared to be mainly due to osmotic swelling of the treated cells. Exposure of red cells to dichlone also resulted in a rapid and extensive formation of methemoglobin as well as a denaturation of hemoglobin. Thus, dichlone not only may be capable of lowering the capacity of erythrocytes to transport oxygen but also alters their permeability.     

Involvement of protons in the ion transport cycle of Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase

The effect of pH on electrogenic sodium transport by the Na⁺,K⁺-ATPase has been studied. Experiments were carried out by admittance recording in a model system consisting of a bilayer lipid membrane with adsorbed membrane fragments containing purified Na⁺,K⁺-ATPase. Changes in the membrane admittance (capacitance and conductance increments in response to photo-induced release of ATP from caged ATP) were measured as function of AC voltage frequency, sodium ion concentration, and pH. In solutions containing 150 mM Na⁺, the frequency dependence of capacitance increments was not significantly dependent on pH in the range between 6 and 8. At a low NaCl concentration (3 mM), the capacitance increments at low frequencies decreased with the increasing pH. In the absence of NaCl, the frequency-dependent capacitance increment at low frequencies was similar to that measured in the presence of 3 mM NaCl. These results may be explained by involvement of protons in the Na⁺,K⁺-ATPase pump cycle, i.e., electroneutral exchange of sodium ions for protons under physiological conditions, electrogenic transport of sodium ions at high pH, and electrogenic transport of protons at low concentrations (and in the absence) of sodium ions.     

Histamine (re)uptake by astrocytes: an experimental and computational study

Astrocytes participate in the clearance of neurotransmitters by their uptake and subsequent enzymatic degradation. Histamine as a polar and/or protonated molecule must use a carrier to be transported across the cell membrane, although a specific histamine transporter has not been elucidated, yet. In this work we upgraded the kinetic studies of histamine uptake into neonatal rat cultured type 1 astrocytes with quantum chemical calculations of histamine pKa values in conjunction with Langevin dipoles solvation model as the first step toward microscopic simulation of transport. Our results indicate that astrocytes transport histamine by at least two carrier mediated processes, a concentration gradient dependent passive and a sodium-dependent and ATP-driven active transport. We also demonstrated that histamine protonation states depend on the polarity of the environment. In conclusion we suggest that histamine, a polar molecule at physiological pH uses at least two different mechanisms for its uptake into astrocytes –an electrodiffusion and Na⁺-dependent and ouabain sensitive active process. We emphasize relevance of knowledge of histamines protonation states at the rate limiting step of its transport for microscopic simulation that will be possible when structure of histamine transporter is known.     

Effect of aldosterone on active and passive conductance andE Na in the toad bladder

Summary It has been demonstrated previously that aldosterone increases the electrical conductance of the toad bladder in association with the stimulation of active sodium transport. In the present study the concurrent measurement of electrical quantities and ion tracer flux distinguishes effects on active and passive pathways. Lack of an effect on passive Na⁺ or Cl^– tracer flux in hemibladders preselected to eliminate large artefactual leaks indicates that aldosterone has no influence on physiological passive conductance. Thus, the enhancement of electrical conductance is entirely attributable to the active pathway. The magnitude of the increase in the active conductance was estimated. The data permitted also the comparison of effects on the flux ratio of Na⁺ at short circuit (f ₀) and the electrical potential difference adequate to abolish active sodium transport (E _Na). Even in membranes with minimal leakage the flux ratio does not reliably reflectE _Na. Aldosterone increased meanf ₀ from 11 to 22, but did not affectE _Na.