Plants do it differently. A new basis for potassium/sodium selectivity in the pore of an ion channel

Understanding of the molecular architecture necessary for selective K(+) permeation through the pore of ion channels is based primarily on analysis of the crystal structure of the bacterial K(+) channel KcsA, and structure:function studies of cloned animal K(+) channels. Little is known about the conduction properties of a large family of plant proteins with structural similarities to cloned animal cyclic nucleotide-gated channels (CNGCs). Animal CNGCs are nonselective cation channels that do not discriminate between Na(+) and K(+) permeation. These channels all have the same triplet of amino acids in the channel pore ion selectivity filter, and this sequence is different from that of the selectivity filter found in K(+)-selective channels. Plant CNGCs have unique pore selectivity filters; unlike those found in any other family of channels. At present, the significance of the unique pore selectivity filters of plant CNGCs, with regard to discrimination between Na(+) and K(+) permeation is unresolved. Here, we present an electrophysiological analysis of several members of this protein family; identifying the first cloned plant channel (AtCNGC1) that conducts Na(+). Another member of this ion channel family (AtCNGC2) is shown to have a selectivity filter that provides a heretofore unknown molecular basis for discrimination between K(+) and Na(+) permeation. Specific amino acids within the AtCNGC2 pore selectivity filter (Asn-416, Asp-417) are demonstrated to facilitate K(+) over Na(+) conductance. The selectivity filter of AtCNGC2 represents an alternative mechanism to the well-known GYG amino acid triplet of K(+) channels that has been identified as the critical basis for K(+) over Na(+) permeation through the pore of ion channels.     

Single channel analysis of conductance and rectification in cation-selective, mutant glycine receptor channels

Members of the ligand-gated ion channel superfamily mediate fast synaptic transmission in the nervous system. In this study, we investigate the molecular determinants and mechanisms of ion permeation and ion charge selectivity in this family of channels by characterizing the single channel conductance and rectification of alpha1 homomeric human glycine receptor channels (GlyRs) containing pore mutations that impart cation selectivity. The A-1'E mutant GlyR and the selectivity double mutant ([SDM], A-1'E, P-2' Delta) GlyR, had mean inward chord conductances (at -60 mV) of 7 pS and mean outward conductances of 11 and 12 pS (60 mV), respectively. This indicates that the mutations have not simply reduced anion permeability, but have replaced the previous anion conductance with a cation one. An additional mutation to neutralize the ring of positive charge at the extracellular mouth of the channel (SDM+R19'A GlyR) made the conductance-voltage relationship linear (14 pS at both 60 and -60 mV). When this external charged ring was made negative (SDM+R19'E GlyR), the inward conductance was further increased (to 22 pS) and now became sensitive to external divalent cations (being 32 pS in their absence). The effects of the mutations to the external ring of charge on conductance and rectification could be fit to a model where only the main external energy barrier height for permeation was changed. Mean outward conductances in the SDM+R19'A and SDM+R19'E GlyRs were increased when internal divalent cations were absent, consistent with the intracellular end of the pore being flanked by fixed negative charges. This supports our hypothesis that the ion charge selectivity mutations have inverted the electrostatic profile of the pore by introducing a negatively charged ring at the putative selectivity filter. These results also further confirm the role of external pore vestibule electrostatics in determining the conductance and rectification properties of the ligand-gated ion channels.     

Molecular Basis for Cation Selectivity in Claudin-2–based Paracellular Pores: Identification of an Electrostatic Interaction Site

Paracellular ion transport in epithelia is mediated by pores formed by members of the claudin family. The degree of selectivity and the molecular mechanism of ion permeation through claudin pores are poorly understood. By expressing a high-conductance claudin isoform, claudin-2, in high-resistance Madin-Darby canine kidney cells under the control of an inducible promoter, we were able to quantitate claudin pore permeability. Claudin-2 pores were found to be narrow, fluid filled, and cation selective. Charge selectivity was mediated by the electrostatic interaction of partially dehydrated permeating cations with a negatively charged site within the pore that is formed by the side chain carboxyl group of aspartate-65. Thus, paracellular pores use intrapore electrostatic binding sites to achieve a high conductance with a high degree of charge selectivity.     

Examination of subconductance levels arising from a single ion channel.

Single-channel records often show frequent currents at a main conductance level and occasional currents at subconductance levels. In some instances, the conductances occur at regular levels that are multiples of a minimum conductance. It is well-appreciated that multiple conductance levels may arise either from the co-operative gating of more than one pore or from changes that occur in a single pore. In this paper, we used theoretical models of ion permeation to examine subconductances arising in a single-pore channel. In particular, the work focuses on the following question: how can an ion channel that provides only one aqueous pore through the membrane produce regular subconductances and a main conductance that all have the same selectivity and the same ion binding affinity? The three types of ion permeation models used in this study showed that a single-pore channel can have subconductances because of long-lived conformational states, because of alterations in rapid fluctuations between conformational states, or because of slight alterations in the electrostatic properties in the channel's entrance vestibules. Regular subconductances with the same selectivity and binding affinity can arise in a single pore even if the energy profile changes do not meet the constant peak offset condition. The results show that the appearance of regular subconductance levels in a single-channel recording is not sufficient evidence to conclude that identical pores have co-operative gating, as would arise in a channel that is a multi-pore complex.     

Modeling ion permeation through batrachotoxin-modified Na+ channels from rat skeletal muscle with a multi-ion pore.

The mechanism of ion permeation through Na+ channels that have been modified by batrachotoxin (BTX) and inserted into planar bilayers has been generally described by models based on single-ion occupancy, with or without an influence of negative surface charge, depending on the tissue source. For native Na+ channels there is evidence suggestive of a multi-ion conduction mechanism. To explore the question of ion occupancy, we have reexamined permeation of Na+, Li+, and K+ through BTX-modified Na+ channels from rat skeletal muscle. Single-channel current-voltage (I-V) behavior was studied in neutral lipid bilayers in the presence of symmetrical Na+ concentrations ranging from 0.5 to 3,000 mM. The dependence of unitary current on the mole fraction of Na+ was also examined in symmetrical mixtures of Na(+)-Li+ and Na(+)-K+ at a constant total ionic strength of 206 and 2,006 mM. The dependence of unitary conductance on symmetrical Na+ concentration does not exhibit Michaelis-Menten behavior characteristic of single-ion occupancy but can be simulated by an Eyring-type model with three barriers and two sites (3B2S) that includes double occupancy and ion-ion repulsion. Best-fit energy barrier profiles for Na+, Li+, and K+ were obtained by nonlinear curve fitting of I-V data using the 3B2S model. The Na(+)-Li+ and Na(+)-K+ mole-fraction experiments do not exhibit an anomalous mole-fraction effect. However, the 3B2S model is able to account for the biphasic dependence of unitary conductance on symmetrical [Na+] that is suggestive of multiple occupancy and the monotonic dependence of unitary current on the mole fraction of Na+ that is compatible with single or multiple occupancy. The best-fit 3B2S barrier profiles also successfully predict bi-ionic reversal potentials for Na(+)-Li+ and Na(+)-K+ in both orientations across the channel. Our experimental and modeling results reconcile the dual personality of ion permeation through Na+ channels, which can display features of single or multiple occupancy under various conditions. To a first approximation, the 3B2S model developed for this channel does not require corrections for vestibule surface charge. However, if negative surface charges of the protein do influence conduction, the conductance behavior in the limit of low [Na+] does not correspond to a Gouy-Chapman model of planar surface charge.     

Breaking the Hydrophobicity of the MscL Pore: Insights into a Charge-Induced Gating Mechanism

The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.     

Pathways and Barriers for Ion Translocation through the 5-HT3A Receptor Channel

Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT₃) or anion selective (e.g., GlyR, GABA_A and GluCl) membrane channels. Among others, 5-HT₃ is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT₃ is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT₃A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT₃A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT₃A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT₃A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested.     

Crucial points within the pore as determinants of K⁺ channel conductance and gating

While selective for K⁺, K⁺ channels vary significantly among their rate of ion permeation. Here, we probe the effect of steric hindrance and electrostatics within the ion conduction pathway on K⁺ permeation in the MthK K⁺ channel using structure-based mutagenesis combined with single-channel electrophysiology and X-ray crystallography. We demonstrate that changes in side-chain size and polarity at Ala88, which forms the constriction point of the open MthK pore, have profound effects on single-channel conductance as well as open probability. We also reveal that the negatively charged Glu92s at the intracellular entrance of the open pore form an electrostatic trap, which stabilizes a hydrated K⁺ and facilitates ion permeation. This electrostatic attraction is also responsible for intracellular divalent blockage, which renders the channel inward rectified in the presence of Ca²⁺. In light of the high structural conservation of the selectivity filter, the size and chemical environment differences within the portion of the ion conduction pathway other than the filter are likely the determinants for the conductance variations among K⁺ channels.     

Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.     

Brownian dynamics simulations of ion transport through the VDAC

It is important to gain a physical understanding of ion transport through the voltage-dependent anion channel (VDAC) because this channel provides primary permeation pathways for metabolites and electrolytes between the cytosol and mitochondria. We performed grand canonical Monte Carlo/Brownian dynamics (GCMC/BD) simulations to explore the ion transport properties of human VDAC isoform 1 (hVDAC1; PDB:2K4T) embedded in an implicit membrane. When the MD-derived, space-dependent diffusion constant was used in the GCMC/BD simulations, the current-voltage characteristics and ion number profiles inside the pore showed excellent agreement with those calculated from all-atom molecular-dynamics (MD) simulations, thereby validating the GCMC/BD approach. Of the 20 NMR models of hVDAC1 currently available, the third one (NMR03) best reproduces both experimental single-channel conductance and ion selectivity (i.e., the reversal potential). In addition, detailed analyses of the ion trajectories, one-dimensional multi-ion potential of mean force, and protein charge distribution reveal that electrostatic interactions play an important role in the channel structure and ion transport relationship. Finally, the GCMC/BD simulations of various mutants based on NMR03 show good agreement with experimental ion selectivity. The difference in ion selectivity between the wild-type and the mutants is the result of altered potential of mean force profiles that are dominated by the electrostatic interactions.     

Influence of pore residues on permeation properties in the Kv2.1 potassium channel. Evidence for a selective functional interaction of K+ with the outer vestibule

The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external [K(+)]. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K(+) and Na(+) permeation. Permeability ratio measurements, measurements of the lowest [K(+)] required for interaction with the selectivity filter, and measurements of macroscopic K(+) and Na(+) conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K(+) to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K(+) permeation properties, Lys 356 appeared to be without effect on Na(+) permeation. This suggests that Lys 356 limited K(+) flux by interfering with a selective K(+) binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K(+) permeation properties. Together, these results indicate that a selective, functional K(+) binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K(+) channels. We suggest that this site is the location of K(+) hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K(+) current magnitude, but did not influence the ability of internal K(+) to enter the pore. These data indicate that in Kv2.1, exit of K(+) from the selectivity filter, rather than entry of internal K(+) into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K(+) channels.     

Molecular dynamics - potential of mean force calculations as a tool for understanding ion permeation and selectivity in narrow channels

Ion channels catalyze the permeation of charged molecules across cell membranes and are essential for many vital physiological functions, including nerve and muscle activity. To understand better the mechanisms underlying ion conduction and valence selectivity of narrow ion channels, we have employed free energy techniques to calculate the potential of mean force (PMF) for ion movement through the prototypical gramicidin A channel. Employing modern all-atom molecular dynamics (MD) force fields with umbrella sampling methods that incorporate one hundred 1-2 ns trajectories, we find that it is possible to achieve semi-quantitative agreement with experimental binding and conductance measurements. We also examine the sensitivity of the MD-PMF results to the choice of MD force field and compare PMFs for potassium, calcium and chloride ions to explore the basis for the valence selectivity of this narrow and uncharged ion channel. A large central barrier is observed for both anions and divalent ions, consistent with lack of experimental conductance. Neither anion or divalent cation is seen to be stabilized inside the channel relative to the bulk electrolyte and each leads to large disruptions to the protein and membrane structure when held deep inside the channel. Weak binding of calcium ions outside the channel corresponds to a free energy well that is too shallow to demonstrate channel blocking. Our findings emphasize the success of the MD-PMF approach and the sensitivity of ion energetics to the choice of biomolecular force field.     

Conformational model for ion permeation in membrane channels: a comparison with multi-ion models and applications to calcium channel permeability.

The permeation properties of ion channels existing in several conductive states were analyzed. Each state was represented by the one-ion model. A special emphasis was placed on features, assumed to be indicative of a multi-ion mode of channel occupancy such as a deviation of concentration dependence of channel conductance from the Michaelis-Menten equation, an anomalous mole fraction effect, a strong voltage dependence of ion block and coupling of unidirectional fluxes (anomalous Ussing flux ratio). The conformational model was shown to have all these properties. The ion permeation through voltage-sensitive calcium channels fulfilled all the characteristics of the model proposed.     

Control of ion conduction in L-type Ca2+ channels by the concerted action of S5-6 regions

Voltage-gated L-type Ca(2+) channels from cardiac (alpha(1C)) and skeletal (alpha(1S)) muscle differ from one another in ion selectivity and permeation properties, including unitary conductance. In 110 mM Ba(2+), unitary conductance of alpha(1S) is approximately half that of alpha(1C). As a step toward understanding the mechanism of rapid ion flux through these highly selective ion channels, we used chimeras constructed between alpha(1C) and alpha(1S) to identify structural features responsible for the difference in conductance. Combined replacement of the four pore-lining P-loops in alpha(1C) with P-loops from alpha(1S) reduced unitary conductance to a value intermediate between those of the two parent channels. Combined replacement of four larger regions that include sequences flanking the P-loops (S5 and S6 segments along with the P-loop-containing linker between these segments (S5-6)) conferred alpha(1S)-like conductance on alpha(1C). Likewise, substitution of the four S5-6 regions of alpha(1C) into alpha(1S) conferred alpha(1C)-like conductance on alpha(1S). These results indicate that, comparing alpha(1C) with alpha(1S), the differences in structure that are responsible for the difference in ion conduction are housed within the S5-6 regions. Moreover, the pattern of unitary conductance values obtained for chimeras in which a single P-loop or single S5-6 region was replaced suggest a concerted action of pore-lining regions in the control of ion conduction.     

Synthetic nanopores as a test case for ion channel theories: the anomalous mole fraction effect without single filing

The predictions of a theory for the anomalous mole fraction effect (AMFE) are tested experimentally with synthetic nanopores in plastic. The negatively charged synthetic nanopores under consideration are highly cation selective and 50 Å in diameter at their smallest point. These pores exhibit an AMFE in mixtures of Ca²⁺ and monovalent cations. An AMFE occurs when the conductance through a pore is lower in a mixture of salts than in the pure salts at the same concentration. For ion channels, the textbook interpretation of the AMFE is that multiple ions move through the pore in coordinated, single-file motion. However, because the synthetic nanopores are so wide, their AMFE shows that single filing is not necessary for the AMFE. It is shown that the AMFE in the synthetic nanopores is explained by a theory of preferential ion selectivity. The unique properties of the synthetic nanopores allow us to experimentally confirm several predictions of this theory. These same properties make synthetic nanopores an interesting new platform to test theories of ion channel permeation and selectivity in general.     

Ion-channel entrances influence permeation. Net charge, size, shape, and binding considerations.

Many ion channels have wide entrances that serve as transition zones to the more selective narrow region of the pore. Here some physical features of these vestibules are explored. They are considered to have a defined size, funnel shape, and net-negative charge. Ion size, ionic screening of the negatively charged residues, cation binding, and blockage of current are analyzed to determine how the vestibules influence transport. These properties are coupled to an Eyring rate theory model for the narrow length of the pore. The results include the following: Wide vestibules allow the pore to have a short narrow region. Therefore, ions encounter a shorter length of restricted diffusion, and the channel conductance can be greater. The potential produced by the net-negative charge in the vestibules attracts cations into the pore. Since this potential varies with electrolyte concentration, the conductance measured at low electrolyte concentrations is larger than expected from measurements at high concentrations. Net charge inside the vestibules creates a local potential that confers some cation vs. anion, and divalent vs. monovalent selectivity. Large cations are less effective at screening (diminishing) the net-charge potential because they cannot enter the pore as well as small cations. Therefore, at an equivalent bulk concentration the attractive negative potential is larger, which causes large cations to saturate sites in the pore at lower concentrations. Small amounts of large or divalent cations can lead to misinterpretation of the permeation properties of a small monovalent cation.     

Novel Residues Lining the CFTR Chloride Channel Pore Identified by Functional Modification of Introduced Cysteines

Substituted cysteine accessibility mutagenesis (SCAM) has been used widely to identify pore-lining amino acid side chains in ion channel proteins. However, functional effects on permeation and gating can be difficult to separate, leading to uncertainty concerning the location of reactive cysteine side chains. We have combined SCAM with investigation of the charge-dependent effects of methanethiosulfonate (MTS) reagents on the functional permeation properties of cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channels. We find that cysteines substituted for seven out of 21 continuous amino acids in the eleventh and twelfth transmembrane (TM) regions can be modified by external application of positively charged [2-(trimethylammonium)ethyl] MTS bromide (MTSET) and negatively charged sodium [2-sulfonatoethyl] MTS (MTSES). Modification of these cysteines leads to changes in the open channel current–voltage relationship at both the macroscopic and single-channel current levels that reflect specific, charge-dependent effects on the rate of Cl⁻ permeation through the channel from the external solution. This approach therefore identifies amino acid side chains that lie within the permeation pathway. Cysteine mutagenesis of pore-lining residues also affects intrapore anion binding and anion selectivity, giving more information regarding the roles of these residues. Our results demonstrate a straightforward method of screening for pore-lining amino acids in ion channels. We suggest that TM11 contributes to the CFTR pore and that the extracellular loop between TMs 11 and 12 lies close to the outer mouth of the pore.     

Non-Equilibrium Dynamics Contribute to Ion Selectivity in the KcsA Channel

The ability of biological ion channels to conduct selected ions across cell membranes is critical for the survival of both animal and bacterial cells. Numerous investigations of ion selectivity have been conducted over more than 50 years, yet the mechanisms whereby the channels select certain ions and reject others are not well understood. Here we report a new application of Jarzynski’s Equality to investigate the mechanism of ion selectivity using non-equilibrium molecular dynamics simulations of Na⁺ and K⁺ ions moving through the KcsA channel. The simulations show that the selectivity filter of KcsA adapts and responds to the presence of the ions with structural rearrangements that are different for Na⁺ and K⁺. These structural rearrangements facilitate entry of K⁺ ions into the selectivity filter and permeation through the channel, and rejection of Na⁺ ions. A mechanistic model of ion selectivity by this channel based on the results of the simulations relates the structural rearrangement of the selectivity filter to the differential dehydration of ions and multiple-ion occupancy and describes a mechanism to efficiently select and conduct K⁺. Estimates of the K⁺/Na⁺ selectivity ratio and steady state ion conductance for KcsA from the simulations are in good quantitative agreement with experimental measurements. This model also accurately describes experimental observations of channel block by cytoplasmic Na⁺ ions, the “punch through” relief of channel block by cytoplasmic positive voltages, and is consistent with the knock-on mechanism of ion permeation.     

Separation and characterization of currents through store-operated CRAC channels and Mg2+-inhibited cation (MIC) channels

Although store-operated calcium release-activated Ca(2+) (CRAC) channels are highly Ca(2+)-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na(+) and Cs(+) with a unitary conductance of approximately 40 pS, and that intracellular Mg(2+) modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg(2+)-inhibited cation (MIC) channels that open as Mg(2+) is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8-10 mM intracellular Mg(2+) to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca(2+) to divalent-free extracellular solution evokes Na(+) current through open CRAC channels (Na(+)-I(CRAC)) that is initially eightfold larger than the preceding Ca(2+) current and declines by approximately 80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs(+) (P(Cs)/P(Na) = 0.13 vs. 1.2 for MIC). Neither the decline in Na(+)-I(CRAC) nor its low Cs(+) permeability are affected by intracellular Mg(2+) (90 microM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca(2+) channels.     

Potassium channel of cardiac sarcoplasmic reticulum is a multi-ion channel

We have characterized mechanisms of ionic permeation in the K channel of canine cardiac sarcoplasmic reticulum (SR K channel). Ionic selectivity, as measured by relative permeabilities, followed Eisenman sequence l, a low field strength sequence. Slope conductance measured in symmetrical solutions across the bilayer followed Eisenman sequence V. In all cases, the selectivity characteristics of the prominent subconductance state (O1) were similar to those of the main-state (O2). Further, our studies have revealed that this channel differs in three significant ways from the highly characterized SR K channel of skeletal muscle. First, the ratio of permeabilities Cs+ to K+ was a complex function of ion concentration. Second, the concentration dependence of conductance was not well described by the Michaelis-Menten formalism. Instead, we modeled the observed relations using a more general approach based on classical rate theory. Third, mole fraction experiments (Cs+ with K+) demonstrated a prominent anomalous effect. Certain of our Cs+ data required the Eyring rate theory approach for adequate interpretation. We adopted a symmetrical energy profile incorporating ion-ion interaction and thereby accounted for much of the data. We conclude that the canine cardiac SR K channel is significantly different from that of skeletal muscle, and it may accommodate more than one ion at a time.