Reconstitution of the influenza virus M2 ion channel in lipid bilayers

M₂, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M₂ baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25–500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25–90 pS). (3) The channels were selective for cations; t _Na = 0.75 when 150 mm NaCl bathed both sides of the membrane. (4) Amantadine reduced the probability of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs ～ Na > Li. The pH activation, amantadine block and ion selectivity of the M₂ protein ion channel in bilayers are consistent with those observed on expression of the M₂ protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M₂ protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

CLIC4 (p64H1) and its putative transmembrane domain form poorly selective, redox-regulated ion channels

Despite being synthesized in the cytosol without a leader sequence, the soluble 253-residue mammalian protein CLIC4 (Chloride Intracellular Channel 4, or p64H1), a structural homologue of Omega-type glutathione-S-transferase, autoinserts into membranes to form an integral membrane protein with ion channel activity. A predicted transmembrane domain (TMD) near the N-terminus of CLIC4 could mediate membrane insertion, and contribute to oligomeric pores, with minimal reorganization of the soluble protein structure. We tested this idea by reconstituting recombinant CLIC4 in planar bilayers containing phosphatidyethanolamine, phosphatidylserine and cholesterol, recording ion channels with a maximum conductance of approximately 15 pS in KCl under both oxidizing and reducing conditions. The channels discriminated poorly between anions and cations, incompatible with the current "CLIC" nomenclature, and their conductance was modified by the trans (external or luminal) redox potential, as previously observed for CLIC1. We then reconstituted a truncated version of the protein, limited to the first 61 residues containing the predicted TMD. This included a single trans cysteine residue in the putative pore-forming subunits, at the external entrance to the pore. The truncated protein formed non-selective channels with a reduced conductance, but they retained their trans-redox sensitivity, and could still be blocked or inactivated by trans (not cis) thiol-reative dithiobisnitrobenzoic acid. We suggest that oligomers containing the putative TMD are essential components of the CLIC4 pore. However, the pore is inherently non-selective, and any ionic selectivity in CLIC4 (and other membrane CLICs) may be attributable to other regions of the protein, including the channel vestibules.     

Electrostatic basis of valence selectivity in cationic channels

We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.     

Electrostatic basis of valence selectivity in cationic channels

We examine how a variety of cationic channels discriminate between ions of differing charge. We construct models of the KcsA potassium channel, voltage gated sodium channel and L-type calcium channel, and show that they all conduct monovalent cations, but that only the calcium channel conducts divalent cations. In the KcsA and sodium channels divalent ions block the channel and prevent any further conduction. We demonstrate that in each case, this discrimination and some of the more complex conductance properties of the channels is a consequence of the electrostatic interaction of the ions with the charges in the channel protein. The KcsA and sodium channels bind divalent ions strongly enough that they cannot be displaced by other ions and thereby block the channel. On the other hand, the calcium channel binds them less strongly such that they can be destabilized by the repulsion of another incoming divalent ion, but not by the lesser repulsion from monovalent ions.     

Properties of three different ion channels in the plasma membrane of the slime mold Dictyostelium discoideum.

'Patch-clamp' experiments in the cell-attached configuration have shown the existence of three distinct types of ion channels in the plasma membrane of Dictyostelium discoideum. Channels DI (slope conductance 11 pS) and DII (slope conductance 6 pS) promote an outward current at depolarizing voltages. A third ion channel (HI, slope conductance 3 pS) opens preferentially at hyperpolarization and promotes inward current flow. It is suggested that under physiological conditions current through the DI and DII channels is carried by K+, whereas Ca2+ may be the current carrier in the HI channel. The density of these ion channels in the membrane of D. discoideum is low: approx. 0.1/micron 2 for the DI and HI channel and 0.02/micron 2 for the DII channel. The gating properties of the ion channels appear to be complicated because openings are grouped into bursts of activity. The probability of the DI channel being in the open state increases with depolarization. The mean channel life-time is about 20 ms and voltage-independent. The burst duration increases with depolarization whereas the interburst time decreases. The minimal kinetic model accounting for the behaviour of the DI channel is a three-state model with two closed and one open state. A detailed analysis of the gating of the DII and the HI channel was prevented by their low rate of occurrence (DII) or fast inactivation (HI). The formation of a seal resistance greater than or equal to 1 G omega depends critically on the composition of the pipette solution. Examination of a series of monovalent and divalent cations as well as different organic and inorganic anions has shown that 'gigaseals' are formed only in the presence of at least 1 mM Ca2+ or Sr2+, whereas Ba2+, Mg2+ and monovalent cations (Li+, Na+, K+, Rb+, Cs+) do not support the formation of high seal resistances. Anions seem not to affect the seal formation.     

Cation permeability and cation-anion interactions in a mutant GABA-gated chloride channel from Drosophila.

To investigate the structural basis of anion selectivity of Drosophila GABA-gated Cl(-) channels, the permeation properties of wild-type and mutant channels were studied in Xenopus oocytes. This work focused on asparagine 319, which by homology is one amino acid away from a putative extracellular ring of charge that regulates cation permeation in nicotinic receptors. Mutation of this residue to aspartate reduced channel conductance, and mutation to lysine or arginine increased channel conductance. These results are consistent with an electrostatic interaction between this site and permeating anions. The lysine mutant, but not the arginine mutant, formed a channel that is permeable to cations, and this cannot be explained in terms of electrostatics. The lysine mutant had a 25-mV reversal potential in solutions with symmetrical Cl(-) and asymmetrical cations. The permeability ratio of K(+) to Cl(-) was determined as 0. 33 from reversal potential measurements in KCl gradients. Experiments with large organic cations and anions showed that cation permeation can only be seen in the presence of Cl(-), but Cl(-) permeation can be seen in the absence of permeant cations. Measurements of permeability ratios of organic anions indicated that the lysine mutant has an increased pore size. The cation permeability of the lysine-containing mutant channel cannot be accounted for by a simple electrostatic interaction with permeating ions. It is likely that lysine substitution causes a structural change that extends beyond this one residue to influence the positions of other channel-forming residues. Thus protein conformation plays an important role in enabling ion channels to distinguish between anions and cations.     

The first extracellular loop domain is a major determinant of charge selectivity in connexin46 channels

Intercellular channels formed of members of the gene family of connexins (Cxs) vary from being substantially cation selective to being anion selective. We took advantage of the ability of Cx46 to function as an unopposed hemichannel to examine the basis of Cx charge selectivity. Previously we showed Cx46 hemichannels to be large pores that predominantly conduct cations and inwardly rectify in symmetric salts, properties suggesting selectivity is influenced by fixed negative charges located toward the extracellular end of the pore. Here we demonstrate that high ionic strength solutions applied to the extracellular, but not the intracellular, side of Cx46 hemichannels substantially reduce the ratio of cation to anion permeability. Substitution of the first extracellular loop (E1) domain of Cx32, an anion-preferring Cx, reduces conductance, converts Cx46 from cation to anion preferring, and changes the I-V relation form inwardly to outwardly rectifying. These data suggest that fixed negative charges influencing selectivity in Cx46 are located in E1 and are substantially reduced and/or are replaced with positive charges from the Cx32 E1 sequence. Extending studies to Cx46 cell-cell channels, we show that they maintain a strong preference for cations, have a conductance nearly that expected by the series addition of hemichannels, but lack rectification in symmetric salts. These properties are consistent with preservation of the fixed charge region in E1 of hemichannels, which upon docking, become symmetrically placed near the center of the cell-cell channel pore. Furthermore, heterotypic cell-cell channels formed by pairing Cx46 with Cx32 or Cx43 rectify in symmetric salts in accordance with the differences in the charges we ascribed to E1. These data are consistent with charged residues in E1 facing the channel lumen and playing an important role in determining Cx channel conductance and selectivity.     

Bis[(benzo-15-crown-5)-15-yl methyl] pimelate forms ion channels in planar lipid bilayer: a novel model ion channel

Some crown ethers translocate cations across the liposomal membrane either by a carrier mechanism or by forming ion channels. We report formation of ion channels in lipid bilayer membranes by bis[(benzo-15-crown-5)-15-yl methyl] pimelate, a crown ether known to form ion inclusion complexes with alkali metal cations. The channels have characteristic long openings lasting several seconds and a low conductance (4 pS in 500 mM KCl and 2.5 pS in 500 mM NaCl). A model of the crown ether channel formed by stacking of four monomers is proposed. A large database of structural information on crown ethers and their ion inclusion complexes as well as large family of crown ethers with a variety of substitutions in the ring are commercially available. Thus the crown ether channel is an attractive model system to study the role of various chemical moieties in ion conduction which may provide deeper insight into understanding the mechanism(s) of selectivity, ion transport, etc. in biological ion channels.     

Divalent cation effects on acetylcholine-activated channels at the frog neuromuscular junction

The effects of the divalent cations Ca and Mg on the properties of ACh-activated channels at the frog neuromuscular junction were studied using a two-microelectrode voltage clamp. The divalent cation concentration was varied from 2 to 40 mM in solutions containing 50% normal Na. The reversal potential was determined by interpolation of the acetylcholine (ACh)-induced current versus voltage relationship. The single-channel conductance and the mean channel lifetime were calculated from fluctuation analysis of the ACh-induced end-plate current. Extracellular Na and/or divalent cations affected the reversal potential of endplate channels in a way that cannot be described by the Goldman-Hodgkin-Katz equation or by a simple two-barrier, one-binding site model of the channel if the assumption was made that permeability ratios were constant and not a function of ion concentrations. Increasing the divalent cation concentration decreased the single-channel conductance to approximately 10 pS in solutions with 50% Na and 40 mM divalent cation concentrations. The effect of the divalent cations Ca and Mg on the mean channel lifetime was complex and dependent on whether the divalent cation was Ca or Mg. The mean channel lifetime was not significantly changed in most solutions with increased Ca concentration, while it was slightly prolonged by increased Mg concentration.     

Anion-cation permeability correlates with hydrated counterion size in glycine receptor channels

The functional role of ligand-gated ion channels depends critically on whether they are predominantly permeable to cations or anions. However, these, and other ion channels, are not perfectly selective, allowing some counterions to also permeate. To address the mechanisms by which such counterion permeation occurs, we measured the anion-cation permeabilities of different alkali cations, Li⁺ Na⁺, and Cs⁺, relative to either Cl⁻ or anions in both a wild-type glycine receptor channel (GlyR) and a mutant GlyR with a wider pore diameter. We hypothesized and showed that counterion permeation in anionic channels correlated inversely with an equivalent or effective hydrated size of the cation relative to the channel pore radius, with larger counterion permeabilities being observed in the wider pore channel. We also showed that the anion component of conductance was independent of the nature of the cation. We suggest that anions and counterion cations can permeate through the pore as neutral ion pairs, to allow the cations to overcome the large energy barriers resulting from the positively charged selectivity filter in small GlyR channels, with the permeability of such ion pairs being dependent on the effective hydrated diameter of the ion pair relative to the pore diameter.     

Interactions of n-3 fatty acids with ion channels in excitable tissues

In summary, we have shown that the conventional explanation for the site of action of a ligand which alters the conductance of a membrane ion channel is that the ligand interacts or binds with the ion channel protein, changing its conductance, is inadequate to explain the primary site of action of the antiarrhythmic n-3 PUFAs. We have shown that when a neutral asparagine is replaced by a positively charged lysine in the N406 amino acid site in the alpha-subunit of the human cardiac sodium channel, the n-3 fatty acids lose their inhibitory action on the sodium current. The inadequacy of this finding to explain the primary site of action of the n-3 PUFAs is demonstrated by the inhibitory effect on all other cardiac ion channels, so far tested. We show that ion channels, which share no amino acid homology with the PUFAs, have their conductance also reduced in the presence of the PUFAs, Thus a more general conceptual framework or paradigm is needed to account for the broad action of the PUFAs on diverse different ion channels lacking amino acid homology. We have been testing the membrane tension hypothesis of Andersen and associates. According to this hypothesis, the fatty acids are not acting directly on the ion channel protein but accumulating in the phospholipid membrane in immediate juxtaposition to the site in the membrane where the ion channel protein penetrates the membrane phospholipid bilayer. This alters membrane tensions exerted by the phospholipid membrane on the ion channel, which in turn causes conformational changes in the ion channel, altering the conductance of the ion channel. Our preliminary data seem to support this membrane tension hypothesis.     

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.     

Ion selectivity and current saturation in inward-rectifier K+ channels

We investigated the features of the inward-rectifier K channel Kir1.1 (ROMK) that underlie the saturation of currents through these channels as a function of permeant ion concentration. We compared values of maximal currents and apparent K(m) for three permeant ions: K(+), Rb(+), and NH(4)(+). Compared with K(+) (i(max) = 4.6 pA and K(m) = 10 mM at -100 mV), Rb(+) had a lower permeability, a lower i(max) (1.8 pA), and a higher K(m) (26 mM). For NH(4)(+), the permeability was reduced more with smaller changes in i(max) (3.7 pA) and K(m) (16 mM). We assessed the role of a site near the outer mouth of channel in the saturation process. This site could be occupied by either permeant ions or low-affinity blocking ions such as Na(+), Li(+), Mg(2+), and Ca(2+) with similar voltage dependence (apparent valence, 0.15-0.20). It prefers Mg(2+) over Ca(2+) and has a monovalent cation selectivity, based on the ability to displace Mg(2+), of K(+) > Li(+) ～ Na(+) > Rb(+) ～ NH(4)(+). Conversely, in the presence of Mg(2+), the K(m) for K(+) conductance was substantially increased. The ability of Mg(2+) to block the channels was reduced when four negatively charged amino acids in the extracellular domain of the channel were mutated to neutral residues. The apparent K(m) for K(+) conduction was unchanged by these mutations under control conditions but became sensitive to the presence of external negative charges when residual divalent cations were chelated with EDTA. The results suggest that a binding site in the outer mouth of the pore controls current saturation. Permeability is more affected by interactions with other sites within the selectivity filter. Most features of permeation (and block) could be simulated by a five-state kinetic model of ion movement through the channel.     

Identification and regulation of whole-cell chloride currents in airway epithelium

We used the whole-cell patch-clamp technique to study membrane currents in human airway epithelial cells. The conductive properties, as described by the instantaneous current-voltage relationship, rectified in the outward direction when bathed in symmetrical CsCl solutions. In the presence of Cl concentration gradients, currents reversed near ECl and were not altered significantly by cations. Agents that inhibit the apical membrane Cl conductance inhibited Cl currents. These conductive properties are similar to the conductive properties of the apical membrane Cl channel studied with the single-channel patch-clamp technique. The results suggest that the outwardly rectifying Cl channel is the predominant Cl-conductive pathway in the cell membrane. The steady-state and non-steady-state kinetics indicate that current flows through ion channels that are open at hyperpolarizing voltages and close with depolarization. These Cl currents were regulated by the cAMP-dependent protein kinase: when the catalytic subunit of cAMP-dependent protein kinase was included in the pipette solution, Cl channel current more than doubled. We also found that reducing extracellular osmolarity by 30% increased Cl current, suggesting that cell-swelling stimulated Cl current. Studies of transepithelial Cl transport in cell monolayers suggest that a reduction in solution osmolarity activates the apical Cl channel: reducing extracellular osmolarity stimulated a short-circuit current that was inhibited by Cl-free solution, by mucosal addition of a Cl channel antagonist, and by submucosal addition of a loop diuretic. These results suggest that apical membrane Cl channels may be regulated by cell volume and by the cAMP-dependent protein kinase.     

Increased salt concentration promotes competitive block of OmpF channel by protons

Porins are channel-forming proteins that are located in the outer membranes (OM) of Gram-negative bacteria and allow the influx of hydrophilic nutrients and the extrusion of waste products. The fine regulation of the ion transport through these wide channels could play an important role in the survival of the bacteria in acidic media. We investigate here the mechanism responsible for the pH sensitivity of the trimeric porin OmpF, of Escherichia coli. Planar lipid bilayer electrophysiology and site-directed mutagenesis were used to study the effect of pH on the ion conductive properties of the OmpF channel in its fully open, "nongated" conformation. At low pH we observe a large drop in the OmpF open channel conductance that is accompanied by a substantial increase of the current noise. These channel features are strongly dependent on the salt concentration and we propose that they are originated by competitive binding of cations and protons occurring in the narrow central constriction of the channel. This subtle mechanism reveals to be capital for the channel function because it not only drives the channel sensitivity to pH but is also indispensable for the particularly efficient permeation mechanism of the channel at physiological conditions (~neutral pH).     

Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity

Summary The E1 subgroup (E1, A, Ib, etc.) of antibacterial toxins called colicins are known to form voltage-dependent channels in planar lipid bilayers. The genes for colicins E1, A and Ib have been cloned and sequenced, making these channels interesting models for the widespread phenomenon of voltage dependence in cellular channels. In this paper we investigate ion selectivity and channel size—properties relevant to model building. Our major finding is that the colicin E1 channel is large, having a diameter ofat least 8 Å at its narrowest point. We established this from measurements of reversal potentials for gradients formed by salts of large cations or large anions. In so doing, we exploited the fact that the colicin channel is permeable to both cations and anions, and its relative selectivity to them is a functions and anions, and its relative selectivity to them is a function of pH. The channel is anion selective (Cl^– over K⁺) in neutral membranes, and the degree of selectivity is highly dependent on pH. In negatively charged membranes, it becomes cation selective at pH's higher than about 5. Experiments with pH gradients cross the membrane suggest that titratable groups both within the channel lumen and near the channel ends affect the selectivity. Individual E1 channels have more than one open conductance state, all displaying comparable ion selectivity. Colicins A and Ib also exhibit pH-dependent ion selectivity, and appear to have even larger lumens than E1.     

Changes in Negative Charge at the Luminal Mouth of the Pore Alter Ion Handling and Gating in the Cardiac Ryanodine-Receptor

We have tested the hypothesis that a high density of negative charge at the luminal mouth of the RyR2 pore plays a pivotal role in the high cation conductance and limited selectivity observed in this channel by introducing into each monomer a double point mutation to neutralize acidic residues in this region of the mouse RyR2 channel. The resultant channel, ED4832AA, is capable of functioning as a calcium-release channel in situ. Consistent with our hypothesis, the ED4832AA mutation altered the ion handling characteristics of single RyR2 channels. The mutant channel retains the ability to discriminate between cations and anions but cation conductance is altered significantly. Unitary K⁺ conductance is reduced at low levels of activity but increases dramatically as activity is raised and shows little sign of saturation. ED4832AA no longer discriminates between divalent and monovalent cations. In addition, the gating characteristics of single RyR2 channels are altered markedly by residue neutralization. Open probability in the ED4832AA channel is substantially higher than that of the wild-type channel. Moreover, at holding potentials in excess of ±50 mV several subconductance states become apparent in ED4832AA and are more prevalent at very high holding potentials. These observations are discussed within the structural framework provided by a previously developed model of the RyR2 pore. Our data indicates that neutralization of acidic residues in the luminal mouth of the pore produces wide-ranging changes in the electric field in the pore, the interaction energies of permeant ions in the pore and the stability of the selectivity filter region of the pore, which together contribute to the observed changes ion handling and gating.     

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.     

Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion

We further investigated the rapid fluctuations between two different conductance levels promoted by protons when monovalent ions carry current through single L-type Ca channels. We tested for voltage dependence of the proton-induced current fluctuations and for accessibility of the protonation site from both sides of the membrane patch. The results strongly suggest an extracellular location of the protonation site. We also studied the dependence of the kinetics of the fluctuations and of the two conductance levels on the concentration of permeant ion and on external ionic strength. We find that saturation curves of channel conductance vs. [K] are similar for the two conductance levels. This provides evidence that protonation does not appreciably change the surface potential near the entry of the permeation pathway. The proton-induced conduction change must therefore result from an indirect interaction between the protonation site and the ion-conducting pathway. Concentration of permeant ion and ionic strength also affect the kinetics of the current fluctuations, in a manner consistent with our previous hypothesis that channel occupancy destabilizes the low conductance channel conformation. We show that the absence of measurable fluctuations with Li and Ba as charge carriers can be explained by significantly higher affinities of these ions for permeation sites. Low concentrations of Li reduce the Na conductance and abbreviate the lifetimes of the low conductance level seen in the presence of Na. We use whole-cell recordings to extrapolate our findings to the physiological conditions of Ca channel permeation and conclude that in the presence of 1.8 mM Ca no proton-induced fluctuations occur between pH 7.5 and 6.5. Finally, we propose a possible physical interpretation of the formal model of the protonation cycle introduced in the companion paper.     

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.