Beta 1 subunits of sodium channels. Studies with subunit-specific antibodies

The purified Na+ channel from rat brain consists of alpha (260 kDa), beta 1 (36 kDa), and beta 2 (33 kDa) subunits. Pure beta 1 subunits were prepared from purified rat brain Na+ channels which had been adsorbed to hydroxylapatite resin and used to prepare specific anti-beta 1 subunit antiserum. Antibodies purified from this antiserum by antigen affinity chromatography immunoprecipitate 125I-labeled, purified beta 1 subunits and purified Na+ channels (measured as high affinity [3H] saxitoxin binding sites) and recognize beta 1 subunits on immunoblots of solubilized rat brain membranes. The affinity-purified anti-beta 1 antibodies recognize beta 1 subunits in rat spinal cord, heart, skeletal muscle, and sciatic nerve, but do not detect immunoreactive beta 1 subunits in eel electroplax or eel brain. The developmental time course of expression of immunoreactive beta 1 subunits in rat forebrain was measured by immunoprecipitation followed by immunoblotting with affinity-purified anti-beta 1 antibodies. The amount of immunoreactive beta 1 subunits increased steadily to adult levels during the first 21 days of postnatal development.     

Stabilization of a sodium channel state with high affinity for saxitoxin by intramolecular cross-linking. Evidence for allosteric effects of saxitoxin binding

Incubation of purified rat brain sodium channels at 37 degrees C or at high ionic strength causes a concomitant loss of saxitoxin-binding activity and dissociation of beta 1 subunits. Reaction with hydrophilic carbodiimides produced a resistance against the loss of saxitoxin binding and caused covalent cross-linking of alpha, beta 1, and beta 2 subunits. In the presence of saxitoxin, this cross-linking reaction led to formation of a state with increased affinity for saxitoxin. However, analysis of the concentration dependence of covalent cross-linking and its inhibition by hydrophilic nucleophiles showed that the stabilization of the saxitoxin-binding activity was due to the formation of a small number of isopeptide bonds in the alpha subunit rather than to cross-linking of alpha and beta 1 subunits. In the presence of amine nucleophiles, carbodiimides caused loss of saxitoxin binding, which was prevented in the presence of the toxin. Nucleophiles yielding positively charged amide products were more effective than those forming uncharged or negatively charged products. Under conditions where saxitoxin protected the binding activity of the sodium channel from inactivation, the overall availability of carboxyl groups for reaction was increased, providing evidence for a toxin-induced conformational change on binding. These results are considered in terms of an allosteric model of saxitoxin binding, in which the functional form of the sodium channel having high affinity for saxitoxin can be stabilized against inactivation by noncovalent interactions with beta 1 subunits, binding of saxitoxin and tetrodotoxin, or intramolecular cross-linking of amino acid residues within the alpha subunit.     

Modulation of the Na,K-pump function by beta subunit isoforms

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.     

Functional properties of rat brain sodium channels lacking the beta 1 or beta 2 subunit

The sodium channel purified from rat brain is a heterotrimeric complex of alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000) subunits. alpha and beta 2 are attached by disulfide bonds. Removal of beta 1 subunits by incubation in 1.0 M MgCl2 followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 2 which did not bind [3H]saxitoxin, mediate veratridine-activated 22Na+ influx, or bind the 125I-labeled alpha-scorpion toxin from Leiurus quinquestriatus (LqTx). In contrast, removal of beta 2 subunits by reduction of disulfide bonds with 1.5 mM dithiothreitol followed by reconstitution into phospholipid vesicles yielded a preparation of alpha beta 1 that retained full sodium channel function. Alpha beta 1 bound [3H]saxitoxin with a KD of 4.1 nM at 36 degrees C. It mediated veratridine-activated 22Na+ influx at a comparable initial rate as intact sodium channels with a K0.5 for veratridine of 46 microM. Tetracaine and tetrodotoxin blocked 22Na+ influx. Like intact sodium channels, alpha beta 1 bound 125I-LqTx in a voltage-dependent manner with a KD of approximately 6 nM at a membrane potential of -60 mV and was specifically covalently labeled by azidonitrobenzoyl 125I-LqTx. When incorporated into planar phospholipid bilayers, alpha beta 1 formed batrachotoxin-activated sodium channels of 24 pS whose voltage-dependent activation was characterized by V50 = -110 mV and an apparent gating charge of 3.3 +/- 0.3. These results indicate that beta 2 subunits are not required for the function of purified and reconstituted sodium channels while a complex of alpha and beta 1 subunits is both necessary and sufficient for channel function in the purified state.     

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+.     

Competitive and synergistic interactions of G protein beta(2) and Ca(2+) channel beta(1b) subunits with Ca(v)2.1 channels, revealed by mammalian two-hybrid and fluorescence resonance energy transfer measurements

Presynaptic Ca2+ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein betagamma subunits modulate the binding of the Ca2+ channel beta subunit on the Ca2+ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2+ channel beta and G protein beta subunits on the two separate binding sites, i.e. the loopI-II and the C terminus, and on the full-length P/Q-type alpha12.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca2+ channel beta1b subunit induces a strong fluorescent signal when interacting with the loopI-II but not with the C terminus. In contrast, the G protein beta subunit induces FRET signals on both the C terminus and loopI-II. Analysis of the interactions on the full-length channel indicates that Ca2+ channel beta1b and G protein beta subunits bind to the alpha1 subunit at the same time. Coexpression of the G protein increases the FRET signal between alpha1/beta1b FRET pairs but not for alpha1/beta1b FRET pairs where the C terminus was deleted from the alpha1 subunit. The results suggest that the G protein alters the orientation and/or association between the Ca2+ channel beta and alpha12.1 subunits, which involves the C terminus of the alpha1 subunit and may corresponds to a new conformational state of the channel.     

All three WW domains of murine Nedd4 are involved in the regulation of epithelial sodium channels by intracellular Na+

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPxY motif which is necessary for interaction with the WW domains of the ubiquitin-protein ligase, Nedd4. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PY motif of either the beta or gamma subunits, results in increased ENaC activity. We have recently shown using the whole-cell patch clamp technique that Nedd4 mediates the ubiquitin-dependent down-regulation of Na+ channel activity in response to increased intracellular Na+. In this paper, we demonstrate that WW domains 2 and 3 bind alpha-, beta-, and gamma-ENaC with varying degrees of affinity, whereas WW domain 1 does not bind to any of the subunits. We further show using whole-cell patch clamp techniques that Nedd4-mediated down-regulation of ENaC in mouse mandibular duct cells involves binding of the WW domains of Nedd4 to three distinct sites. We propose that Nedd4-mediated down-regulation of Na+ channels involves the binding of WW domains 2 and 3 to the Na+ channel and of WW domain 1 to an unknown associated protein.     

Beta 2 subunits of sodium channels from vertebrate brain. Studies with subunit-specific antibodies

The sodium channel purified from rat brain is composed of three subunits: alpha (Mr 260,000), beta 1 (Mr 36,000), and beta 2 (Mr 33,000). alpha and beta 2 subunits are linked through disulfide bonds. Procedures are described for preparative isolation of the beta 1 and beta 2 subunits under native conditions. Pure beta 2 subunits obtained by this procedure were used to prepare a specific anti-beta 2 subunit antiserum. Antibodies purified from this serum by antigen affinity chromatography recognize only disulfide-linked alpha beta 2 complexes and beta 2 subunits in immunoblots, and immunoprecipitate 32P-labeled alpha subunits of purified sodium channels having intact disulfide bonds, but not those of sodium channels from which beta 2 subunits have been detached by reduction of disulfide bonds. These antibodies also immunoprecipitate 89% of the high affinity saxitoxin-binding sites from rat brain membranes, indicating that nearly all sodium channels in rat brain have disulfide-linked alpha beta 2 subunits. Approximately 22% of beta 2 subunits in adult rat brain are not disulfide-linked to alpha subunits. Anti-beta 2 subunit antibodies are specific for sodium channels in the central nervous system and will not cross-react with sodium channels in skeletal muscle or sciatic nerve. The brains of a broad range of vertebrate species, including electric eel, are shown to express sodium channels with disulfide-linked alpha beta 2 subunits.     

Structural basis for ligand selectivity of heteromeric olfactory cyclic nucleotide-gated channels

In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca(2+) entry and depolarization of the cell. These CNG channels are composed of alpha subunits and at least two types of beta subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the beta subunits (CNG5, CNCalpha4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with alpha subunit homomultimers (alpha channels), channels composed of alpha and beta subunits (alpha+beta channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the alpha subunit and a chimeric beta subunit with the alpha subunit cyclic nucleotide-binding region (CNBR) (alpha+beta-CNBRalpha channels) were restored to near the values for alpha channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the beta subunit to a glutamic acid as in the alpha subunit (beta-M475E) reverted the K(1/2,cAMP)/K(1/2,cGMP) and K(1/2, cIMP)/K(1/2,cGMP) ratios of alpha+beta-M475E channels to be very similar to those of alpha channels. In addition, comparison of alpha+beta-CNBRalpha channels with alpha+beta-M475E channels suggests that the CNBR of the beta subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type beta subunit, the chimeric beta/alpha subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for alpha+beta channels, block of alpha+betaCNBRalpha channels by internal Mg(2+) was not steeply voltage-dependent (zdelta approximately 1e(-)) as compared to block of alpha channels (zdelta 2.7e(-)). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the beta subunit alters ligand selectivity of the heteromeric channels.     

Up-regulation of cell-surface alpha4beta2 neuronal nicotinic receptors by lower temperature and expression of chimeric subunits.

The predominant nicotinic acetylcholine receptor (nAChR) expressed in vertebrate brain is a pentamer containing alpha4 and beta2 subunits. In this study we have examined how temperature and the expression of subunit chimeras can influence the efficiency of cell-surface expression of the rat alpha4beta2 nAChR. Functional recombinant alpha4beta2 nAChRs, showing high affinity binding of nicotinic radioligands (K(d) = 41 +/- 22 pM for [(3)H]epibatidine), are expressed in both stably and transiently transfected mammalian cell lines. Despite this, only very low levels of alpha4beta2 nAChRs can be detected on the cell surface of transfected mammalian cells maintained at 37 degrees C. At 30 degrees C, however, cells expressing alpha4beta2 nAChRs show a 12-fold increase in radioligand binding (with no change in affinity), and a 5-fold up-regulation in cell-surface receptors with no increase in total subunit protein. In contrast to "wild-type" alpha4 and beta2 subunits, chimeric nicotinic/serotonergic subunits ("alpha4chi" and "beta2chi") are expressed very efficiently on the cell surface (at 30 degrees C or 37 degrees C), either as hetero-oligomeric complexes (e.g. alpha4chi+beta2 or alpha4chi+beta2chi) or when expressed alone. Compared with alpha4beta2 nAChRs, expression of complexes containing chimeric subunits typically results in up to 20-fold increase in nicotinic radioligand binding sites (with no change in affinity) and a similar increase in cell-surface receptor, despite a similar level of total chimeric and wild-type protein.     

Palmitylation, sulfation, and glycosylation of the alpha subunit of the sodium channel. Role of post-translational modifications in channel assembly

Antibodies to the alpha and beta 2 subunits and site-directed antibodies that distinguish alpha subunits of the RI and RII subtypes have been used to study the biosynthesis and assembly of sodium channels. The RII sodium channel subtype is preferentially expressed in rat brain neurons in primary cell culture. Post-translational processing of alpha subunits includes incorporation of palmityl residues in thioester linkage and sulfate residues attached to oligosaccharides. The incorporation of [3H] palmitate into alpha subunits is inhibited by tunicamycin, indicating that it occurs in the early stages of biosynthesis but after co-translational glycosylation. Mature alpha subunits are attached to beta 2 subunits through disulfide bonds within 1 h after synthesis and up to 30% can be specifically immunoprecipitated from the cell surface with antibodies against the beta 2 subunits by 4 h after synthesis. The remaining alpha subunits remain in an intracellular pool. The alpha subunits synthesized in the presence of castanospermine and swainsonine have reduced apparent size. Castanospermine prevents incorporation of approximately 81% of the sialic acid of the alpha subunit and inhibits sulfation but not palmitylation. Although inhibition of glycosylation with tunicamycin blocks assembly of functional sodium channels, castanospermine and swainsonine do not prevent the covalent assembly of alpha and beta 2 subunits or the transport of alpha beta 2 complexes to the cell surface, and sodium channels synthesized under these conditions have normal affinity for saxitoxin. Thus, the extensive processing and terminal sialylation of oligosaccharide chains during maturation of the alpha subunit is not essential. A kinetic model for biosynthesis, processing, and assembly of sodium channel subunits is presented.     

Identification and characterization of Mg2+ binding sites in isolated alpha and beta subunits of H(+)-ATPase from Bacillus PS3

The properties of the nucleotide binding sites in the isolated beta and alpha subunits of H(+)-ATPase from Bacillus PS3 (TF1) have been examined by studying the EPR properties of bound VO(2+), which is a paramagnetic probe for the native Mg2+ cation cofactor. The amino acid ligands of the VO2+ complexes with the isolated beta subunit, with the isolated alpha subunit, with different mixtures of both alpha and beta subunits, and with the catalytic alpha 3 beta 3 gamma subcomplex have been characterized by a combination of EPR, ESEEM, and HYSCORE spectroscopies. The EPR spectrum of the isolated beta subunit with bound VO2+ (1 VO2+/beta) is characterized by (51)V hyperfine coupling parameters (A( parallel) = 168 x 10(-)(4) cm(-)(1) and A( perpendicular) = 60 x 10(-)(4) cm(-)(1)) that suggest that VO2+ binds to the isolated beta subunit with at least one nitrogen ligand. Results obtained for the analogous VO2+ complex with the isolated alpha subunit are virtually identical. ESEEM and HYSCORE spectra are also reported and are similar for both complexes, indicating a very similar coordination scheme for VO2+ bound to isolated alpha and beta subunits. In the isolated beta (or alpha) subunit, the bound VO2+ cation is coordinated by one nitrogen ligand with hyperfine coupling parameters A( parallel)((14)N) = 4.44 MHz, and A( perpendicular)((14)N) = 4.3 MHz and quadrupole coupling parameters e(2)()qQ approximately 3.18 MHz and eta approximately 1. These are typical for amine-type nitrogen ligands equatorial to the VO2+ cation; amino acid residues in the TF1 beta and alpha subunits with nitrogen donors that may bind VO2+ are reviewed. VO2+ bound to a mixture of alpha and beta subunits in the presence of 200 mM Na2SO4 to promote the formation of the alpha 3 beta 3 hexamer has a second nitrogen ligand with magnetic properties similar to those of a histidine imidazole. This situation is analogous to that in the alpha 3 beta 3 gamma subcomplex and in the whole TF1 enzyme [Buy, C., Matsui, T., Andrianambinintsoa, S., Sigalat, C., Girault, G., and Zimmermann, J.-L. (1996) Biochemistry 35, 14281-14293]. These data are interpreted in terms of only partially structured nucleotide binding sites in the isolated beta and alpha subunits as compared to fully structured nucleotide binding sites in the alpha 3 beta 3 heterohexamer, the alpha 3 beta 3 gamma subcomplex, and the whole TF1 ATPase.     

Photoaffinity labeling of (Na+K+)-ATPase with [125I]iodoazidocymarin

A radioiodinated, photoactive cardiac glycoside derivative, 4'-(3-iodo-4-azidobenzene sulfonyl)cymarin (IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC inhibited the activity of (Na+K+)-ATPase in electroplax microsomes from Electrophorus electricus with the same I50 as cymarin. [125I]IAC binding, in the presence of Mg2+ and Pi, was specific, of high affinity (KD = 0.4 microM), and reversible (k-1 = 0.11 min-1) at 30 degrees C. At 0 degree C, the complex was stable for at least 3 h, thus permitting washing before photolysis. Analysis of [125]IAC photolabeled electroplax microsomes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (7-14%) showed that most of the incorporated radioactivity was associated with the alpha (Mr = 98,000) and beta (Mr = 44,000) subunits of the (Na+K+)-ATPase (ratio of alpha to beta labeling = 2.5). A higher molecular weight peptide (100,000), similar in molecular weight to the brain alpha(+) subunit, and two lower molecular weight peptides (12,000-15,000), which may be proteolipid, were also labeled. Two-dimensional gel electrophoresis (isoelectric focusing then SDS-PAGE, 10%) resolved the beta subunit into 12 labeled peptides ranging in pI from 4.3 to 5.5. When (Na+K+)-ATPase in synaptosomes from monkey brain cortex was photolabeled and analyzed by SDS-PAGE (7-14%), specific labeling of the alpha(+), alpha, and beta subunits could be detected (ratio of alpha(+) plus alpha to beta labeling = 35). The results show that [125I]IAC is a sensitive probe of the cardiac glycoside binding site of (Na+K+)-ATPase and can be used to detect the presence of the alpha(+) subunit in crude membrane fractions from various sources.     

Functional consequences of lidocaine binding to slow-inactivated sodium channels

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use- dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to - 20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow- inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow- inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.     

Identification of amino acid residues of GABA(A) receptor subunits contributing to the formation and affinity of the tert-butylbicyclophosphorothionate binding site

A chimeric GABA(A) receptor subunit was constructed that contained the beta3 sequence from the N-terminus to the first two amino acids of the second transmembrane (TM2) domain. The remaining part of this chimera had the sequence of the alpha1 subunit. On co-expression with alpha1 subunits, this chimera was able to form heterooligomeric channels that were open in the absence of GABA. Picrotoxin and tert-butylbicyclophosphorothionate (TBPS) were able to block these channels with low potency. These channels exhibited high-affinity [3H]muscimol but no high-affinity [35S]TBPS binding sites. Introduction of V251, A252, and L253 of the beta3 subunit into the chimera resulted in the formation of closed channels that could be opened by GABA. The introduction of A252 and L253 of the beta3 subunit into this chimera was sufficient to reconstitute the specific high-affinity [35S]TBPS binding site in receptors composed of the chimera and alpha1 subunits. Replacement of other amino acids of the TM2 region of the chimera with corresponding amino acids of the beta3 subunit modulated the affinity of this [35S]TBPS binding site. Results obtained provide important information on the structure-function relationship of GABA(A) receptors.     

Interaction of charybdotoxin S10A with single maxi-K channels: kinetics of blockade depend on the presence of the beta 1 subunit

The maxi-K channel from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine ChTX-S10A blocking kinetics of single maxi-K channels in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, maxi-K channels from smooth muscle showed ChTX-S10A block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, ChTX-S10A block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle maxi-K channel. Increasing the external ionic strength caused a decrease in ChTX-S10A block times for maxi-K channel complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native maxi-K channels by monitoring the kinetics of ChTX blockade of single channels, and they suggest that maxi-K channels in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-IbTX-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-IbTX-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-IbTX-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of ChTX block of single maxi-K channels provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.     

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.     

Recombinant addition of N-glycosylation sites to the basolateral Na,K-ATPase beta1 subunit results in its clustering in caveolae and apical sorting in HGT-1 cells

In most polarized cells, the Na,K-ATPase is localized on the basolateral plasma membrane. However, an unusual location of the Na,K-ATPase was detected in polarized HGT-1 cells (a human gastric adenocarcinoma cell line). The Na,K-ATPase alpha1 subunit was detected along with the beta2 subunit predominantly on the apical membrane, whereas the Na,K-ATPase beta1 subunit was not found in HGT-1 cells. However, when expressed in the same cell line, a yellow fluorescent protein-linked Na,K-ATPase beta1 subunit was localized exclusively to the basolateral surface and resulted in partial redistribution of the endogenous alpha1 subunit to the basolateral membrane. The human beta2 subunit has eight N-glycosylation sites, whereas the beta1 isoform has only three. Accordingly, up to five additional N-glycosylation sites homologous to the ones present in the beta2 subunit were successively introduced in the beta1 subunit by site-directed mutagenesis. The mutated beta1 subunits were detected on both apical and basolateral membranes. The fraction of a mutant beta1 subunit present on the apical membrane increased in proportion to the number of glycosylation sites inserted and reached 80% of the total surface amount for the beta1 mutant with five additional sites. Clustered distribution and co-localization with caveolin-1 was detected by confocal microscopy for the endogenous beta2 subunit and the beta1 mutant with additional glycosylation sites but not for the wild type beta1 subunit. Hence, the N-glycans linked to the beta2 subunit of the Na,K-ATPase contain apical sorting information, and the high abundance of the beta2 subunit isoform, which is rich in N-glycans, along with the absence of the beta1 subunit, is responsible for the unusual apical location of the Na,K-ATPase in HGT-1 cells.