Structural correlates of selectivity and inactivation in potassium channels

Potassium channels are involved in a tremendously diverse range of physiological applications requiring distinctly different functional properties. Not surprisingly, the amino acid sequences for these proteins are diverse as well, except for the region that has been ordained the "selectivity filter". The goal of this review is to examine our current understanding of the role of the selectivity filter and regions adjacent to it in specifying selectivity as well as its role in gating/inactivation and possible mechanisms by which these processes are coupled. Our working hypothesis is that an amino acid network behind the filter modulates selectivity in channels with the same signature sequence while at the same time affecting channel inactivation properties. This article is part of a Special Issue entitled: Membrane protein structure and function.     

Free energy analysis of conductivity and charge selectivity of M2GlyR-derived synthetic channels

Significant progresses have been made in the design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the second transmembrane domain of the α-subunit of the glycine receptor (GlyR). The latest designs, including p22 (KKKKP ARVGL GITTV LTMTT QS), are highly soluble in water with minimal aggregation propensity and insert efficiently into cell membranes to form highly conductive ion channels. The last obstacle to a potential lead sequence for channel replacement treatment of CF patients is achieving adequate chloride selectivity. We have performed free energy simulation to analyze the conductance and charge selectivity of M2GlyR-derived synthetic channels. The results reveal that the pentameric p22 pore is non-selective. Moderate barriers for permeation of both K⁺ and Cl⁻ are dominated by the desolvation cost. Despite previous evidence suggesting a potential role of threonine side chains in anion selectivity, the hydroxyl group is not a good surrogate of water for coordinating these ions. We have also tested initial ideas of introducing additional rings of positive changes to various positions along the pore to increase anion selectivity. The results support the feasibility of achieving anion selectivity by modifying the electrostatic properties of the pore, but at the same time suggest that the peptide assembly and pore topology may also be dramatically modified, which could abolish the effects of modified electrostatics on anion selectivity. This was confirmed by subsequent two-electrode voltage clamp measurements showing that none of the tested mono-, di- and tri-Dap substituted sequences was selective. The current study thus highlights the importance of controlling channel topology besides modifying pore electrostatics for achieving anion selectivity. Several strategies are now being explored in our continued efforts to design an anion selective peptide channel with suitable biophysical, physiological and pharmacological properties as a potential treatment modality for channel replacement therapy. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Defective interactions of protein partner with ion channels and transporters as alternative mechanisms of membrane channelopathies

The past twenty years have revealed the existence of numerous ion channel mutations resulting in human pathology. Ion channels provide the basis of diverse cellular functions, ranging from hormone secretion, excitation–contraction coupling, cell signaling, immune response, and trans-epithelial transport. Therefore, the regulation of biophysical properties of channels is vital in human physiology. Only within the last decade has the role of non-ion channel components come to light in regard to ion channel spatial, temporal, and biophysical regulation in physiology. A growing number of auxiliary components have been determined to play elemental roles in excitable cell physiology, with dysfunction resulting in disorders and related manifestations. This review focuses on the broad implications of such dysfunction, focusing on disease-causing mutations that alter interactions between ion channels and auxiliary ion channel components in a diverse set of human excitable cell disease. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé     

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.     

Threonine in the selectivity filter of the acetylcholine receptor channel.

The acetylcholine receptor (AChR) is a cation selective channel whose biophysical properties as well as its molecular composition are fairly well characterized. Previous studies on the rat muscle alpha-subunit indicate that a threonine residue located near the cytoplasmic side of the M2 segment is a determinant of ion flow. We have studied the role of this threonine in ionic selectivity by measuring conductance sequences for monovalent alkali cations and bionic reversal potentials of the wild type (alpha beta gamma delta channel) and two mutant channels in which this threonine was replaced by either valine (alpha T264V) or glycine (alpha T264G). For the wild type channel we found the selectivity sequence Rb greater than Cs greater than K greater than Na. The alpha T264V mutant channel had the sequence Rb greater than K greater than Cs greater than Na. The alpha T264G mutant channel on the other hand had the same selectivity sequence as the wild type, but larger permeability ratios Px/PNa for the larger cations. Conductance concentration curves indicate that the effect of both mutations is to change both the maximum conductance as well as the apparent binding constant of the ions to the channel. A difference in Mg2+ sensitivity between wild-type and mutant channels, which is a consequence of the differences in ion binding, was also found. The present results suggest that alpha T264 form part of the selectivity filter of the AChR channel were large ions are selected according to their dehydrated size.     

Patch clamp studies of the amphibian nephron.

Transepithelial transport, intracellular ion activities and membrane potentials are all affected by changes in the conductive properties of the membranes of polarised epithelial cells. Conventional electrophysiological techniques have already determined the major conductances of the apical and basolateral membranes of the various nephron segments. These conductances are presently being studied at the molecular level with the aid of the patch clamp technique. In the case of the amphibian nephron, single-channel studies have been carried out in the proximal and early distal (diluting) segments. Almost all of the channels described so far have been selective for potassium, and the properties of these channels are described in this review. In addition, the basic electrophysiological and transport properties of these two general nephron segments are briefly described. From the physiological stand-point, the results of single-channel studies are providing us with information concerning the regulation of the conductances by intracellular mediators, allowing us to make predictions about the effects of various perturbations on cell membrane conductances. On the other hand, biophysical analysis is giving information ranging from the voltage dependence and ion selectivity of the channels to clues concerning their submicroscopic structure.     

VDAC structure, selectivity, and dynamics

VDAC channels exist in the mitochondrial outer membrane of all eukaryotic organisms. Of the different isoforms present in one organism, it seems that one of these is the canonical VDAC whose properties and 3D structure are highly conserved. The fundamental role of these channels is to control the flux of metabolites between the cytosol and mitochondrial spaces. Based on many functional studies, the fundamental structure of the pore wall consists of one α helix and 13 β strands tilted at a 46° angle. This results in a pore with an estimated internal diameter of 2.5nm. This structure has not yet been resolved. The published 3D structure consists of 19 β strands and is different from the functional structure that forms voltage-gated channels. The selectivity of the channel is exquisite, being able to select for ATP over molecules of the same size and charge. Voltage gating involves two separate gating processes. The mechanism involves the translocation of a positively charged portion of the wall of the channel to the membrane surface resulting in a reduction in pore diameter and volume and an inversion in ion selectivity. This mechanism is consistent with experiments probing changes in selectivity, voltage gating, kinetics and energetics. Other published mechanisms are in conflict with experimental results. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.     

Defining the factors that affect solute permeation of gap junction channels

This review focuses on the biophysical properties and structure of the pore and vestibule of homotypic gap junction channels as they relate to channel permeability and selectivity. Gap junction channels are unique in their sole role to connect the cytoplasm of two adjacent cells. In general, these channels are considered to be poorly selective, possess open probabilities approximating unity, and exhibit mean open times ranging from milliseconds to seconds. These properties suggest that such channels can function as delivery pathways from cell to cell for solutes that are significantly larger than monovalent ions. We have taken quantitative data from published works concerning unitary conductance, ion flux, and permeability for homotypic connexin 43 (Cx43), Cx40, Cx26, Cx50, and Cx37, and performed a comparative analysis of conductance and/or ion/solute flux versus diffusion coefficient. The analysis of monovalent cation flux portrays the pore as equivalent to an aqueous space where hydrogen bonding and weak interactions with binding sites dominate. For larger solutes, size, shape and charge are also significant components in determining the permeation rate. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Ion channels in smooth muscle: regulators of intracellular calcium and contractility

Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.     

Ankyrin-based cellular pathways for cardiac ion channel and transporter targeting and regulation

The coordinate activities of ion channels and transporters regulate myocyte membrane excitability and normal cardiac function. Dysfunction in cardiac ion channel and transporter function may result in cardiac arrhythmias and sudden cardiac death. While the past fifteen years have linked defects in ion channel biophysical properties with human disease, more recent findings illustrate that ion channel and transporter localization within cardiomyocytes is equally critical for normal membrane excitability and tissue function. Ankyrins are a family of multifunctional adapter proteins required for the expression, membrane localization, and regulation of select cardiac ion channels and transporters. Notably, loss of ankyrin expression in mice, and ankyrin loss-of-function in humans is now associated with defects in myocyte excitability and cardiac physiology. Here, we provide an overview of the roles of ankyrin polypeptides in cardiac physiology, as well as review other recently identified pathways required for the membrane expression and regulation of key cardiac ion channels and transporters.     

Computational recognition of potassium channel sequences

MOTIVATION: Potassium channels are mainly known for their role in regulating and maintaining the membrane potential. Since this is one of the key mechanisms of signal transduction, malfunction of these potassium channels leads to a wide variety of severe diseases. Thus potassium channels are priority targets of research for new drugs, despite the fact that this protein family is highly variable and closely related to other channels, which makes it very difficult to identify new types of potassium channel sequences. RESULTS: Here we present a new method for identifying potassium channel sequences (PSM, Property Signature Method), which-in contrast to the known methods for protein classification-is directly based on physicochemical properties of amino acids rather than on the amino acids themselves. A signature for the pore region including the selectivity filter has been created, representing the most common physicochemical properties of known potassium channels. This string enables genome-wide screening for sequences with similar features despite a very low degree of amino acid similarity within a protein family.     

Properties of gramicidin A channels in erythrocyte membranes

Studies for the cation permeability properties of the gramicidin A channel in erythrocyte membranes are presented. It is shown that gramicidin A interacts with the membrane in a cooperative manner, creating aggregates of the antibiotic molecules in the lipid lattice of the membrane. Cationic channels exist in these aggregates with the following order of selectivity: Rb+ greater than Cs+ greater K+ greater than Na+. The cation permeability of the channels depends on the media surrounding the membrane. This finding has been explained on the basis of Hodgkin-Keynes theory for single-file ion diffusion through extra-narrow pores.     

A novel technique to study pore-forming peptides in a natural membrane

The biophysical characteristics and the pore formation dynamics of synthetic or naturally occurring peptides forming membrane-spanning channels were investigated by using isolated photoreceptor rod outer segments (OS) recorded in whole-cell configuration. Once blocking the two OS endogenous conductances (the cGMP channels by light and the Na⁺:Ca²⁺,K⁺ exchanger by removing one of the transported ion species from both sides of the membrane, i.e. K⁺, Na⁺ or Ca²⁺), the OS membrane resistance (R _m ) was typically larger than 1 GΩ in the presence of 1 mM external Ca²⁺. Therefore, any exogenous current could be studied down to the single channel level. The peptides were applied to (and removed from) the extracellular OS side in ∼50 ms with a computer-controlled microperfusion system, in which every perfusion parameter, as the rate of solution flow, the temporal sequence of solution changes or the number of automatic, self-washing cycles were controlled by a user-friendly interface. This technique was then used to determine the biophysical properties and the pore formation dynamics of antibiotic peptaibols, as the native alamethicin mixture, the synthesized major component of the neutral fraction (F50/5) of alamethicin, and the synthetic trichogin GA IV.     

The purification of ion channels from excitable cells

Conclusion It should be emphasized that the problem of isolating a gene for which the gene product has been defined solely by genetic criteria is not trivial, but this approach is potentially very powerful. Not only does it provide a means for determining the sequence and structure of ion channels for which there are no biochemical probes, but it also provides a system in which specific mutations in the channel gene can be correlated with changes in channel function. Although this approach is limited to organisms that are readily manipulated by genetic methods, the results of these studies should be widely applicable. The biophysical properties of ion channels are generally similar from one species to another, suggesting that the structures of the channels have been highly conserved. The combination of biochemical, biophysical and genetic techniques will undoubtedly be exploited more fully in future studies of ion channel structure and function.     

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.     

Determinants of cation transport selectivity: Equilibrium binding and transport kinetics

ConclusionThe equilibrium ion-binding properties of ion channels and transporters can be difficult to discern from crystal structures alone, as proteins often adopt different lowest energy states depending on the ions bound. In cases where transport is slow, their inherent ion-binding preferences can be used to infer their transport preferences. However, in cases where transport is fast, the transport selectivity can hide their equilibrium preferences by accentuating the kinetics of ions hopping through a channel over its inherent ion-binding preferences. Thus, depending on the arrangement of ion-binding sites in a channel’s selectivity filter, one can achieve either selective or nonselective ion transport.The equilibrium K⁺ selectivity of some nonselective channels suggests a potential mechanism whereby they could evolve into a fast K⁺-selective channel. K⁺ channels and nonselective channels like CNG and HCN are related to one another in both sequence and structure, suggesting an evolutionary link between them. Swap experiments show that only a few mutations separate a nonselective channel from a K⁺-selective channel. One might imagine an evolutionary path between these channels in which the equilibrium preference for a K⁺ ion in a nonselective channel evolves into a K⁺-selective channel through these few mutations to create the selective ion queue. Alternatively, a slow single-ion channel with an equilibrium and transport preference for K⁺ ions could be transformed into a fast multi-ion channel through mutations that create a queue of K⁺-selective ion-binding sites, as is seen in most K⁺ channels studied to date.In the case of multi-ion selectivity filters, such as those found in K⁺ channels, the selectivity filter can be viewed as the active site that interacts with different queues of ions and water molecules. At least three properties emerge from multi-ion queues: (1) high conductance by reducing the affinity of multiple bound ions versus single ions; (2) high selectivity by allowing disfavored ions time to dissociate back into solution; and, consequently, (3) robust selectivity in an environment where ion concentrations can change. For transporters and carriers, the equilibrium preference and slow transport naturally create robust selectivity. In all these cases, equilibrium-based ion selectivity is achieved by slowing transport enough so that the disfavored ion is able to dissociate back into solution before transport takes place.     

Use of optical biosensors to detect modulation of Slack potassium channels by G protein-coupled receptors

Ion channels control the electrical properties of neurons and other excitable cell types by selectively allowing ion to flow through the plasma membrane. To regulate neuronal excitability, the biophysical properties of ion channels are modified by signaling proteins and molecules, which often bind to the channels themselves to form a heteromeric channel complex. Traditional assays examining the interaction between channels and regulatory proteins generally provide little information on the time-course of interactions in living cells. We have now used a novel label-free technology to detect changes in the distribution of mass close to the plasma membrane following modulation of potassium channels by G protein-coupled receptors (GPCRs). This technology uses optical sensors embedded in microplates to detect changes in the refractive index at the surface of cells. Although the activation of GPCRs has been studied with this system, protein-protein interactions due to modulation of ion channels have not yet been characterized. Here we present data that the characteristic pattern of mass distribution following GPCR activation is significantly modified by the presence of a sodium-activated potassium channel, Slack-B, a channel that is known to be potently modulated by activation of these receptors.     

The M1 and pre-M1 segments contribute differently to ion selectivity in ASICs and ENaCs

The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid–based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.     

Two decades with dimorphic Chloride Intracellular Channels (CLICs)

Plasma membrane channels have been extensively studied, and their physiological roles are well established. In contrast, relatively little information is available about intracellular ion channels. Chloride Intracellular Channel (CLICs) proteins are a novel class of putative intracellular ion channels. They are widely expressed in different intracellular compartments, and possess distinct properties such as the presence of a single transmembrane domain, and a dimorphic existence as either a soluble or membranous form. How these soluble proteins unfold, target to, and auto-insert into the intracellular membranes to form functional integral ion channels is a complex biological question. Recent information from studies of their crystal structures, biophysical characterization and functional roles has provoked interest in these unusual channels.     

Isolation and characterization of I(Kr) in cardiac myocytes by Cs+ permeation

Isolation of the rapidly activating delayed rectifier potassium current (I(Kr)) from other cardiac currents has been a difficult task for quantitative study of this current. The present study was designed to separate I(Kr) using Cs+ in cardiac myocytes. Cs+ have been known to block a variety of K+ channels, including many of those involved in the cardiac action potential such as inward rectifier potassium current I(K1) and the transient outward potassium current I(to). However, under isotonic Cs+ conditions (135 mM Cs+), a significant membrane current was recorded in isolated rabbit ventricular myocytes. This current displayed the voltage-dependent onset of and recovery from inactivation that are characteristic to I(Kr). Consistently, the current was selectively inhibited by the specific I(Kr) blockers. The biophysical and pharmacological properties of the Cs+-carried human ether-a-go-go-related gene (hERG) current were very similar to those of the Cs+-carried I(Kr) in ventricular myocytes. The primary sequence of the selectivity filter in hERG was in part responsible for the Cs+ permeability, which was lost when the sequence was changed from GFG to GYG, characteristic of other, Cs+-impermeable K+ channels. Thus the unique high Cs+ permeability in I(Kr) channels provides an effective way to isolate I(Kr) current. Although the biophysical and pharmacological properties of the Cs+-carried I(Kr) are different from those of the K+-carried I(Kr), such an assay enables I(Kr) current to be recorded at a level that is large enough and sufficiently robust to evaluate any I(Kr) alterations in native tissues in response to physiological or pathological changes. It is particularly useful for exploring the role of reduction of I(Kr) in arrhythmias associated with heart failure and long QT syndrome due to the reduced hERG channel membrane expression.