The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter

In Arabidopsis thaliana, the SOS1 (Salt Overly Sensitive 1) locus is essential for Na(+) and K(+) homeostasis, and sos1 mutations render plants more sensitive to growth inhibition by high Na(+) and low K(+) environments. SOS1 is cloned and predicted to encode a 127-kDa protein with 12 transmembrane domains in the N-terminal part and a long hydrophilic cytoplasmic tail in the C-terminal part. The transmembrane region of SOS1 has significant sequence similarities to plasma membrane Na(+)/H(+) antiporters from bacteria and fungi. Sequence analysis of various sos1 mutant alleles reveals several residues and regions in the transmembrane as well as the tail parts that are critical for SOS1 function in plant salt tolerance. SOS1 gene expression in plants is up-regulated in response to NaCl stress. This up-regulation is abated in sos3 or sos2 mutant plants, suggesting that it is controlled by the SOS3/SOS2 regulatory pathway.     

Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana,by SOS2 and SOS3

Maintaining low levels of sodium ions in the cell cytosol is critical for plant growth and development. Biochemical studies suggest that Na(+)/H(+) exchangers in the plasma membrane of plant cells contribute to cellular sodium homeostasis by transporting sodium ions out of the cell; however, these exchangers have not been identified at the molecular level. Genetic analysis has linked components of the salt overly sensitive pathway (SOS1-3) to salt tolerance in Arabidopsis thaliana. The predicted SOS1 protein sequence and comparisons of sodium ion accumulation in wild-type and sos1 plants suggest that SOS1 is involved directly in the transport of sodium ions across the plasma membrane. To demonstrate the transport capability of SOS1, we studied Na(+)/H(+)-exchange activity in wild-type and sos plants using highly purified plasma membrane vesicles. The results showed that plasma membrane Na(+)/H(+)-exchange activity was present in wild-type plants treated with 250 mM NaCl, but this transport activity was reduced by 80% in similarly treated sos1 plants. In vitro addition of activated SOS2 protein (a protein kinase) increased Na(+)/H(+)-exchange activity in salt-treated wild-type plants 2-fold relative to transport without added protein. However, the addition of activated SOS2 did not have any stimulatory effect on the exchange activity in sos1 plants. Although vesicles of sos2 and sos3 plants had reduced plasma membrane Na(+)/H(+)-exchange activity, transport activity in both increased with the addition of activated SOS2 protein. These results demonstrate that SOS1 contributes to plasma membrane Na(+)/H(+) exchange and that SOS2 and SOS3 regulate SOS1 transport activity.     

Mouse Na+/K+-ATPase beta1-subunit has a K+-dependent cell adhesion activity for beta-GlcNAc-terminating glycans

A 48-kDa beta-N-acetylglucosamine (GlcNAc)-binding protein was isolated from mouse brain by GlcNAc-agarose column chromatography. The N-terminal amino acid residues showed the protein to be a mouse Na(+)/K(+)-ATPase beta1-subunit. When the recombinant FLAG-beta1-subunit expressed in Sf-9 cells was applied to a GlcNAc-agarose column, only the glycosylated 38- and 40-kDa proteins bound to the column. In the absence of KCl, little of the proteins bound to a GlcNAc-agarose column, but the 38- and 40-kDa proteins bound in the presence of KCl at concentrations above 1 mM. Immunohistochemical study showed that the beta1-subunit and GlcNAc-terminating oligosaccharides are at the cell contact sites. Inclusion of anti-beta1-subunit antibody or chitobiose in cell aggregation assays using mouse neural cells resulted in inhibition of cell aggregation. These results indicate that the Na(+)/K(+)-ATPase beta1-subunit is a potassium-dependent lectin that binds to GlcNAc-terminating oligosaccharides: it may be involved in neural cell interactions.     

The functional effect of adenoviral Na+/Ca2+ exchanger overexpression in rabbit myocytes depends on the activity of the Na+/K+-ATPase

OBJECTIVES: The functional consequences of Na+/Ca2+ exchanger (NCX) overexpression in heart failure have been controversially discussed. NCX function strongly depends on intracellular sodium which has been shown to be increased in heart failure. METHODS AND RESULTS: We investigated the Na+/K+-ATPase (NKA) inhibitor ouabain (0.5-16 micromol/l) in electrically stimulated, isotonically contracting adult rabbit cardiocytes overexpressing NCX after adenoviral gene transfer (Ad-NCX-GFP, 48 h culture time). Myocytes transfected with adenovirus encoding for green fluorescent protein (Ad-GFP) served as a control. Contractions were analyzed by video-edge detection. In the Ad-NCX-GFP group, the maximum inotropic response was significantly reduced by 50.7% (P<0.05). This was a result of an enhanced susceptibility to contracture after exposure to the drug (median concentration (25-75%): 4 (4-8) vs. 8 (6-16) micromol/l, P<0.05). When analyzing relaxation before contracture, the maximum relaxation velocity was reduced (0.15+/-0.04 vs. 0.27+/-0.04 microm/s, P<0.05) and the time from peak shortening to 90% of relaxation was increased (298+/-39 vs. 185+/-15 ms, P<0.05). No differences in systolic and diastolic parameters were observed with the Na+ channel modulator BDF9198 (1 micromol/l). CONCLUSIONS: Inhibition of NKA by ouabain induces a combined diastolic and systolic dysfunction in NCX overexpressing rabbit myocytes. This may be the consequence of cytoplasmic Ca2+ overload due to inhibition of forward mode or induction of reverse mode Na+/Ca2+ exchange. In end-stage failing human myocardium and during digitalis treatment this mechanism may be of major importance.     

Phospholemman phosphorylation mediates the protein kinase C-dependent effects on Na+/K+ pump function in cardiac myocytes

Because phospholemman (PLM) regulates the Na(+)/K(+) pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na(+)](i) decline (fluorescence measurements) and current (I(pump)) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, I(pump) (both at 10 and 100 mmol/L Na(+) in the pipette solution) and maximal NKA-mediated Na(+) extrusion rate (V(max)) from 7.9+/-1.1 to 12.7+/-1.9 mmol.L(-1) per minute without altering NKA affinity for internal Na(+) (K(0.5)). In PLM knockout mice, PDBu had no effect on either V(max) or K(0.5). After pretreatment with isoproterenol (ISO) (1 mumol/L), PDBu still increased the NKA V(max) and PLM phosphorylation at Ser63 and Ser68. Conversely, after pretreatment with PDBu, ISO further increased the Na(+) affinity of NKA and phosphorylation at Ser68, as it did alone without PDBu. The final NKA activity was independent of the application sequence. The NKA activity in PLM knockout myocytes, after normalizing the protein level, was similar to that after PDBu and ISO treatment. We conclude that (1) PLM mediates the PKC-dependent activation of NKA function in cardiac myocytes, (2) PDBu and ISO effects are additive in the mouse (affecting mainly V(max) and K(0.5), respectively), and (3) PDBu and ISO combine to activate NKA in wild-type to the level found in the PLM knockout mouse.     

Na+/K+-ATP酶ATP1A2基因突变与偏头痛

偏头痛是一种常见的反复发作性神经疾病。病因涉及遗传和环境因素,其发病机制尚未完全阐明。家族性偏瘫型偏头痛(familial hemiplegic migraine,FHM)中Na /K ATP酶α2亚基ATP1A2基因突变的发现,是偏头痛遗传学研究的重大突破。目前已鉴定出25种ATP1A2基因突变,且绝大部分为错义突变,这些突变或者引起Na /K -ATP酶α2亚基单个等位基因的功能丧失,或者引起钠钾泵的动力学改变。不过,ATP1A2基因与普通偏头痛的关系尚不明确,有待进一步研究。     

Angiotensin II modulates the activity of the Na+/K+ATPase in eel kidney

We have previously shown that angiotensin II (Ang II) has a role at the level of the eel gill chloride cell regulating sodium balance, and therefore osmoregulation; the purpose of the present study was to extend these findings to another important osmoregulatory organ, the kidney. By catalytic histochemistry Na(+)/K(+)ATPase activity was found in both sea water (SW)- and freshwater (FW)-adapted eel kidney, particularly at the level of both proximal and distal tubules. Quantitation of tubular cell Na(+)/K(+)ATPase activity, by imaging, gave values in SW-adapted eels which were double those found in FW-adapted eels (Student's t-test: P<0.0001). This was due to a reduced number of positive tubules present in FW-adapted eels compared with SW-adapted eels. By conventional enzymatic assay, the Na(+)/K(+)ATPase activity in isolated tubular cells from SW-adapted eels showed values 1.85-fold higher those found in FW-adapted eels (Student's ttest: P<0.0001). Perfusion of kidney for 20 min with 100 nM Ang II provoked a significant increase (1.8-fold) in Na(+)/K(+)ATPase activity in FW, due to up-regulation of Na(+)/K(+)ATPase activity in a significantly larger number of tubules (Student's t-test: P<0.0001). The effect of 100 nM Ang II in SW-adapted kidneys was not significant. Stimulation with increasing Ang II concentrations was performed on isolated kidney tubule cells: Ang II provoked a dose-dependent stimulation of the Na(+)/K(+)ATPase activity in FW-adapted eels, reaching a maximum at 100 nM (1.82-fold stimulation), but no significant effect was found in SW-adapted eels (ANOVA: P<0.001 and P>0.05 respectively). Isolated tubule cells stimulated with 100 nM Ang II showed a significant generation of inositol trisphosphate (InsP(3)) and an increment in calcium release from intracellular stores. In conclusion, our results suggest that tubular Na(+)/K(+)ATPase is modulated by environmental salinity, and that Ang II has a role in regulating its activity in FW-adapted eels, probably through an InsP(3)-dependent mechanism.     

Parathyroid hormone stimulation of the Na+/K+ pump in rat clonal osteosarcoma cells

A clonal line of osteoblastic cells from a rat osteogenic sarcoma (UMR 106-06), known to possess parathyroid hormone (PTH)-responsive adenylate cyclase, has been shown to increase its rate of K+ uptake mediated by a Na+/K+ pump after exposure to the hormone. The increase in pump activity was not associated with significant changes in K+ efflux or Na+ influx and would therefore be expected to alter intracellular levels of both Na+ and K+. The maximal (75%) increase in pump activity was noted at a PTH concentration of 100 micrograms/l and half-maximal stimulation at 1.9 micrograms/l. The effect appeared to be independent of the adenylate cyclase system, since a synthetic peptide antagonist of PTH activation of adenylate cyclase failed to prevent stimulation of the Na+/K+ pump. Similarly, prostaglandin E2, an alternative agonist of adenylate cyclase in these cells, had no effect on the Na+/K+ pump. This novel action of PTH on monovalent cation transport in osteoblast-like cells should provide a clearer insight into the mechanisms of hormone-induced bone resorption.     

A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana

Electroneutral monovalent cation/proton antiport across the chloroplast envelope has been shown previously to have an important regulatory effect on stromal pH and thereby on photosynthetic carbon reduction. Here we report that an Arabidopsis nuclear gene, AtCHX23, encodes a putative Na(+)(K(+))/H(+) exchanger and functions in the adjustment of pH in the cytosol and possibly in maintaining a high pH level in the chloroplast stroma. The AtCHX23 protein is localized in the chloroplast envelope. Plastids from chx23 mutants had straight thylakoids but lacked grana lamellae. chx23 mutant leaves were pale yellow and had a much reduced chlorophyll content. The chlorophyll content of chx23 was increased by growing in medium at low (4.0) pH and decreased by growing at high (7.0) pH. The cytosolic pH in the leaves of the mutant was significantly higher than that in the wild type. chx23 mutants displayed a high sensitivity to NaCl. Together, these data indicate that CHX23 is a probable chloroplast Na(+)(K(+))/H(+) exchanger important for pH homeostasis and chloroplast development and function.     

Energy-dispersive,bulk specimen X-ray microanalytical measurement of the intracellular Na+/K+ ratio in human laryngeal tumors

Summary Energy-dispersive X-ray microanalysis was performed on human biopsy materials taken during laryngoscopic intervention in 18 cases. The removed tissue pieces were divided into two parts. One of them was used for pathohistological studies, the other was processed for X-ray microanalysis by the freeze-fracture freeze-drying method. Of the cases investigated 4 proved to be of benign character, whereas the rest contained carcinoma planocellulare keratoides or nonkeratoides. Bulk specimen energy-dispersive X-ray microanalysis of 135 cells from the benign tissue samples revealed an average Na⁺/K⁺ molar ratio of 0.13±0.01 (SEM) in the intracellular space, with a regular Gaussian distribution. In the cases of carcinomas 641 cells were measured, the average of the same ratio was 0.67±0.03 (SEM) due mostly to an increase in the Na⁺ content. The distribution of data was apparently not normal in the cancerous samples. These observations and some theoretical considerations support the notion that the intracellular Na⁺/K⁺ ratio correlates with the proliferative capacity of tissues. The relevance of some recent biochemical results is also discussed in this respect.     

The Arabidopsis thaliana HY1 locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases

The hy1 mutants of Arabidopsis thaliana fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXalpha, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the HY1 gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of hy1 contain DNA lesions within the HY1 coding region, and a genomic sequence spanning the HY1 locus complements the hy1-1 mutation. HY1 is a member of a gene family and is expressed in a variety of A. thaliana tissues. Based on its homology, we propose that HY1 encodes a higher-plant heme oxygenase, designated AtHO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXalpha.     

Expression of the Na+/H+ exchanger regulatory protein family in genetically hypertensive rats

OBJECTIVE: To examine a possible involvement of a regulatory protein of Na+/H+ exchanger (NHE) in the increased renal NHE activity in spontaneously hypertensive rats (SHR), we investigated mRNA expression of inhibitory members of the NHE regulatory protein family, NHERF1 and NHERF2, in the kidney. DESIGN: Prehypertensive 4-week-old and hypertensive 11-week-old SHR and age-matched Wistar-Kyoto (WKY) rats were used to determine the changes in NHE activity and NHERF family expression in the kidney. Dahl salt sensitive (DS) and resistant rats were also used to examine whether these changes are specific for SHR. METHODS: mRNA expression in the kidney was quantified by RNase protection assay. The NHE activity in primary cultured proximal tubular cells was measured as Na-dependent pHi recovery rate by the NH4Cl prepulse technique with 2'7'-bis-(2-carboxyethyl)-5.6-carboxyfluorescein (BCECF). RESULTS: NHERF1 mRNA expression was significantly decreased in both prehypertensive and hypertensive SHR in comparison with age-matched WKY rats, whereas NHERF2 mRNA expression was significantly increased in SHR only in the hypertensive period. Antihypertensive treatment did not abolish these changes seen in control SHR. On the other hand, hypertensive DS rats fed a high-salt diet showed significant decreases in NHE activity and NHE3 mRNA expression compared with normotensive DS rats fed a low-salt diet, without significant changes in NHERF1 and NHERF2 mRNA expression. CONCLUSION: These results suggest that decreased expression of NHERF1 may be related to the enhanced NHE activity in SHR and that these changes are likely to be genetically determined, whereas the increased NHERF2 expression may be induced as a compensatory mechanism.     

Energy landscape of the reactions governing the Na+ deeply occluded state of the Na+/K+-ATPase in the giant axon of the Humboldt squid

The Na(+)/K(+) pump is a nearly ubiquitous membrane protein in animal cells that uses the free energy of ATP hydrolysis to alternatively export 3Na(+) from the cell and import 2K(+) per cycle. This exchange of ions produces a steady-state outwardly directed current, which is proportional in magnitude to the turnover rate. Under certain ionic conditions, a sudden voltage jump generates temporally distinct transient currents mediated by the Na(+)/K(+) pump that represent the kinetics of extracellular Na(+) binding/release and Na(+) occlusion/deocclusion transitions. For many years, these events have escaped a proper thermodynamic treatment due to the relatively small electrical signal. Here, taking the advantages offered by the large diameter of the axons from the squid Dosidicus gigas, we have been able to separate the kinetic components of the transient currents in an extended temperature range and thus characterize the energetic landscape of the pump cycle and those transitions associated with the extracellular release of the first Na(+) from the deeply occluded state. Occlusion/deocclusion transition involves large changes in enthalpy and entropy as the ion is exposed to the external milieu for release. Binding/unbinding is substantially less costly, yet larger than predicted for the energetic cost of an ion diffusing through a permeation pathway, which suggests that ion binding/unbinding must involve amino acid side-chain rearrangements at the site.     

Steroid regulation of Na+/K+-ATPase activity in the rat kidney: effect of a new 19-nor-progestagen

The effects of Nomegestrol acetate (17 alpha-acetoxy-6-methyl-19-nor-4,6-pregnadiene-3,20-dione), a new 19-nor-progesterone derivative, on renal Na+/K+-ATPase activity were assessed in normal and adrenalectomized rats, and compared with the stimulatory or inhibitory actions produced by other steroids. This compound displayed an inhibitory effect which was similar to, but smaller than, that induced by progesterone and quite distinct from the stimulation produced by 19-nor-progesterone and corticosteroids. In addition, unlike progesterone, it did not antagonize the effect of aldosterone in adrenalectomized rats. This result, together with previous in-vivo and in-vitro observations on this compound indicates that additional modifications introduced in the molecular structure of 19-nor-progesterone produces a potent progestagenic substance virtually devoid of effects on renal Na+/K+-ATPase activity and sodium loss in urine.     

Conductance selectivity of Na+ across the K+ channel via Na+ trapped in a tortuous trajectory

Ion selectivity of the potassium channel is crucial for regulating electrical activity in living cells; however, the mechanism underlying the potassium channel selectivity that favors large K⁺ over small Na⁺ remains unclear. Generally, Na⁺ is not completely excluded from permeation through potassium channels. Herein, the distinct nature of Na⁺ conduction through the prototypical KcsA potassium channel was examined. Single-channel current recordings revealed that, at a high Na⁺ concentration (200 mM), the channel was blocked by Na⁺, and this blocking was relieved at high membrane potentials, suggesting the passage of Na⁺ across the channel. At a 2,000 mM Na⁺ concentration, single-channel Na⁺ conductance was measured as one-eightieth of the K⁺ conductance, indicating that the selectivity filter allows substantial conduit of Na⁺. Molecular dynamics simulations revealed unprecedented atomic trajectories of Na⁺ permeation. In the selectivity filter having a series of carbonyl oxygen rings, a smaller Na⁺ was distributed off-center in eight carbonyl oxygen-coordinated sites as well as on-center in four carbonyl oxygen-coordinated sites. This amphipathic nature of Na⁺ coordination yielded a continuous but tortuous path along the filter. Trapping of Na⁺ in many deep free energy wells in the filter caused slow elution. Conversely, K⁺ is conducted via a straight path, and as the number of occupied K⁺ ions increased to three, the concerted conduction was accelerated dramatically, generating the conductance selectivity ratio of up to 80. The selectivity filter allows accommodation of different ion species, but the ion coordination and interactions between ions render contrast conduction rates, constituting the potassium channel conductance selectivity.

Ion selectivity is a fundamental property of ion channels that generate the physiological functions of the cell membrane. Ion selectivity determines the direction of net currents through a channel under a given ionic composition of intracellular and extracellular solutions (1). Potassium channels carry outward K⁺ currents even in the presence of abundant Na⁺ ions in the extracellular solution. The selective passage of large K⁺ ions (ionic radius of 1.3 Å) over smaller Na⁺ ions (1.0 Å) (2–5) is a feature of the potassium channel that distinguishes it from other channels. The molecular mechanisms underlying this selectivity have been studied extensively for decades (2, 4, 6–8) but remain to be fully elucidated (6, 9–11). To examine the selectivity mechanism, the KcsA potassium channel has been applied as a prototypical channel; a broad spectrum of data related to ion permeation and selectivity has been accumulated (8, 12–17). The crystal structure of the KcsA channel revealed that its selectivity filter is narrow (3 Å in diameter) and short (12 Å in length), which is common for all potassium channels.Generally, potassium channels share a typical selectivity feature for monovalent cations, and the permeability ratio relative to K⁺, which is the most frequently used parameter for the ion selectivity, is plotted here as a function of the ionic radius of the relevant ion species (Fig. 1) (1, 4). The channel allows the permeation of ionic species in a limited window with respect to ion size. Ionic species, with ionic radii ranging from 0.9 to 1.7 Å, including K⁺, Tl⁺, Rb⁺, and Na⁺ as a limiting case, are permissible for conduction; however, larger and even a smaller ion (Li⁺) are rejected from permeation. This feature of the potassium channel has been explained by the classical and static concept of the snug fit (3, 18), wherein an ion species within a limited ion size is selected through matching to the cavity size in the pore via formation of a host-guest complex (19). However, molecular dynamics (MD) simulation has demonstrated that the filter structure of the potassium channel is intrinsically flexible, dismissing the strict cavity size (14). Moreover, the crystal structure revealed that even the nonconducting Li⁺ is bound in the selectivity filter (12). A more dynamic picture of the cavity, such as that based on the concept of strain energy, was proposed (10, 14, 19).Open in a separate windowFig. 1.Ion selectivity of potassium channels. Permeability ratios of monovalent cations relative to K⁺ as a function of ionic radius are shown for various types of potassium channels. The data were collected from literature (SI Appendix, Table S1) and are presented as a box plot, where the box covers the 25th to 75th percentiles, with the center line indicating the median. The potassium channel selectivity is characterized as a band-pass filter with a limited ion-size window (broken green line). The band-pass is arbitrarily decomposed into plausible small-pass (or low-pass; broken blue line) and large-pass (or high-pass; broken red line) filters. The lines do not have physical meanings.In the selectivity filter, K⁺ is coordinated to eight carbonyl oxygens (cage configuration), whereas Na⁺ is coordinated to four carbonyl oxygens (plane configuration) (9, 12, 20–24). High-affinity K⁺ binding to the filter deduced from the equilibrium crystal structure and spectroscopy has been interpreted as the basis for strict K⁺ selectivity (25–28). However, a comparison between the affinity and conductance revealed that the high affinity of an ion is not a determinant of its selectivity (11, 29). Recently, data supporting the low-affinity binding of K⁺ to the filter have been accumulated (30–32), and a dynamic principle has been proposed as an alternative mechanism for the selectivity (20, 33). Simulation studies revealed differential conduction of K⁺ and Na⁺ under different energy profiles, and Burykin et al. demonstrated that a different effective charge–charge dielectric for the Na⁺ and K⁺ was a unifying idea of the origin of the selectivity (34–36). The distinct nature of Na⁺ from that of K⁺ under the collective dynamics of ions and water within the selectivity filter has been studied extensively (9, 12, 20, 21, 33, 37–40) (see references in the following review articles: refs. 6, 9–11, 33, 37).Here, we consider that the selectivity arises dynamically and that traveling ions undergo multiple selection steps along the passage of the entire pore. To delineate these selectivity processes, we describe the unique selectivity of potassium channels as a feature of a band-pass filter. The band-pass filter is an electrical device, allowing the passage of signals within a limited frequency range by rejecting low- and high-frequency signals (41). By analogy, potassium channel selectivity is featured as a band-pass filter with respect to the ion size (broken green line, Fig. 1). Generally, a band-pass filter is fabricated from a combination of low- and high-pass filters. Accordingly, the band-pass feature of ion selectivity can be deconvoluted into two types of successive filtering processes along the pore. As small-pass (low-pass) filters (broken blue line, Fig. 1), potassium channels allow the passage of small ionic species with a size cutoff of ∼1.7 Å. The working principle of the small-pass filter is shared with that of other types of channels and is determined by the geometrical pore size (1, 42). Simultaneously, potassium channels impose a unique large-pass (high-pass) filter (broken red line, Fig. 1) that rejects small ions; this is the main issue addressed and studied herein.Na⁺ is a small ion that can serve as a signature ion to characterize the features of conduction through potassium channels. Consequently, in this study, Na⁺ conduction was examined using single-channel current recordings and MD simulations for the KcsA potassium channel. To characterize the selectivity, the permeability ratio obtained from experimentally measured reversal potentials has frequently been used, as in Fig. 1. Although it is an experimentally feasible parameter, the underlying Nernst–Planck equation assumes independent ion diffusion across a homogeneous membrane phase rather than through a structured pore (1, 43). The reversal potential for calculating the permeability ratio is simply the membrane potential at which inward currents and outward currents are balanced (zero-current potential). Alternatively, the single-channel conductance ratio at a specific membrane potential for different ion species is more straightforward and contrasts the integrated permeation kinetics of different ion species through the pore (4). However, the single-channel conductance of Na⁺ through the potassium channel has not been measured (12, 20, 33) due to experimental difficulty. Instead, indirect evidence of Na⁺ conduction through a potassium channel at the single-channel level has been reported as a punchthrough (12, 13). In the previous simulation, Na⁺ and K⁺ conduction through the KcsA channel was performed, and the conductance ratio was theoretically predicted (34).In the present study, the intracellular Na⁺ concentration was increased, exceeding the physiological concentration range, and the distinct Na⁺ processes in the pore, which involved blocking and a single-channel Na⁺ current through the potassium channel, were resolved. MD simulations revealed an intricate process of Na⁺ conductance at an atomic scale across the channel. Accordingly, critical permeation processes of the large-pass filter for Na⁺ conduction have been highlighted here.     

Coexpression of alpha 1 with putative beta 3 subunits results in functional Na+/K+ pumps in Xenopus oocytes.

The active Na+/K+ pump is composed of an alpha and a beta subunit. Until now, three putative isoforms of the beta subunit have been identified that share sequence similarity. We have expressed the beta 1 and beta 3 isoforms of Xenopus laevis Na+/K(+)-ATPase in Xenopus oocytes to compare functional properties of the Na+/K+ pump, including either of these two isoforms. Na+/K+ pump current, estimated as K(+)-induced outward current in voltage-clamped oocytes, was doubled by coexpression of alpha 1 subunits with either isoform of the beta subunit compared to expression of alpha 1 subunits alone. The kinetics of activation by external K+ and the voltage dependence of the electrogenic activity of the Na+/K+ pump were similar with both beta isoforms, indicating that both beta 1 and beta 3 isoforms can support expression at the oocyte surface of an active Na+/K+ pump with similar functional properties.