Correlated ion flux through parallel pores: Application to channel subconductance states

Summary Many ion channels that normally gate fully open or shut have recently been observed occasionally to display well-defined subconductance states with conductances much less than those of the fully open channel. One model of this behavior is a channel consisting of several parallel pores with a strong correlation between the flux in each pore such that, normally, they all conduct together but, under special circumstances, the pores may transfer to a state in which only some of them conduct. This paper introduces a general technique for modeling correlated pores, and explores in detail by computer simulation a particular model based upon electric interaction between the pores. Correlation is obtained when the transient electric field of ions passing through the pores acts upon a common set of ionizable residues of the channel protein, causing transient changes in their effective pK and hence in their charged state. The computed properties of such a correlated parallel pore channel with single occupation of each pore are derived and compared to those predicted for a single pore that can contain more than one ion at a time and also to those predicted for a model pore with fluctuating barriers. Experiments that could distinguish between the present and previous models are listed.R.M.B. is grateful to the S.E.R.C. for the award of a graduate studentship.     

A molecular model of the inner pore of the Ca channel in its open state

Structure of the Ca channel open pore is unlikely to be the same as that of the K channel because Ca channels do not contain the hinge residues Gly or Pro. The Ca channel does not have a wide entry into the inner pore, as is found in K channels. First we sought to simulate the open state of the Ca channel by modeling forced opening of the KcsA channel using a procedure of restrained minimization with distance constraints at the level of the α-helical bundle, corresponding to segments Thr-107-Val-115. This produced an intermediate open state, which was populated by amino acid residues of Ca channels and then successively optimized until the opening of the pore reached a diameter of about 10 Å, large enough to allow verapamil to enter and block the Ca channel from inside. Although this approach produced a sterically plausible structure, it was in significant disagreement with the MTSET accessibility data for single cysteine mutations of S6 segments of the P/Q channel¹ that do not fit with an α-helical pattern. Last we explored the idea that the four S6 segments of Ca channels may contain intra-molecular deformations that lead to reorientation of its side chains. After introduction of π-bulges, the model agreed with the MTSET accessibility data. MTSET modification of a cysteine at the C-end of only one S6 could produce physical occlusion and block of the inner pore of the open Ca channel, as observed experimentally, and as expected if the pore opening is narrower than that of K channels.Key words: calcium channels, homology modeling, π-bulges, restrained minimization     

Structural and functional role of the extracellular s5-p linker in the HERG potassium channel

C-type inactivation in the HERG channel is unique among voltage-gated K channels in having extremely fast kinetics and strong voltage sensitivity. This suggests that HERG may have a unique outer mouth structure (where conformational changes underlie C-type inactivation), and/or a unique communication between the outer mouth and the voltage sensor. We use cysteine-scanning mutagenesis and thiol-modifying reagents to probe the structural and functional role of the S5-P (residues 571-613) and P-S6 (residues 631-638) linkers of HERG that line the outer vestibule of the channel. Disulfide formation involving introduced cysteine side chains or modification of side chain properties at "high-impact" positions produces a common mutant phenotype: disruption of C-type inactivation, reduction of K+ selectivity, and hyperpolarizing shift in the voltage-dependence of activation. In particular, we identify 15 consecutive positions in the middle of the S5-P linker (583-597) where side chain modification has marked impact on channel function. Analysis of the degrees of mutation-induced perturbation in channel function along 583-597 reveals an alpha-helical periodicity. Furthermore, the effects of MTS modification suggest that the NH2-terminal of this segment (position 584) may be very close to the pore entrance. We propose a structural model for the outer vestibule of the HERG channel, in which the 583-597 segment forms an alpha-helix. With the NH2 terminus of this helix sitting at the edge of the pore entrance, the length of the helix (approximately 20 A) allows its other end to reach and interact with the voltage-sensing domain. Therefore, the "583-597 helix" in the S5-P linker of the HERG channel serves as a bridge of communication between the outer mouth and the voltage sensor, that may make important contribution to the unique C-type inactivation phenotype.     

A comparison of sodium channel kinetics in the squid axon,the frog node and the frog node with BTX using the “silent gate” model

In this paper it is shown that the very different kinetics measured for the rise of the sodium current which follows a depolarization of the membrane in the squid giant axon, the frog node and the frog node treated with Batrachotoxin may be accurately predicted using only the measured equilibrium and static characteristics for the three preparations and the kinetics measured for the gating charge transfer.The kinetic predictions follow the use of the silent gate model for ion channel gating. The model is electrostatic and its chief assumptions are that the channel gate, called here the N-system, has fast kinetics and responds to the gating charge that transfers but not directly to the trans-membrane voltage applied. Because channel gating, corresponding here to the motion of the N-system, does not change its energy in the trans-membrane applied electric field the gating is electrically silent as far as gating charge transfer measurement is concerned. However the probability of gating rises with the quantity of gating charge that transfers due to the electrostatic interaction between the N-system and the gating charge, redistributed under the influence of the applied trans-membrane electric field. With these assumptions the kinetics of sodium channel gating are predictable using only the static and equilibrium characteristics of gating charge and channel activation measured as a function of membrane voltage, and the kinetics of the gating charge transfer. Because of the fast kinetics assumed for the N-system the predicted kinetics are the same for channels with any number of equivalent and independent N-systems or gates acting in parallel.The model predictions for sodium permeability kinetics are compared in detail with those recently measured for the frog node treated with Batrachotoxin and excellent agreement is obtained.     

Mechanism of luminal Ca<Superscript>2+</Superscript> and Mg<Superscript>2+</Superscript> action on the vacuolar slowly activating channels

The non-selective slow vacuolar (SV) channel can dominate tonoplast conductance, making it necessary to tightly control its activity. Applying the patch-clamp technique to vacuoles from sugar beet (Beta vulgaris L.) taproots we studied the effect of divalent cations on the vacuolar side of the SV channel. Our results show that the SV channel has two independent binding sites for vacuolar divalent cations, (i) a less selective one, inside the channel pore, binding to which impedes channel conductance, and (ii) a Ca²⁺-selective one outside the membrane-spanning part of the channel protein, binding to which stabilizes the channels closed conformations. Vacuolar Ca²⁺ and Mg²⁺ almost indiscriminately blocked ion fluxes through the open channel pore, decreasing measured single-channel current amplitudes. This low-affinity block displays marked voltage dependence, characteristic of a permeable blocker. Vacuolar Ca²⁺—with a much higher affinity than Mg²⁺—slows down SV channel activation and shifts the voltage dependence to more (cytosol) positive potentials. A quantitative analysis results in a model that exactly describes the Ca²⁺-specific effects on the SV channel activation kinetics and voltage gating. According to this model, multiple (approximately three) divalent cations bind with a high affinity at the luminal interface of the membrane to the channel protein, favoring the occupancy of one of the SV channels closed states (C2). Transition to another closed state (C1) diminishes the effective number of bound cations, probably due to mutual repulsion, and channel opening is accompanied by a decrease of binding affinity. Hence, the open state (O) is destabilized with respect to the two closed states, C1 and C2, in the presence of Ca²⁺ at the vacuolar side. The specificity for Ca²⁺ compared to Mg²⁺ is explained in terms of different binding affinities for these cations. In this study we demonstrate that vacuolar Ca²⁺ is a crucial regulator to restrict SV channel activity to a physiologically meaningful range, which is less than 0.1% of maximum SV channel activity.Abbreviation SV Slow vacuolar     

Computational Observation of an Ion Permeation Through a Channel Protein

The ion permeation process, driven by a membrane potential through an outer membrane protein, OmpF porin of Escherichia coli, was simulated by molecular dynamics. A Na⁺ ion, initially placed in the solvent region at the outer side of the porin channel, moved along the electric field passing through the porin channel in a 1.3 nsec simulation; the permeation rate was consistent with the experimentally estimated channel activity (10⁸10⁹/sec). In this simulation, it was indicated that the ion permeation through the porin channel proceeds by a push-out mechanism, and that Asp113 is an important residue for the channel activity.     

Size and selectivity of gap junction channels formed from different connexins

Gap junction channels have long been viewed as static structures containing a large-diameter, aqueous pore. This pore has a high permeability to hydrophilic molecules of 900 daltons in molecular weight and a weak ionic selectivity. The evidence leading to these conclusions is reviewed in the context of more recent observations primarily coming from unitary channel recordings from transfected connexin channels expressed in communication-deficient cell lines. What is emerging is a more diverse view of connexin-specific gap junction channel structure and function where electrical conductance, ionic selectivity, and dye permeability vary by one full order of magnitude or more. Furthermore, the often held contention that channel conductance and ionic or molecular selectivity are inversely proportional is refuted by recent evidence from five distinct connexin channels. The molecular basis for this diversity of channel function remains to be identified for the connexin family of gap junction proteins.     

Ion channels in the plasma membrane ofAmaranthus protoplasts: One cation and one anion channel dominate the conductance

Summary This report details preliminary findings for ion channels in the plasma membrane of protoplasts derived from the cotyledons ofAmaranthus seedlings. The conductance properties of the membrane can be described almost entirely by the behavior of two types of ion channel observed as single channels in attached and detached patches. The first is a cation-selective outward rectifier, and the second a multistate anion-selective channel which, under physiological conditions, acts as an inward rectifier.The cation channel has unit conductance of approx. 30 pS (symmetrical 100 K⁺) and relative permeability sequence K⁺>Na⁺>Cl^– (10.160.03); whole-cell currents activate in a time-dependent manner, and both activation and deactivation kinetics are voltage dependent. The anion channel opens for hyperpolarized membrane potentials, has a full-level conductance of approx. 200 pS and multiple subconductance states. The number of sub-conductances does not appear to be fixed. When activated the channel is open for long periods, though shuts if the membrane potential (V _m ) is depolarized; at millimolar levels of [Ca²⁺]_cyt this voltage dependency disappears. Inward current attributable to the anion channel is not observed in whole-cell recordings when MgATP (2mm) is present in the intracellular solution. By contrast the channel is active in most detached patches, whether MgATP is present or not on the cytoplasmic face of the membrane. The anion channel has a significant permeability to cations, the sequence being NO ₃ ^– >Cl^–>K⁺>Aspartate (2.0410.18 to 0.090.04). The relative permeability for K⁺ decreased at progressively lower conductance states. In the absence of permeant anions this channel could be mistaken for a cation inward rectifier. The anion and cation channels could serve to clampV _m at a preferred value in the face of events which would otherwise perturbV _m .     

Alkali metal ion selectivity of the hemocyanin channel

Summary The selectivity of the hemocyanin channel was measured for alkali metal ions and ammonium. Permeability ratios relative to K⁺ measured from biionic potentials were: NH ₄ ⁺ (1.52)>Rb⁺ (1.05)>K⁺ (1.0)>Cs⁺ (0.89)>Na⁺ (0.81)>Li⁺ (0.35). Single-channel ion conductance was a saturating function of ion concentration regardless of the cation present in the bathing medium. Maximal conductances were 270, 267, 215, 176, 170 and 37 ps for K⁺, Rb⁺, NH ₄ ⁺ , Cs⁺, Na⁺ and Li⁺, respectively. Current-voltage curves for the different monovalent cations were measured and described using a threebarrier model previously used to explain the voltage dependence of the instantaneous channel conductance (Cecchi, Alvarez & Latorre, 1981). In this way, binding and peak energies were estimated for the different ions. Considering the energy peaks as transition states between the ion and the channel, it is concluded that they follow Eisenman's selectivity sequences XI (cis peak, i.e., Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺; highest field strength), VII (central peak) and II (trans peak). The cis side was that to which hemocyanin was added and was electrically ground. The binding energies, on the other hand, follow Eisenman's series XI for strong electric field sites. Binding of NH ₄ ⁺ to the cis-well suggests that the orientation of the ligands in the site is tetrahedric.     

The barium site in a potassium channel by x-ray crystallography

X-ray diffraction data were collected from frozen crystals (100 degrees K) of the KcsA K(+) channel equilibrated with solutions containing barium chloride. Difference electron density maps (F(barium) - F(native), 5.0 A resolution) show that Ba(2+) resides at a single location within the selectivity filter. The Ba(2+) blocking site corresponds to the internal aspect (adjacent to the central cavity) of the "inner ion" position where an alkali metal cation is found in the absence of the blocking Ba(2+) ion. The location of Ba(2+) with respect to Rb(+) ions in the pore is in good agreement with the findings on the functional interaction of Ba(2+) with K(+) (and Rb(+)) in Ca(2+)-activated K(+) channels (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-567). Taken together, these structural and functional data imply that at physiological ion concentrations a third ion may interact with two ions in the selectivity filter, perhaps by entering from one side and displacing an ion on the opposite side.     

Watching small molecules move: Interrogating ionic channels using neutral solutes

Whether they are small enough to wriggle through the current-carrying part of an ionic channel or big enough to be kept outside and thus able to exert an osmotic stress on the channel space, polymers interact with channels in several instructive ways. The osmotic stress of excluded polymers allows one to measure the number of water molecules that come out of the channel in transitions between various open to closed states. The loss of osmotic activity, due to the partial or completely unrestricted admission of small polymers becomes a measure of the transfer probabilities of polymers from solution to small cavities; it provides an opportunity to study polymer conformation in a perfectly sieved preparation. Current fluctuations due to the partial blockage by a transient polymer are converted into estimates of times of passage and diffusion constants of polymers in channels. These estimates show how a channel whose functional states last for milliseconds is able to average over the interactions with polymers, interactions that last only microseconds. One sees clearly that in this averaging, the macromolecular channel is large enough to react like a macroscopic object to the chemical potentials of the species that modulate its activity.     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

The influence of static magnetic fields on mechanosensitive ion channel activity in artificial liposomes

The influence of static magnetic fields (SMFs) on the activity of recombinant mechanosensitive ion channels (the bacterial mechanosensitive ion channel of large conductance—MscL) following reconstitution into artificial liposomes has been investigated. Preliminary findings suggest that exposure to 80-mT SMFs does not induce spontaneous MscL activation in the absence of mechanical stimulation. However, SMFs do appear to influence the open probability and single channel kinetics of MscL exposed to negative pipette pressure. Typical responses include an overall reduction in channel activity or an increased likelihood of channels becoming trapped open in sub-conducting states following exposure to SMFs. There is a delay in the onset of this effect and it is maintained throughout exposure. Generally, channel activity showed slow or limited recovery following removal of the magnetic field and responses to the magnetic were often reduced or abolished upon subsequent exposures. Pre-exposure of the liposomes to SMFs resulted in reduced sensitivity of MscL to negative pipette pressure, with higher pressures required to activate the channels. Although the mechanisms of this effect are not clear, our initial observations appear to support previous work showing that the effects of SMFs on ion channels may be mediated by changes in membrane properties due to anisotropic diamagnetism of lipid molecules.     

Surface potentials near the mouth of the large-conductance K+ channel from Chara australis: A new method of testing for diffusion-limited ion flow

The kinetics of single K⁺ channels were derived for patch-clamp recordings of membrane patches excised from cytoplasmic drops from the plant, Chara australis R. Br. Specifically, the tilt effect model of MacKinnon, Latorre and Miller (1989. Biochemistry 28:8092–8099) has been used to measure the electrostatic potential (surface PD) and fixed charge at the entrances of the channel. The surface PD is derived from the difference between the trans-pore potential difference (PD) and that between the two bulk phases. The trans-pore PD is probed using three voltage-dependent properties of the channel. These are (1) the association and dissociation rates of Ca²⁺ binding to the channel, from both the cytoplasmic and vacuolar solutions. These were determined from the mean blocked and unblocked durations of the channel in the presence of either 20 mmol liter^–1 vacuolar or 1 mmol liter^–1 cytoplasmic Ca²⁺; (2) the closing rate of the channel's intrinsic gating process. This was determined from the mean channel open time in the absence of vacuolar Ca²⁺ at membrane PDs more negative than –100 mV; and (3) the effect of Mg²⁺ on channel conductance when added to solutions initially containing 3 mmol liter^–1 KCl.The voltage dependence of properties 1 and 2 shifts along the voltage axis according to the ionic strength of the bathing media, consistent with the presence of negative charge in the channel vestibules. Furthermore, the magnitude of this shift depends on the current in a manner consistent with diffusion-limited ion flow in the channel (i.e., the rate of ion diffusion in the external electrolyte limits the channel conductance). Mg²⁺ on either side of the membrane alters channel conductance in a voltage-dependent way. A novel feature of the Mg²⁺ effect is that it reverses, from a block to an enhancement, when the membrane PD is more negative than –70 mV. This reversal only appears in solutions of low ionic strength. The attenuating effect is due to voltage-dependent binding of Mg²⁺ within the pore, which presumably plugs the channel. The enhancing effect is due to screening by Mg²⁺ of surface potentials arising from diffusion-limited flow of K⁺.     

Patch clamp analysis of chemically activated and modulated ionic channels in isolated mammalian cardiomyocytes

Techniques routinely utilized in this laboratory for recording currents through single ionic channels of isolated atrial and ventricular rat cardiomyocytes are described. Emphasis is placed in two main areas: first, on methods for obtaining a sufficient yield of Ca⁺⁺-tolerant myocytes suitable for patch clamp experiments, and secondly, on methods for analyzing the temporal characteristics of patched ionic channels. These methods were used on acetylcholine activated K⁺ channels in isolated atrial myocytes and on an inwardly-rectifying K⁺ channel in ventricular myocytes. The latter is an example of a hormonally modulated K⁺ channel, since its activity could be substantially increased by norepinephrine. Analysis of the closed and open time distributions suggested that one of the closed states of this channel is markedly abbreviated by norepinephrine, whereas the open state is nearly unaffected. Norepinephrine was effective when channel activity was recorded from on-cell patches and the hormone was added to the solution bathing the cell membrane outside of the patched area. This indicates that a second messenger substance is probably mediating the action of norepinephrine.     

A site accessible to extracellular TEA+ and K+ influences intracellular Mg2+ block of cloned potassium channels

The members of the RCK family of cloned voltage-dependent K⁺ channels are quite homologous in primary structure, but they are highly diverse in functional properties. RCK4 channels differ from RCK1 and RCK2 channels in inactivation and permeation properties, the sensitivity to external TEA, and to current modulation by external K⁺ ions. Here we show several other interesting differences: While RCK1 and RCK2 are blocked in a voltage and concentration dependent manner by internal Mg²⁺ ions, RCK4 is only weakly blocked at very high potentials. The single-channel current-voltage relations of RCK4 are rather linear while RCK2 exhibits an inwardly rectifying single-channel current in symmetrical K⁺ solutions. The deactivation of the channels, measured by tail current protocols, is faster in RCK4 by a factor of two compared with RCK2. In a search for the structural motif responsible for these differences, point mutants creating homology between RCK2 and RCK4 in the pore region were tested. The single-point mutant K533Y in the background of RCK4 conferred the properties of Mg²⁺ block, tail current kinetics, and inward ion permeation of RCK2 to RCK4. This mutant was previously shown to be responsible for the alterations in external TEA sensitivity and channel regulation by external K⁺ ions. Thus, this residue is expected to be located at the external side of the pore entrance. The data are consistent with the idea that the mutation alters the channel occupancy by K⁺ and thereby indirectly affects internal Mg²⁺ block and channel closing.Abbreviations TEA tetraethylammonium - EGTA Ethylene glycol-bis (-aminoethyl ether) N,N,N,N-tetraacetic acid - 2S3B model 2-site 3-barrier model Correspondence to: S. H. Heinemann     

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles

RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A.     

Protein electrostriction: a possibility of elastic deformation of the α-hemolysin channel by the applied field

While conformational flexibility of proteins is widely recognized as one of their functionally crucial features and enjoys proper attention for this reason, their elastic properties are rarely discussed. In ion channel studies, where the voltage-induced or ligand-induced conformational transitions, gating, are the leading topic of research, the elastic structural deformation by the applied electric field has never been addressed at all. Here we examine elasticity using a model channel of known crystal structure—Staphylococcus aureus -hemolysin. Working with single channels reconstituted into planar lipid bilayers, we first show that their ionic conductance is asymmetric with voltage even at the highest salt concentration used where the static charges in the channel interior are maximally shielded. Second, choosing 18-crown-6 as a molecular probe whose size is close to the size of the narrowest part of the -hemolysin pore, we analyze the blockage of the channel by the crown/K⁺ complex. Analysis of the blockage within the framework of the Woodhull model in its generalized form demonstrates that the model is able to correctly describe the crown effect only if the parameters of the model are considered to be voltage-dependent. Specifically, one has to include either a voltage-dependent barrier for crown release to the cis side of the channel or voltage-dependent interactions between the binding site and the crown. We suggest that the voltage sensitivity of both the ionic conductance of the channel seen at the highest salt concentration and its blockage by the crown reflects a field-induced deformation of the pore.     

Asymmetric block of a monovalent cation-selective channel of rabbit cardiac sarcoplasmic reticulum by succinyl choline

Summary We have investigated the effect of the skeletal muscle relaxant succinyl choline (SC) on the conduction of potassium ions through a monovalent cation-selective channel present in the cardiac muscle sarcoplasmic reticulum membrane (CSR). This channel has been studied under voltage-clamp conditions following the fusion of purified CSR membrane vesicles with preformed planar phospholipid bilayers. The channel assumes a fixed orientation in the bilayer and displays two conducting states (B. Tomlins, A.J. Williams & R.A.P. Montgomery, 1984,J. Membrane Biol. 80: 191–199). SC blocks potassium conductance through the channel in a voltage-dependent manner. Block occurs from both sides of the channel, in both conducting states and is resolved as discrete flickering events. Although SC is capable of blocking potassium conductance from both sides of the membrane, block is asymmetric. The zero-voltage dissociation constant for block from the cis side of the membrane is approximately threefold lower than that from thetrans side. Block from thecis side displays a linear dependence on SC concentration for both open states and is competitive with potassium ions at saturating potassium activities, consistent with a singlesite blocking model. The degree of SC-induced block is also influenced by membrane surface charge. SC block differs from that previously described for bis quaternary ammonium (bis Qn) compounds such as decamethonium in that SC blocks preferentially from thecis side of the channel.