The proton ladder,a static mechanism for ion/proton coports and counterports

An ion/proton counterport is formed simply by locating a chain of ionizable residues connected by a proton conducting path near a passive ion pore which spans the membrane. The electric coupling between the ion in transit through the pore and the residues can ensure that for each ion passing through the pore in one direction a proton is driven along the chain of ionizable residues (the proton ladder) in the same or in the opposite direction. The mechanism is symmetrical in that a trans-membrane ion gradient may drive protons against their electrochemical potential gradient or a proton gradient may drive ions against theirs. The mechanism is applicable to cation or anion channels and to coports or counterports. No mechanical motion is required other than the motion of the ions and the protons. Monte Carlo computer simulations are performed on the model and its predicted properties are listed. The new type of counterport model is compared with currently used models. Offprint requests to: D. T Edmonds     

Charge selectivity of the designed uncharged peptide ion channel Ac-(LSSLLSL)3-CONH2.

Charge selectivity in ion channel proteins is not fully understood. We have studied charge selectivity in a simple model system without charged groups, in which an amphiphilic helical peptide, Ac-(Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2, forms ion channels across an uncharged phospholipid membrane. We find these channels to conduct both K+ and Cl-, with a permeability ratio (based on reversal potentials) that depends on the direction of the KCl concentration gradient across the membrane. The channel shows high selectivity for K+ when [KCl] is lowered on the side of the membrane that is held at a positive potential (the putative C-terminal side), but only modest K+ selectivity when [KCl] is lowered on the opposite side (the putative N-terminal side). Neither a simple Nernst-Planck electrodiffusion model including screening of the helix dipole potential, nor a multi-ion, state transition model allowing simultaneous cation and anion occupancy of the channel can satisfactorily fit the current-voltage curves over the full range of experimental conditions. However, the C-side/N-side dilution asymmetry in reversal potentials can be simulated with either type of model.     

Insights into the biophysical properties of GABA(A) ion channels: modulation of ion permeation by drugs and protein interactions

The fundamental properties of ion channels assure their selectivity for a particular ion, its rapid permeation through a central pore and that such electrical activity is modulated by factors that control the opening and closing (gating) of the channel. All cell types possess ion channels and their regulated flux of ions across the membrane play critical roles in all steps of life. An ion channel does not act alone to control cell excitability but rather forms part of larger protein complexes. The identification of protein interaction partners of ion channels and their influence on both the fundamental biophysical properties of the channel and its expression in the membrane are revealing the many ways in which electrical activity may be regulated. Highlighted here is the novel use of the patch clamp method to dissect out the influence of protein interactions on the activity of individual GABA(A) receptors. The studies demonstrate that ion conduction is a dynamic property of a channel and that protein interactions in a cytoplasmic domain underlie the channel's ability to alter ion permeation. A structural model describing a reorganisation of the conserved cytoplasmic gondola domain and the influence of drugs on this process are presented.     

Regulation of single sodium channels in renal tissue: a role in sodium homeostasis

The kidney is responsible for the maintenance of an organism's body solute and water balance (i.e., Na+ homeostasis). The distal nephron and the cortical collecting duct (CCD) (an example of a tight epithelium) are important sites of regulatory control over the rate of Na+ reabsorption. The Na+ channel, a specialized protein located in the apical membrane of CCD cells, is the specific site of transepithelial Na+ movement. Na+ entry into the cell across the apical membrane occurs by passive diffusion of Na+ down an electrochemical gradient. We have used the patch-voltage clamp method to examine single-channel conductance events of the amiloride-sensitive apical Na+ channel in A6 cells, a model of CCD. Two types of Na+ channel were identified. One type was characterized by low selectivity (Na+ to K+) and high conductance, the other by high selectivity and low conductance. The type and frequency of channel observed depended on the transporting state of the epithelium. In a tissue with poor transport rates, the low-selectivity type of channel was prevalent (the other type of channel was present, but in a very low density). Therefore, the poorly transporting tissue had an overall low apical Na+ conductance. In a tissue with high transport rates, the highly selective channel appeared to be predominant. In this case the net result was a highly Na+ conductive apical 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.     

An electrostatic model of a membrane ion pump

The model is based upon an ion channel with an electric dipolar structure. With simplifying assumptions it is possible to calculate that a typical channel, 1 nm in diameter and 5 nm long, could contain at most two or three univalent cations at a time. The channel ion binding sites have an effective affinity for ions from the fluid bathing the negative end of the channel, several orders of magnitude higher than their affinity for ions from the fluid bathing the positive end of the channel. The approach of an external, positively charged body to the negative end of the channel, is sufficient to convert the two- or three-channel ion sites with high affinity for ions from the fluid bathing this end into very low affinity sites for the same ions that now have access only to the fluid bathing the other end of the channel. The change in affinity and fluid access requires no molecular or electrical change in the channel structure other than the passive superposition of the electrostatic potential of the dipolar channel and that of the charged body. An oscillating electric field externally applied to an electric dipolar channel is shown to result in the unidirectional pumping of cations in the direction of the channel dipole even against large adverse ion concentration gradients. The energy required must be supplied by the sources of the electric field. By using two such channels in close proximity, one selective for K+ ions with its dipole moment pointing into a cell and the other selective for Na+ ions with its dipole moment pointing out from the cell, it is possible to construct a model pump with calculated properties that simulate many of those measured for Na+-K+-ATPase, with both physiological and artificial ionic concentrations.     

Members of the Arabidopsis AtTPK/KCO family form homomeric vacuolar channels in planta

The Arabidopsis thaliana K⁺ channel family of AtTPK/KCO proteins consists of six members including a 'single-pore' (K_ir-type) and five 'tandem-pore' channels. AtTPK4 is currently the only ion channel of this family for which a function has been demonstrated in planta . The protein is located at the plasma membrane forming a voltage-independent K⁺ channel that is blocked by extracellular calcium ions. In contrast, AtTPK1 is a tonoplast-localized protein, that establishes a K⁺-selective, voltage-independent ion channel activated by cytosolic calcium when expressed in a heterologous system, i.e. yeast. Here, we provide evidence that other AtTPK/KCO channel subunits, i.e. AtTPK2, AtTPK3, AtTPK5 and AtKCO3, are also targeted to the vacuolar membrane, opening the possibility that they interact at the target membrane to form heteromeric ion channels. However, when testing the cellular expression patterns of AtTPK / KCO genes we observed distinct expression domains that overlap in only a few tissues of the Arabidopsis plant, making it unlikely that different channel subunits interact to form heteromeric channels. This conclusion was substantiated by in planta expression of combinations of selected tonoplast AtTPK/KCO proteins. Fluorescence resonance energy transfer assays indicate that protein interaction occurs between identical channel subunits (most efficiently between AtTPK1 or AtKCO3) but not between different channel subunits. The finding could be confirmed by bimolecular fluorescence complementation assays. We conclude that tonoplast-located AtTPK/KCO subunits form homomeric ion channels in vivo .     

Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG

Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca2+-dependent K+ current, (ii) a transient K+ current inhibited by 4-AP, (iii) an inwardly rectifying K+ current blocked by Ba2+ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl- current, (v) a TTX-sensitive Na+ conductance and (vi) a L-type Ca2+ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca2+ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca2+-dependent K+ influx against the electrochemical gradient, probably via an ouabain-sensitive Na+-K+ pump.     

Hysteresis in voltage-gated channels

Ion channels constitute a superfamily of membrane proteins found in all living creatures. Their activity allows fast translocation of ions across the plasma membrane down the ion's transmembrane electrochemical gradient, resulting in a difference in electrical potential across the plasma membrane, known as the membrane potential. A group within this superfamily, namely voltage-gated channels, displays activity that is sensitive to the membrane potential. The activity of voltage-gated channels is controlled by the membrane potential, while the membrane potential is changed by these channels' activity. This interplay produces variations in the membrane potential that have evolved into electrical signals in many organisms. These signals are essential for numerous biological processes, including neuronal activity, insulin release, muscle contraction, fertilization and many others. In recent years, the activity of the voltage-gated channels has been observed not to follow a simple relationship with the membrane potential. Instead, it has been shown that the activity of voltage-gated channel displays hysteresis. In fact, a growing number of evidence have demonstrated that the voltage dependence of channel activity is dynamically modulated by activity itself. In spite of the great impact that this property can have on electrical signaling, hysteresis in voltage-gated channels is often overlooked. Addressing this issue, this review provides examples of voltage-gated ion channels displaying hysteretic behavior. Further, this review will discuss how Dynamic Voltage Dependence in voltage-gated channels can have a physiological role in electrical signaling. Furthermore, this review will elaborate on the current thoughts on the mechanism underlying hysteresis in voltage-gated channels.     

Ionic Blockage of Sodium Channels in Nerve

Increasing the hydrogen ion concentration of the bathing medium reversibly depresses the sodium permeability of voltage-clamped frog nerves. The depression depends on membrane voltage: changing from pH 7 to pH 5 causes a 60% reduction in sodium permeability at +20 mV, but only a 20% reduction at +180 mV. This voltage-dependent block of sodium channels by hydrogen ions is explained by assuming that hydrogen ions enter the open sodium channel and bind there, preventing sodium ion passage. The voltage dependence arises because the binding site is assumed to lie far enough across the membrane for bound ions to be affected by part of the potential difference across the membrane. Equations are derived for the general case where the blocking ion enters the channel from either side of the membrane. For H⁺ ion blockage, a simpler model, in which H⁺ enters the channel only from the bathing medium, is found to be sufficient. The dissociation constant of H⁺ ions from the channel site, 3.9 x 10^-6 M (pK_a 5.4), is like that of a carboxylic acid. From the voltage dependence of the block, this acid site is about one-quarter of the way across the membrane potential from the outside. In addition to blocking as described by the model, hydrogen ions also shift the responses of sodium channel "gates" to voltage, probably by altering the surface potential of the nerve. Evidence for voltage-dependent blockage by calcium ions is also presented.     

Potassium channels as multi-ion single-file pores

A literature review reveals many lines of evidence that both delayed rectifier and inward rectifier potassium channels are multi-ion pores. These include unidirectional flux ratios given by the 2--2.5 power of the electrochemical activity ratio, very steeply voltage-dependent block with monovalent blocking ions, relief of block by permeant ions added to the side opposite from the blocking ion, rectification depending on E--EK, and a minimum in the reversal potential or conductance as external K+ ions are replaced by an equivalent concentration of T1+ ions. We consider a channel with a linear sequence of energy barriers and binding sites. The channel can be occupied by more than one ion at a time, and ions hop in single file into vacant sites with rate constants that depend on barrier heights, membrane potential, and interionic repulsion. Such multi-ion models reproduce qualitatively the special flux properties of potassium channels when the barriers for hopping out of the pore are larger than for hopping between sites within the pore and when there is repulsion between ions. These conditions also produce multiple maxima in the conductance-ion activity relationship. In agreement with Armstrong's hypothesis (1969. J. Gen. Physiol. 54:553--575), inward rectification may be understood in terms of block by an internal blocking cation. Potassium channels must have at least three sites and often contain at least two ions at a time.     

Repulsion between tetraethylammonium ions in cloned voltage-gated potassium channels.

Tetraethylammonium ion (TEA+) blocks voltage-gated K+ channels by acting at two sites located at opposite ends of the aqueous pore. This allowed us to test two predictions made by models of ion permeation, namely that K+ channels can be simultaneously occupied by multiple ions and that the ions repel each other. We show that externally applied TEA+ antagonize block by internal TEA+ and vice versa. The antagonism is less than predicted for competitive binding, hence TEA+ may occupy both sites simultaneously. External TEA+ and internal TEA+ reduce each others affinity 4- to 5-fold. In addition, K+ antagonizes block by TEA+ at the opposite side of the membrane, and external TEA+ antagonizes is block by internal Ba2+. The antagonism between ions applied at opposite sides of the membrane may be common to all cations binding to K+ channels.     

Channels produced by spider venoms in bilayer lipid membrane: mechanisms of ion transport and toxic action

The selectivity of ion channels produced by latrotoxin obtained from a black widow spider venom and by venom from the spider Steatoda paykulliana in bilayer phospholipid membrane was studied. Experimental current-voltage curves of these channels were used for the estimation of parameters of a two barrier model of their energy profiles. Selectivities of both types of channels are similar. Alkaline earth cations are permeable, the permeability increasing in the order Mg2+ less than Ca2+ less than Sr2+ less than Ba2+. In contrast transition metal cations block the channel, their efficiency decreases in the order: Cd2+ greater than or equal to Ni2+ greater than Zn2+ greater than Co2+ greater than Mn2+ (Steatoda paykulliana spider venom) and Cd2+ greater than Co2+ greater than Ni2+ greater than Zn2+ greater than Mn2+ (latrotoxin). Amplitudes of current carried by corresponding ions are mainly determined by the depth of the potential well for this ion, i.e., by its affinity to the cation binding site in the channel. The channels are also permeable to monovalent cations but they do not bind them. Selectivity for monovalent cations depends on Ca2+ concentration at the cis-side of membrane in the micromolar range. However, the addition of Ca2+ to the trans-side up to 10 mM does not affect currents carried by monovalent ions. It is suggested that venom-induced calcium channels have two conformational states with different selectivities which interconvert upon binding one calcium ion. Possible general schemes for the organisation of calcium channels in excitable membranes are also discussed. Finally, using a mathematical model of synaptic transmission, possible mechanisms of toxic action of spider venoms are considered.     

Uptake of Magnesium by Chromaffin Granules In Vitro: Role of the Proton Electrochemical Gradient

Abstract: The chromaffin granule membrane in vitro is impermeable to protons as well as to Mg²⁺; however, when granules are incubated in the presence of the proton ionophore carbonyl cyanide p -trifluoromethoxy-phenylhydrazone or an inhibitor of the granule membrane Mg²⁺-dependent ATPase, the metal ion is accumulated inside the granules. This accumulation is dependent upon the granule transmembrane potential. The simultaneous presence of the ATPase inhibitor and the proton ionophore markedly increases metal ion incorporation. Mg²⁺ incorporation is also promoted by nigericin in the presence of potassium or sodium ions, indicating that Mg²⁺ accumulation is also dependent upon the transmembrane pH gradient. Concomitant with the Mg²⁺ accumulation, there is a significant loss of endogenous catecholamines. It is concluded that Mg²⁺ accumulation is determined by the electrochemical gradient maintained across the membrane. Once the metal ion has accumulated into the granules it displaces catecholamines from their storage sites.     

A binding-block ion selective mechanism revealed by a Na/K selective channel

Mechanosensitive (MS) channels are extensively studied membrane protein for maintaining intracellular homeostasis through translocating solutes and ions across the membrane, but its mechanisms of channel gating and ion selectivity are largely unknown. Here, we identified the YnaI channel as the Na⁺/K⁺ cation-selective MS channel and solved its structure at 3.8 Å by cryo-EM single-particle method. YnaI exhibits low conductance among the family of MS channels in E. coli, and shares a similar overall heptamer structure fold with previously studied MscS channels. By combining structural based mutagenesis, quantum mechanical and electrophysiological characterizations, we revealed that ion selective filter formed by seven hydrophobic methionine (YnaI^Met158) in the transmembrane pore determined ion selectivity, and both ion selectivity and gating of YnaI channel were affected by accompanying anions in solution. Further quantum simulation and functional validation support that the distinct binding energies with various anions to YnaI^Met158 facilitate Na⁺/K⁺ pass through, which was defined as bindingblock mechanism. Our structural and functional studies provided a new perspective for understanding the mechanism of how MS channels select ions driven by mechanical force.     

Effects of membrane lipids on ion channel structure and function

Biologic membranes are not simply inert physical barriers, but complex and dynamic environments that affect membrane protein structure and function. Residing within these environments, ion channels control the flux of ions across the membrane through conformational changes that allow transient ion flux through a central pore. These conformational changes may be modulated by changes in transmembrane electrochemical potential, the binding of small ligands or other proteins, or changes in the local lipid environment. Ion channels play fundamental roles in cellular function and, in higher eukaryotes, are the primary means of intercellular signaling, especially between excitable cells such as neurons. The focus of this review is to examine how the composition of the bilayer affects ion channel structure and function. This is an important consideration because the bilayer composition varies greatly in different cell types and in different organellar membranes. Even within a membrane, the lipid composition differs between the inner and outer leaflets, and the composition within a given leaflet is both heterogeneous and highly dynamic. Differential packing of lipids (and proteins) leads to the formation of microdomains, and lateral diffusion of these microdomains or "lipid rafts" serve as mobile platforms for the clustering and organization of bilayer constituents including ion channels. The structure and function of these channels are sensitive to specific chemical interactions with neighboring components of the membrane and also to the biophysical properties of their membrane microenvironment (e.g., fluidity, lateral pressure profile, and bilayer thickness). As specific examples, we have focused on the K+ ion channels and the ligand-gated nicotinicoid receptors, two classes of ion channels that have been well-characterized structurally and functionally. The responsiveness of these ion channels to changes in the lipid environment illustrate how ion channels, and more generally, any membrane protein, may be regulated via cellular control of membrane composition.     

The molecular chaperone hsp70 interacts with the cytosolic II-III loop of the Cav2.3 E-type voltage-gated Ca2+ channel.

Multiple types of voltage-activated Ca2+ channels (T, L, N, P, Q, R type) coexist in excitable cells and participate in synaptic differentiation, secretion, transmitter release, and neuronal plasticity. Ca2+ ions entering cells trigger these events through their interaction with the ion channel itself or through Ca2+ binding to target proteins initiating signalling cascades at cytosolic loops of the ion conducting subunit (Cava1). These loops interact with target proteins in a Ca2+-dependent or independent manner. In Cav2.3-containing channels the cytosolic linker between domains II and III confers a novel Ca2+ sensitivity to E-type Ca2+ channels including phorbol ester sensitive signalling via protein kinase C (PKC) in Cav2.3 transfected HEK-293 cells. To understand Ca2+ and phorbol ester mediated activation of Cav2.3 Ca2+ channels, protein interaction partners of the II-III loop were identified. FLAG-tagged II-III - loop of human Cav2.3 was over-expressed in HEK 293 cells, and the molecular chaperone hsp70, which is known to interact with PKC, was identified as a novel functional interaction partner. Immunopurified II-III loop-protein of neuronal and endocrine Cav2.3 splice variants stimulate autophosphorylation of PKCa, leading to the suggestion that hsp70--binding to the II-III loop--may act as an adaptor for Ca2+ dependent targeting of PKC to E-type Ca2+ channels.     

The Excitable Membrane: A Physiochemical Model

The model of the excitable membrane assumes common channels for Na⁺ and K⁺; the two ion species interact within the pores through their electrostatic forces. The electric field varies across the membrane and with time, as a result of ionic redistribution. Ionic flow is primarily controlled by energy barriers at the two interfaces and by Ca⁺⁺ adsorption at the external interface. When the membrane is polarized, the high electric field at the external interface acting on the membrane fixed charge keeps the effective channel diameter small, so that only dihydrated ions can cross the interface. The higher energy required to partially dehydrate Na⁺ accounts for its lower permeability when polarized. Depolarized, the channel entrance can expand, permitting quadrihydrated ions to pass; the large initial Na⁺ flow is the result of the large concentration ratio across the interface. The effect at the internal interface is symmetric; Na⁺ crosses with greater difficulty when the membrane is depolarized. Na⁺ inactivation occurs when the ion distribution within the membrane has assumed its new steady-state value. Calculations based on parameters consistent with physicochemical data agree generally with a wide range of experiments. The model does not obey the two fundamental Hodgkin-Huxley (HH) postulates (independence principle, ion flow proportional to thermodynamic potential). In several instances the model predicts experimental results which are not predicted by the HH equations.     

Gadolinium-sensitive, voltage-dependent calcium release channels in the endoplasmic reticulum of a higher plant mechanoreceptor organ.

The lipid bilayer technique was adapted to the functional reconstitution of ion channels from the endoplasmic reticulum of a higher plant. This was obtained at high purity from touch-sensitive tendrils of Bryonia dioica. In this preparation, a calcium-selective strongly rectifying channel is prevailing whose single-channel properties have been characterized. The single-channel conductance is 29 pS in 50 mM CaCl2. The Ca2+: K+ selectivity was determined to be approximately 6.6. The channel is voltage-gated and, more importantly, the gating voltage is strongly shifted towards more negative voltages when a transmembrane Ca2+ gradient is applied. Thus, at physiological voltages across the endoplasmic reticulum membrane, the channel's open probability will be governed largely by the chemical potential gradient of Ca2+, generated by the Ca(2+)-ATPase in that same membrane. The calcium release channel described here is effectively blocked by Gd3+ which also completely suppresses a tendril's reaction to touch, suggesting that this channel could be a key element of calcium signaling in higher plant mechanotransduction. Its molecular characteristics and inhibitor data show it to be the first known member of a hitherto unrecognized class of calcium channels.