Point mutations in the post-M2 region of human alpha-ENaC regulate cation selectivity

We tested the hypothesis that an arginine-rich region immediately following the second transmembrane domain may constitute part of the inner mouth of the epithelial Na+ channel (ENaC) pore and, hence, influence conduction and/or selectivity properties of the channel by expressing double point mutants in Xenopus oocytes. Double point mutations of arginines in this post-M2 region of the human alpha-ENaC (alpha-hENaC) led to a decrease and increase in the macroscopic conductance of alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, respectively, but had no effect on the single-channel conductance of either double point mutant. However, the apparent equilibrium dissociation constant for Na+ was decreased for both alphaR586E,R587Ebetagamma- and alphaR589E,R591Ebetagamma-hENaC, and the maximum amiloride-sensitive Na+ current was decreased for alphaR586E,R587Ebetagamma-hENaC and increased for alphaR589E,R591Ebetagamma-hENaC. The relative permeabilities of Li+ and K+ vs. Na+ were increased 11.25- to 27.57-fold for alphaR586E,R587Ebetagamma-hENaC compared with wild type. The relative ion permeability of these double mutants and wild-type ENaC was inversely related to the crystal diameter of the permeant ions. Thus the region of positive charge is important for the ion permeation properties of the channel and may form part of the pore itself.     

Molecular architecture of the voltage-dependent Na channel: functional evidence for alpha helices in the pore

The permeation pathway of the Na channel is formed by asymmetric loops (P segments) contributed by each of the four domains of the protein. In contrast to the analogous region of K channels, previously we (Yamagishi, T., M. Janecki, E. Marban, and G. Tomaselli. 1997. Biophys. J. 73:195-204) have shown that the P segments do not span the selectivity region, that is, they are accessible only from the extracellular surface. The portion of the P-segment NH(2)-terminal to the selectivity region is referred to as SS1. To explore further the topology and functional role of the SS1 region, 40 amino acids NH(2)-terminal to the selectivity ring (10 in each of the P segments) of the rat skeletal muscle Na channel were substituted by cysteine and expressed in tsA-201 cells. Selected mutants in each domain could be blocked with high affinity by externally applied Cd(2)+ and were resistant to tetrodotoxin as compared with the wild-type channel. None of the externally applied sulfhydryl-specific methanethiosulfonate reagents modified the current through any of the mutant channels. Both R395C and R750C altered ionic selectivity, producing significant increases in K(+) and NH(4)(+) currents. The pattern of side chain accessibility is consistent with a pore helix like that observed in the crystal structure of the bacterial K channel, KcsA. Structure prediction of the Na channel using the program PHDhtm suggests an alpha helix in the SS1 region of each domain channel. We conclude that each of the P segments undergoes a hairpin turn in the permeation pathway, such that amino acids on both sides of the putative selectivity filter line the outer mouth of the pore. Evolutionary conservation of the pore helix motif from bacterial K channels to mammalian Na channels identifies this structure as a critical feature in the architecture of ion selective pores.     

Functional influence of the pore helix glutamate in the KcsA K+ channel

The E71 residue is buried near the selectivity filter in the KcsA K+ channel and forms a carboxyl-carboxylate bridge with D80. We have investigated the importance of E71 by examining neutralization mutants at this position using biochemical and electrophysiological methods. E71 mutations differentially destabilize the detergent-solubilized tetramer; among them, the E71V neutralization mutant has a relatively subtle effect. The E71V channel displays electrical activity when reconstituted into planar lipid bilayers. In single channel recordings, the mutant retains K+/Na+ selectivity, and its conductance in the outward direction is unaltered. Some conduction properties are changed: inward conductance is increased. Our results show that that the E71 side chain is not a primary determinant of ion selectivity or conduction in the wild-type channel, either directly or through the E71:D80 carboxyl-carboxylate bridge.     

Topology of the P segments in the sodium channel pore revealed by cysteine mutagenesis.

The P segments of the voltage-dependent Na+ channel line the outer mouth and selectivity filter of the pore. The residues that form the cytoplasmic mouth of the pore of the channel have not been identified. To study the structure of the inner pore mouth, the presumed selectivity filter residues (D400, E755, K1237, and A1529), and three amino acids just amino-terminal to each of these residues in the rat skeletal muscle Na+ channel, were mutated to cysteine and expressed in tsA 201 cells. These amino acids are predicted (by analogy to K+ channels) to be on the cytoplasmic side of the putative selectivity filter residues. Inward and outward Na+ currents were measured with the whole-cell configuration of the patch-clamp technique. Cysteinyl side-chain accessibility was gauged by sensitivity to Cd2+ block and by reactivity with methanethiosulfonate (MTS) reagents applied to both the inside and the outside of the cell. Outward currents through the wild-type and all of the mutant channels were unaffected by internal Cd2+ (100 microM). Similarly, 1 mM methanethiosulfonate ethylammonium (MTSEA) applied to the inside of the membrane did not affect wild-type or mutant outward currents. However, two mutants amino-terminal to the selectivity position in domain III (F1236C and T1235C) and one in domain IV (S1528C) were blocked with high affinity by external Cd2+. The Na+ current through F1236C and S1528C channels was inhibited by MTSEA applied to the outside of the cell. The accessibility of these mutants to externally applied cysteinyl ligands indicates that the side chains of the mutated residues face outward rather than inward. The K+ channel model of the P segments as protein loops that span the selectivity region is not applicable to the Na+ channel.     

Ultra-slow inactivation in mu1 Na+ channels is produced by a structural rearrangement of the outer vestibule

While studying the adult rat skeletal muscle Na+ channel outer vestibule, we found that certain mutations of the lysine residue in the domain III P region at amino acid position 1237 of the alpha subunit, which is essential for the Na+ selectivity of the channel, produced substantial changes in the inactivation process. When skeletal muscle alpha subunits (micro1) with K1237 mutated to either serine (K1237S) or glutamic acid (K1237E) were expressed in Xenopus oocytes and depolarized for several minutes, the channels entered a state of inactivation from which recovery was very slow, i.e., the time constants of entry into and exit from this state were in the order of approximately 100 s. We refer to this process as "ultra-slow inactivation". By contrast, wild-type channels and channels with the charge-preserving mutation K1237R largely recovered within approximately 60 s, with only 20-30% of the current showing ultra-slow recovery. Coexpression of the rat brain beta1 subunit along with the K1237E alpha subunit tended to accelerate the faster components of recovery from inactivation, as has been reported previously of native channels, but had no effect on the mutation-induced ultra-slow inactivation. This implied that ultra-slow inactivation was a distinct process different from normal inactivation. Binding to the pore of a partially blocking peptide reduced the number of channels entering the ultra-slow inactivation state, possibly by interference with a structural rearrangement of the outer vestibule. Thus, ultra-slow inactivation, favored by charge-altering mutations at site 1237 in micro1 Na+ channels, may be analogous to C-type inactivation in Shaker K+ channels.     

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein.     

Ion binding to KcsA: implications in ion selectivity and channel gating

Binding of K+ and Na+ to the potassium channel KcsA has been characterized from the stabilization observed in the heat-induced denaturation of the protein as the ion concentration is increased. KcsA thermal denaturation is known to include (i) dissociation of the homotetrameric channel into its constituent subunits and (ii) protein unfolding. The ion concentration-dependent changes in the thermal stability of the protein, evaluated as the Tm value for thermal-induced denaturation of the protein, may suggest the existence of both high- and low-affinity K+ binding sites of KcsA, which lend support to the tenet that channel gating may be governed by K+ concentration-dependent transitions between different affinity states of the channel selectivity filter. We also found that Na+ binds to KcsA with a KD similar to that estimated electrophysiologically from channel blockade. Therefore, our findings on ion binding to KcsA partly account for K+ over Na+ selectivity and Na+ blockade and argue against the strict “snug fit” hypothesis used initially to explain ion selectivity from the X-ray channel structure. Furthermore, the remarkable effects of increasing the ion concentration, K+ in particular, on the Tm of the denaturation process evidence that synergistic effects of the metal-mediated intersubunit interactions at the channel selectivity filter are a major contributor to the stability of the tetrameric protein. This observation substantiates the notion of a role for ions as structural “effectors” of ion channels.     

Threonine in the selectivity filter of the acetylcholine receptor channel.

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

Selectivity filter residues contribute unequally to pore stabilization in voltage-gated sodium channels

Mutations in the putative selectivity filter region of the voltage-gated Na+ channel, the so-called DEKA-motif, not only affect selectivity but also alter the channel's gating properties, suggesting functional coupling between permeation and gating. We have previously reported that charge-altering mutations at position 1237 in the P-loop of domain III (position K of the DEKA-motif in the adult rat skeletal muscle Na+ channel, rNa(v)1.4) dramatically enhanced entry to an inactivated state from which the channels recovered with a very slow time constant on the order of approximately 100 s (Todt, H., Dudley, S. C. J., Kyle, J. W., French, R. J., and Fozzard, H. A. (1999) Biophys. J. 76, 1335-1345). This state, termed "ultra-slow inactivation", may reflect a complex molecular rearrangement of the channel's pore region that involves both the extracellular and the cytoplasmic pore. Here, we tested whether charged DEKA-motif residues other than K1237 were also important determinants of a channel's gating properties. Therefore, we constructed the charge-neutralizing mutations D400A, E755A, and K1237A and studied the effects of these mutations on I(US). We found that, compared to wild-type rNa(v)1.4 channels, mutant D400A and K1237A but not E755A channels exhibited enhanced entry into ultra-slow inactivation. Selectivity for Na+ over K+, as judged from shifts in reversal potentials, was preserved in D400A, reduced in E755A, and completely lost in K1237A. These data suggest that an electrostatic interaction between the positively charged residue K1237 and the negatively charged residue D400 stabilizes the structure of the pore and thereby prevents I(US). Moreover, the interaction between K1237 and E755 appears to provide the basis for selective permeation of Na+ over K+.     

Molecular Motions of the Outer Ring of Charge of the Sodium Channel: Do They Couple to Slow Inactivation?

In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain beta1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the alpha carbons of these residues of no more than 7 A. The redox catalyst Cu(II) (1,10-phenanthroline)3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.     

The pre-transmembrane 1 domain of acid-sensing ion channels participates in the ion pore

The acid-sensing ion channel (ASIC) subunits ASIC1, ASIC2, and ASIC3 are members of the amiloride-sensitive Na+ channel/degenerin family of ion channels. They form proton-gated channels that are expressed in the central nervous system and in sensory neurons, where they are thought to play an important role in pain accompanying tissue acidosis. A splice variant of ASIC2, ASIC2b, is not active on its own but modifies the properties of ASIC3. In particular, whereas most members of the amiloride-sensitive Na+ channel/degenerin family are highly selective for Na+ over K+, ASIC3/ASIC2b heteromultimers show a nonselective component. Chimeras of the two splice variants allowed identification of a 9-amino acid region preceding the first transmembrane (TM) domain (pre-TM1) of ASIC2 that is involved in ion permeation and is critical for Na+ selectivity. Three amino acids in this region (Ile-19, Phe-20, and Thr-25) appear to be particularly important, because channels mutated at these residues discriminate poorly between Na+ and K+. In addition, the pH dependences of the activity of the F20S and T25K mutants are changed as compared with that of wild-type ASIC2. A corresponding ASIC3 mutant (T26K) also has modified Na+ selectivity. Our results suggest that the pre-TM1 region of ASICs participates in the ion pore.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

Single Na+ channels activated by veratridine and batrachotoxin

Voltage-sensitive Na+ channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX). Both of these toxins are known to cause persistent activation of Na+ channels. With BTX as the channel activator, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s. Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-11-alpha-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators. Under symmetrical solution conditions, the single channel conductance for Na+ is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na+ greater than Li+ greater than K+) than for VT (Na+ greater than K+ greater than Li+). Open channel current-voltage curves in symmetrical Na+ and Li+ are roughly linear, while those in symmetrical K+ are inwardly rectifying. Na+ currents are blocked asymmetrically by K+ with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT. The results show that the alkaloid neurotoxins not only alter the gating process of the Na+ channel, but also affect the structure of the open channel. We further conclude that the rate-determining step for conduction by Na+ does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.     

Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase.

Previous studies showed that the alpha 1, alpha 2, and alpha 3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for the ligands ATP, Na(+), and K(+). For the rat isoforms transfected into HeLa cells, K'(ATP) for ATP binding at its low affinity site is lower for alpha 2 and alpha 3 compared with alpha 1; relative to alpha 1 and alpha 2, alpha 3 has a higher K'(Na) and lower K'(K) (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925--16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B, and Blostein, R. (1994) J. Biol. Chem. 269, 16668--16676). The experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that alpha 2 differs from alpha1 primarily by a shift in the E(1) E(2) equilibrium in favor of E(1) form(s) as evidenced by (i) a approximately 20-fold increase in IC(50) for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K'(ATP) compared with alpha 1, the E(1) E(2) poise of alpha 3 is not shifted toward E(1). Distinct intrinsic interactions with Na(+) ions are underscored by the marked selectivity for Na(+) over Li(+) of alpha 3 compared with either alpha1 or alpha 2 and higher K'(Na) for cytoplasmic Na(+), which persists over a 100-fold range in proton concentration, independent of the presence of K(+). The kinetic analysis also suggests alpha 3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na(+) and K(+).     

Purified epithelial Na+ channel complex contains the pertussis toxin-sensitive G alpha i-3 protein.

We have recently demonstrated that the amiloride-sensitive Na+ channel in the apical membrane of the renal epithelial cell line, A6, is modulated by the alpha i-3 subunit of the Gi-3 protein. We also showed that a 700-kDa protein complex can be purified from the membranes of A6 epithelia which (a) can reconstitute the amiloride-sensitive Na+ influx in liposomes and planar bilayer membranes and (b) consists of six major protein bands observed on reducing sodium dodecyl sulfate-polyacrylamide gels with molecular masses ranging from 35 to 320 kDa. The present study was undertaken to determine if the alpha i-3 subunit was a member of this Na+ channel complex. G alpha i structure and function were identified by Western blotting with specific G alpha i subunit antibodies and Na+ channel antibodies, through ADP-ribosylation with pertussis toxin, and by immunocytochemical localization of the Na+ channel and G alpha i proteins. We demonstrate that two protein substrates are ADP-ribosylated in the 700-kDa complex in the presence of pertussis toxin and are specifically immunoprecipitated with an anti-Na+ channel polyclonal antibody. One of these substrates, a 41-kDa protein, was identified as the alpha i-3 subunit of the Gi-3 protein on Western blots with specific antibodies. Na+ channel antibodies do not recognize G alpha i-3 on Western blots of Golgi membranes which contain alpha i-3 but not Na+ channel proteins, nor do they immunoprecipitate alpha i-3 from solubilized Golgi membranes; however, alpha i-3 is coprecipitated as part of the Na+ channel complex from A6 cell membranes by polyclonal Na+ channel antibodies. Both alpha i-3 and the Na+ channel have been localized in A6 cells by confocal imaging and immunofluorescence with specific antibodies and are found to be in distinct but adjacent domains of the apical cell surface. In functional studies, alpha i-3, but not alpha i-2, stimulates Na+ channel activity. These data are therefore consistent with the localization of Na+ channel activity and modulatory alpha i-3 protein at the apical plasma membrane, which together represent a specific signal transduction pathway for ion channel regulation.     

Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the alpha-subunit of the rat kidney Na+,K(+)-ATPase.

Site-specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the alpha 1-subunit of the ouabain resistant rat kidney Na+,K(+)-ATPase. cDNAs encoding either of the Na+,K(+)-ATPase mutants Pro328-->Ala and Leu332-->Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS-1 cells. Ouabain-resistant clones growing in the presence of 10 microM ouabain were isolated, and the Na+,K+, ATP and pH dependencies of the Na+,K(+)-ATPase activity measured in the presence of 10 microM ouabain were analysed. Under these conditions the exogenous expressed Na+,K(+)-ATPase contributed more than 95% of the Na+,K(+)-ATPase activity. The Pro328-->Ala mutant displayed a reduced apparent affinity for Na+ (K0.5 (Na+) 13.04 mM), relative to the wild type (K0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu332-->Ala mutant was increased (K0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K0.5 (K+) 2.46 mM for Pro328-->Ala and 1.97 mM for Leu332-->Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K0.5 (ATP) 0.086 mM for Pro328-->Ala and 0.042 mM for Leu332-->Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)-->E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.     

Contribution of ion binding affinity to ion selectivity and permeation in KcsA, a model potassium channel

Ion permeation and selectivity, key features in ion channel function, are believed to arise from a complex ensemble of energetic and kinetic variables. Here we evaluate the contribution of pore cation binding to ion permeation and selectivity features of KcsA, a model potassium channel. For this, we used E71A and M96V KcsA mutants in which the equilibrium between conductive and nonconductive conformations of the channel is differently shifted. E71A KcsA is a noninactivating channel mutant. Binding of K(+) to this mutant reveals a single set of low-affinity K(+) binding sites, similar to that seen in the binding of K(+) to wild-type KcsA that produces a conductive, low-affinity complex. This seems consistent with the observed K(+) permeation in E71A. Nonetheless, the E71A mutant retains K(+) selectivity, which cannot be explained on the basis of just its low affinity for this ion. At variance, M96V KcsA is a rapidly inactivating mutant that has lost selectivity for K(+) and also conducts Na(+). Here, low-affinity binding and high-affinity binding of both cations are detected, seemingly in agreement with both being permeating species in this mutant channel. In conclusion, binding of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation properties. Ion binding stabilizes greatly the channel and, depending upon ion type and concentration, leads to different conformations and ion binding affinities. High-affinity states guarantee binding of specific ions and mediate ion selectivity but are nonconductive. Conversely, low-affinity states would not discriminate well among different ions but allow permeation to occur.     

A point mutation in the pore region alters gating, Ca(2+) blockage, and permeation of olfactory cyclic nucleotide-gated channels

Upon stimulation by odorants, Ca(2+) and Na(+) enter the cilia of olfactory sensory neurons through channels directly gated by cAMP. Cyclic nucleotide-gated channels have been found in a variety of cells and extensively investigated in the past few years. Glutamate residues at position 363 of the alpha subunit of the bovine retinal rod channel have previously been shown to constitute a cation-binding site important for blockage by external divalent cations and to control single-channel properties. It has therefore been assumed, but not proven, that glutamate residues at the corresponding position of the other cyclic nucleotide-gated channels play a similar role. We studied the corresponding glutamate (E340) of the alpha subunit of the bovine olfactory channel to determine its role in channel gating and in permeation and blockage by Ca(2+) and Mg(2+). E340 was mutated into either an aspartate, glycine, glutamine, or asparagine residue and properties of mutant channels expressed in Xenopus laevis oocytes were measured in excised patches. By single-channel recordings, we demonstrated that the open probabilities in the presence of cGMP or cAMP were decreased by the mutations, with a larger decrease observed on gating by cAMP. Moreover, we observed that the mutant E340N presented two conductance levels. We found that both external Ca(2+) and Mg(2+) powerfully blocked the current in wild-type and E340D mutants, whereas their blockage efficacy was drastically reduced when the glutamate charge was neutralized. The inward current carried by external Ca(2+) relative to Na(+) was larger in the E340G mutant compared with wild-type channels. In conclusion, we have confirmed that the residue at position E340 of the bovine olfactory CNG channel is in the pore region, controls permeation and blockage by external Ca(2+) and Mg(2+), and affects channel gating by cAMP more than by cGMP.     

Na,K-ATPase mutations in familial hemiplegic migraine lead to functional inactivation

The Na,K-ATPase is an ion-translocating transmembrane protein that actively maintains the electrochemical gradients for Na+ and K+ across the plasma membrane. The functional protein is a heterodimer comprising a catalytic alpha-subunit (four isoforms) and an ancillary beta-subunit (three isoforms). Mutations in the alpha2-subunit have recently been implicated in familial hemiplegic migraine type 2, but almost no thorough studies of the functional consequences of these mutations have been provided. We investigated the functional properties of the mutations L764P and W887R in the human Na,K-ATPase alpha2-subunit upon heterologous expression in Xenopus oocytes. No Na,K-ATPase-specific pump currents could be detected in cells expressing these mutants. The binding of radiolabelled [3H]ouabain to intact cells suggested that this could be due to a lack of plasma membrane expression. However, plasma membrane isolation showed that the mutated pumps are well expressed at the plasma membrane. 86Rb+-flux and ATPase activity measurements demonstrated that the mutants are inactive. Therefore, the primary disease-causing mechanism is loss-of-function of the Na,K-ATPase alpha2-isoform.     

Steric selectivity in Na channels arising from protein polarization and mobile side chains

Monte Carlo simulations of equilibrium selectivity of Na channels with a DEKA locus are performed over a range of radius R and protein dielectric coefficient epsilon(p). Selectivity arises from the balance of electrostatic forces and steric repulsion by excluded volume of ions and side chains of the channel protein in the highly concentrated and charged (approximately 30 M) selectivity filter resembling an ionic liquid. Ions and structural side chains are described as mobile charged hard spheres that assume positions of minimal free energy. Water is a dielectric continuum. Size selectivity (ratio of Na+ occupancy to K+ occupancy) and charge selectivity (Na+ to Ca2+) are computed in concentrations as low as 10(-5) M Ca2+. In general, small R reduces ion occupancy and favors Na+ over K+ because of steric repulsion. Small epsilon(p) increases occupancy and favors Na+ over Ca2+ because protein polarization amplifies the pore's net charge. Size selectivity depends on R and is independent of epsilon(p); charge selectivity depends on both R and epsilon(p). Thus, small R and epsilon(p) make an efficient Na channel that excludes K+ and Ca2+ while maximizing Na+ occupancy. Selectivity properties depend on interactions that cannot be described by qualitative or verbal models or by quantitative models with a fixed free energy landscape.