System y+L-like Activities Account for High and Low Amino-Acid Transport Phenotypes in Chicken Erythrocytes

The functional properties of the transport of lysine across the chicken erythrocyte membrane were investigated. The animal population studied (male Leghorn chickens, 6–14 weeks old) was found to consist of two groups presenting either low (LT, 19 individuals) or high transport rates (HT, 20 individuals). The rates of influx in the two groups, measured at a concentration of l-lysine of 1 μm, differed by a factor of 34. The transport activities observed in LT and HT erythrocytes were compatible with the general features of system y⁺L, but showed some differences in specificity. The transporter in the LT group was found to bind l-lysine, l-leucine, l-methionine and l-glutamine with high affinity, in the presence of sodium, as described for system y⁺L in human erythrocytes. The activity present in HT erythrocytes exhibited a much lower affinity for l-leucine, but was able to interact strongly with l-glutamine and l-methionine. The specificity pattern of the HT transporter, has not been described in other cell types. In other respects, the properties of the two systems were similar. Sodium replacement with potassium, drastically reduced the affinity for l-leucine, without affecting lysine transport. Both transporters function as tightly coupled exchangers, are inactivated by p-chloromercuribenzene sulfonate and resistant to N-ethylmaleimide. These findings explain previous results obtained in selective breeding experiments of chicken with high and low amino-acid transport activity. Received: 12 February 2001/Revised: 11 June 2001     

Amino acid transport system y+L of human erythrocytes: Specificity and cation dependence of the translocation step

The transport specificity of system y⁺L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of l-[¹⁴C]-lysine. Some neutral amino acids, l-lysine and l-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external l-leucine and l-lysine increased the rate by 5.3±0.63 and 6.2±0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (l-tryptophan, l-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (l-alanine, l-serine). Half-saturation constants (±sem) calculated from this effect (l-lysine, 10.32±0.49 m and l-leucine, 11.50±0.50 m) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na⁺, Li⁺ or K⁺ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na⁺ or Li⁺ were replaced by K⁺. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.We wish to thank Dr C.A.R. Boyd for helpful discussions and Prof. H.N. Christensen for sharing with us very relevant bibliographic material. We are grateful to FONDECYT (1282/91) and DTI (B 2674) (Chile) for financial assistance.     

Nucleotide Composition Bias Affects Amino Acid Content in Proteins Coded by Animal Mitochondria

We show that in animal mitochondria homologous genes that differ in guanine plus cytosine (G + C) content code for proteins differing in amino acid content in a manner that relates to the G + C content of the codons. DNA sequences were analyzed using square plots, a new method that combines graphical visualization and statistical analysis of compositional differences in both DNA and protein. Square plots divide codons into four groups based on first and second position A + T (adenine plus thymine) and G + C content and indicate differences in amino acid content when comparing sequences that differ in G + C content. When sequences are compared using these plots, the amino acid content is shown to correlate with the nucleotide bias of the genes. This amino acid effect is shown in all protein-coding genes in the mitochondrial genome, including cox I, cox II, and cyt b, mitochondrial genes which are commonly used for phylogenetic studies. Furthermore, nucleotide content differences are shown to affect the content of all amino acids with A + T- and G + C-rich codons. We speculate that phylogenetic analysis of genes so affected may tend erroneously to indicate relatedness (or lack thereof) based only on amino acid content. Received: 3 July 1996 / Accepted: 6 November 1996     

Na+-independent Lysine Transport in Human Intestinal Caco-2 Cells

The nature of transepithelial and cellular transport of the dibasic amino acid lysine in human intestinal epithelial Caco-2 cells has been characterized. Intracellular accumulation of lysine across both the apical and basolateral membranes consists of a Na⁺-independent, membrane potential-sensitive uptake. Na⁺-independent lysine uptake at the basolateral membrane exceeds that at the apical membrane. Lysine uptake consists of both saturable and nonsaturable components. Na⁺-independent lysine uptake at both membranes is inhibited by lysine, arginine, alanine, histidine, methionine, leucine, cystine, cysteine and homoserine. In contrast, proline and taurine are without inhibitory effects at both membranes. Fractional Na⁺-independent lysine efflux from preloaded epithelial layers is greater at the basolateral membrane and shows trans-stimulation across both epithelial borders by lysine, arginine, alanine, histidine, methionine, and leucine but not proline and taurine. Na⁺-independent lysine influx (10 μm) in the presence of 10 mm homoserine shows further concentration dependent inhibition by lysine. Taken together, these data are consistent with lysine transport being mediated by systems b^o,+, y⁺ and a component of very low affinity (nonsaturable) at both membranes. The relative contribution to lysine uptake at each membrane surface (at 10 μm lysine), normalized to total apical uptake (100%), is apical b^o,+ (47%), y⁺ (27%) and the nonsaturable component (26%), and basal b^o,+ (446%), y⁺ (276%) and the nonsaturable component (20%). Northern analysis shows hybridization of Caco-2 poly(A)⁺RNA with a human rBAT cDNA probe. Received: 3 July 1995/Revised: 6 February 1996     

Identification of Important Amino Acid Residues of the Na+-Ca2+ Exchanger Inhibitory Peptide,XIP

The Na⁺-Ca²⁺ exchanger plays an important role in cardiac contractility by moving Ca²⁺ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects of modified peptides on Na⁺-Ca²⁺ exchange activity were determined. Exchange activity was assessed as ⁴⁵Ca²⁺ uptake into Na⁺-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule, is required for optimal inhibitory function. Received: 19 September 1996/Revised: 20 November 1996     

Na+-Coupled Alanine Transport in LLC-PK1 Cells: The Relationship Between the K m for Na+ at Low [Alanine] and Potential Dependence for the System

Analysis of the mechanistic basis by which sodium-coupled transport systems respond to changes in membrane potential is inherently complex. Algebraic expressions for the primary kinetic parameters (K _m and V _max ) consist of multiple terms that encompass most rate constants in the transport cycle. Even for a relatively simple cotransport system such as the Na⁺/alanine cotransporter in LLC-PK₁ cells (1:1 Na⁺ to substrate coupling, and an ordered binding sequence), the algebraic expressions for K _m for either substrate includes ten of the twelve rate constants necessary for modeling the full transport cycle. We show here that the expression of K _m of the first-bound substrate (Na⁺) simplifies markedly if the second-bound substrate (alanine) is held at a low concentration so that its' binding becomes the rate limiting step. Under these conditions, the expression for the K ^Na _m includes rate constants for only two steps in the full cycle: (i) binding/dissociation of Na⁺, and (ii) conformational `translocation' of the substrate-free protein. The influence of imposed changes in membrane potential on the apparent K ^Na _m for the LLC-PK₁ alanine cotransporter at low alanine thus provides insight to potential dependence at these sites. The data show no potential dependence for K ^Na _m at 5 μm alanine, despite marked potential dependence at 2 mm alanine when the full algebraic expression applies. The results suggest that neither translocation of the substrate-free form of the transporter nor binding/dissociation of extracellular sodium are potential dependent events for this transport system. Received: 10 April 1998/Revised: 6 July 1998     

Coordinated Amino Acid Changes in the Evolution of Mammalian Defensins

The mammalian defensin molecule is a short, highly cationic peptide cytotoxic to both microbial and mammalian cells which is cleaved from a precursor including a signal peptide and a highly anionic propiece. A phylogenetic analysis of 28 complete sequences from five mammalian species (mouse, rat, guinea pig, rabbit, and human) showed species-specific clusters of sequences, indicating that the genes duplicated after divergence of these species. Comparison of rates of synonymous and nonsynonymous nucleotide substitution suggested that gene duplication has often been followed by a period in which diversification of the mature defensins at the amino acid level has been selectively favored. In some comparisons, it appeared that amino acid differences in this region have appeared in a nonrandom fashion so as to change the pattern of residue charges. Because it has been hypothesized that the negative charge in the propiece serves to balance the positive charge in the mature defensin and thus to prevent cytotoxicity prior to cleavage, we used a maximum likelihood method of reconstructing ancestral states in order to test whether this balance has been maintained over evolutionary time in spite of rapid diversification of the mature defensin at the amino acid level. Reconstructed ancestral sequences always maintained a charge balance between mature defensin and propiece, and changes in the net positive charge of the mature defensin were balanced by corresponding changes in the propiece. The results support the hypothesis that, in the evolution of these proteins, amino acid changes have occurred in a coordinated fashion so as to preserve an adaptive phenotype. Received: 23 October 1996 / Accepted: 7 January 1997     

The Action of Blocking Agents Applied to the Inner Face of Ca2+-activated K+ Channels from Human Erythrocytes

The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate conductance calcium-activated potassium (IK_Ca) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba²⁺ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed by TEA at its binding site (δ) of 0.18 and a K _d at 0 mV of 20.5 mm. Ba²⁺ was a very potent blocker of the channel, breaking the single channel activity up into bursts, interspersed with silent periods lasting several seconds. The effect of Ba²⁺ was very voltage sensitive, δ= 0.44, and a K _d at 0 mV of 0.15 μm. Clotrimazole applied to the inner face of the membrane at a concentration ≤1 μm produced a slow block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the channel. A high concentration of cetiedil (100 μm) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular site on the channel. Received; 18 February 1998/Received: 5 June 1998     

Effects of Permeant Cations on K+ Channel Gating in Nerve Axons Revisited

An increase in extracellular potassium ion concentration, K _o , significantly slows the potassium channel deactivation rate in squid giant axons, as previously shown. Surprisingly, the effect does not occur in all preparations which, coupled with the voltage independence of this result in preparations in which it does occur, suggests that it is mediated at a site outside of the electric field of the channel, and that this site is accessible to potassium ions in some preparations, but not in others. In other words, the effect does not appear to be related to occupancy of the channel by potassium ions. This conclusion is supported by a four-barrier, three-binding site model of single file diffusion through the channel in which one site, at most, is unoccupied by a potassium ion (single-vacancy model). The model is consistent with current-voltage relations with various levels of K _o , and, by definition, with multiple occupancy by K⁺. The model predicts that occupancy of any given site is essentially independent of K _o (or K _i ). The effects of extracellular Rb⁺ and Cs⁺ on gating are strongly voltage dependent, and they were observed in all preparations investigated. Consequently, the mechanism underlying these results would appear to be different from that which underlies the effect of K⁺ on gating. In particular, the effect of Rb⁺ on gating is reduced by strong hyperpolarization, which in the context of the occupancy hypothesis, is consistent with the voltage dependence of the current-voltage relation in the presence of Rb⁺. The primary, novel, finding in this study is that the effects of Cs⁺ are counterintuitive in this regard. Specifically, the slowing of channel deactivation rate by Cs⁺ is also reduced by hyperpolarization, similar to the Rb⁺ results, whereas blockade is enhanced, which is seemingly inconsistent with the concept that occupancy of the channel by Cs⁺ underlies the effect of this ion on gating. This result is further elucidated by barrier modeling of the current-voltage relation in the presence of Cs⁺. Received: 19 December 1995/Revised: 10 June 1996     

Plastid Genome Phylogeny and a Model of Amino Acid Substitution for Proteins Encoded by Chloroplast DNA

Maximum likelihood (ML) phylogenies based on 9,957 amino acid (AA) sites of 45 proteins encoded in the plastid genomes of Cyanophora, a diatom, a rhodophyte (red algae), a euglenophyte, and five land plants are compared with respect to several properties of the data, including between-site rate variation and aberrant amino acid composition in individual species. Neighbor-joining trees from AA LogDet distances and ML analyses are seen to be congruent when site rate variability was taken into account. Four feasible trees are identified in these analyses, one of which is preferred, and one of which is almost excluded by statistical criteria. A transition probability matrix for the general reversible Markov model of amino acid substitutions is estimated from the data, assuming each of these four trees. In all cases, the tree with diatom and rhodophyte as sister taxa was clearly favored. The new transition matrix based on the best tree, called cpREV, takes into account distinct substitution patterns in plastid-encoded proteins and should be useful in future ML inferences using such data. A second rate matrix, called cpREV*, based on a weighted sum of rate matrices from different trees, is also considered. Received: 3 June 1999 / Accepted: 26 November 1999     

Variations of Intracellular pH in Human Erythrocytes via K+(Na+)/H+ Exchange Under Low Ionic Strength Conditions

The change of intracellular pH of erythrocytes under different experimental conditions was investigated using the pH-sensitive fluorescent dye BCECF and correlated with (ouabain + bumetanide + EGTA)-insensitive K⁺ efflux and Cl⁻ loss. When human erythrocytes were suspended in a physiological NaCl solution (pH _o = 7.4), the measured pH _i was 7.19 ± 0.04 and remained constant for 30 min. When erythrocytes were transferred into a low ionic strength (LIS) solution, an immediate alkalinization increased the pH _i to 7.70 ± 0.15, which was followed by a slower cell acidification. The alkalinization of cells in LIS media was ascribed to a band 3 mediated effect since a rapid loss of approximately 80% of intracellular Cl⁻ content was observed, which was sensitive to known anion transport inhibitors. In the case of cellular acidification, a comparison of the calculated H⁺ influx with the measured unidirectional K⁺ efflux at different extracellular ionic strengths showed a correlation with a nearly 1:1 stoichiometry. Both fluxes were enhanced by decreasing the ionic strength of the solution resulting in a H⁺ influx and a K⁺ efflux in LIS solution of 108.2 ± 20.4 mmol (l _cells hr)⁻¹ and 98.7 ± 19.3 mmol (l _cells hr)⁻¹, respectively. For bovine and porcine erythrocytes, in LIS media, H⁺ influx and K⁺ efflux were of comparable magnitude, but only about 10% of the fluxes observed in human erythrocytes under LIS conditions. Quinacrine, a known inhibitor of the mitochondrial K⁺(Na⁺)/H⁺ exchanger, inhibited the K⁺ efflux in LIS solution by about 80%. Our results provide evidence for the existence of a K⁺(Na⁺)/H⁺ exchanger in the human erythrocyte membrane. Received: 22 December 1999/Revised: 10 April 2000     

Two Novel Toxins from the Venom of the Scorpion Pandinus imperator Show that the N-terminal Amino Acid Sequence is Important for their Affinities towards Shaker B K+ Channels

Two novel peptides were purified from the venom of the scorpion Pandinus imperator, and were named Pi2 and Pi3. Their complete primary structures were determined and their blocking effects on Shaker B K⁺ channels were studied. Both peptides contain 35 amino acids residues, compacted by three disulfide bridges, and reversibly block the Shaker B K⁺ channels. They have only one amino acid changed in their sequence, at position 7 (a proline for a glutamic acid). Whereas peptide Pi2, containing the Pro7, binds the Shaker B K⁺ channels with a K _d of 8.2 nm, peptide Pi3 containing the Glu7 residue has a much lower affinity of 140 nm. Both peptides are capable of displacing the binding of ¹²⁵I-noxiustoxin to brain synaptosome membranes. Since these two novel peptides are about 50% identical to noxiustoxin, the present results support previous data published by our group showing that the amino-terminal region of noxiustoxin, and also the amino-terminal sequence of the newly purified homologues: Pi2, and Pi3, are important for the recognition of potassium channels. Received: 13 November 1995/Revised: 11 March 1996     

A 59 Amino Acid Insertion Increases Ca2+ Sensitivity of rbslo1, a Ca2+-Activated K+ Channel in Renal Epithelia

We previously cloned a MaxiK channel α-subunit isoform, rbslo1, from rabbit kidney with an amino acid sequence highly homologous to mslo but with a 59 amino acid insertion between S8 and S9 (Morita et al., 1997. Am. J. Physiol. 273:F615–F624). rbslo1 activation properties differed substantially from mslo with much greater Ca²⁺ sensitivity, half-activation potential of −49 mV in 1 μm Ca²⁺. We now report single-channel analysis of rbslo1 and delA, a construct produced by removal of the 59 amino acid insertion at site A. delA is identical to mslo from upstream of S1 to downstream of S10 with the exception of 8 amino acids. Slope of the steady-state Boltzmann voltage activation curve was 8.1 mV per e-fold change in probability of opening for both rbslo1 and delA. The apparent [Ca²⁺] _i properties in delA were more like mslo but the voltage-activation properties remained distinctly rbslo1. Ca²⁺ affinity decreased and transmembrane voltage effects on apparent Ca²⁺ affinity increased in delA. The differences between rbslo1 and other cloned channels appear to be localized at insertion site A with both the insertion sequence and amino acid substitutions near site A being important. The steeper activation slope makes the channel more responsive to small changes in transmembrane voltage while the insertion sequence makes the channel functional at physiological low levels of [Ca²⁺] _i . Received: 23 August 1999     

The Basis of Higher Na+ Transport by Inner Medullary Collecting Duct Cells from Dahl Salt-Sensitive Rats: Implicating the Apical Membrane Na+ Channel

The present experiments were designed to examine the function of Na/K pumps from Dahl salt-sensitive (S) and salt-resistant (R) rats. Previous reports have suggested that there is a difference in primary sequence in the α₁ subunit, the major Na/K pump isoform in the kidney. This sequence difference might contribute to differences in NaCl excretion in these two strains which in turn could influence the systemic blood pressure. Using ``back-door' phosphorylation of pumps isolated from basolateral membranes of kidney cortex, we found no differences between S and R strains. We also examined the Na/K pumps from cultured inner medullary collecting duct (IMCD) cells. This approach takes advantage of the fact that monolayers cultured from S rats transport about twice as much Na⁺ as monolayers cultured from R rats. In cells whose apical membrane was made permeable with amphotericin B, comparison of the affinities for ouabain, Na⁺, and K⁺, respectively, showed only small or no differences between S and R monolayers. Ouabain binding showed no difference in the number of Na/K pumps on the basolateral membrane of cultured cells, despite a 2-fold difference in Na⁺ transport rates. The analysis of the steady-state Na⁺ transport indicates that Na/K pumps in IMCD monolayers from S rats operate at a higher fraction of their maximum capacity than do pumps in monolayers from R rats. The results, taken together, suggest that the major reason for the higher rate of Na⁺ transport in S monolayers is because of a primary increase in the conductive permeability of the apical membrane to Na⁺. They suggest that the epithelial Na⁺ channel is intrinsically different or differently regulated in S and R rats. Received: 6 May 1996/Revised: 16 October 1996     

The Sensitivity of the Human Kv1.3 (hKv1.3) Lymphocyte K+ Channel to Regulation by PKA and PKC is Partially Lost in HEK 293 Host Cells

cDNA encoding the full-length hKv1.3 lymphocyte channel and a C-terminal truncated (Δ459-523) form that lacks the putative PKA Ser⁴⁶⁸ phosphorylation site were stably transfected in human embryonic kidney (HEK) 293 cells. Immunostaining of the transfected cells revealed a distribution at the plasma membrane that was uniform in the case of the full-length channel whereas clustering was observed in the case of the truncated channel. Some staining within the cell cytoplasm was found in both instances, suggesting an active process of biosynthesis. Analyses of the K⁺ current by the patch-clamp technique in the whole cell configuration showed that depolarizing steps to 40 mV from a holding potential (HP) of −80 mV elicited an outward current of 2 to 10 nA. The current threshold was positive to −40 mV and the current amplitude increased in a voltage-dependent manner. The parameters of activation were −5.7 and −9.9 mV (slope factor) and −35 mV (half activation, V _0.5) in the case of the full-length and truncated channels, respectively. The characteristics of the inactivation were 14.2 and 24.6 mV (slope factor) and −17.3 and −39.0 mV (V _0.5) for the full-length and truncated channels, respectively. The activation time constant of the full-length channel for potentials ranging from −30 to 40 mV decreased from 18 to 12 msec whereas the inactivation time constant decreased from 6600 msec at −30 mV to 1800 msec at 40 mV. The unit current amplitude measured in cells bathing in 140 mm KCl was 1.3 ± 0.1 pA at 40 mV, the unit conductance, 34.5 pS and the zero current voltage, 0 mV. Both forms of the channels were inhibited by TEA, 4-AP, Ni²⁺ and charybdotoxin. In contrast to the native (Jurkat) lymphocyte Kv1.3 channel that is fully inhibited by PKA and PKC, the addition of TPA resulted in 34.6 ± 7.3% and 38.7 ± 9.4% inhibition of the full-length and the truncated channels, respectively. 8-BrcAMP induced a 39.4 ± 5.4% inhibition of the full-length channel but had no effect (8.6 ± 8.3%) on the truncated channel. Cell dialysis with alkaline phosphatase had no effects, suggesting that the decreased sensitivity of the transfected channels to PKA and PKC was not due to an already phosphorylated channel. Patch extract experiments suggested that the hKv1.3 channel was partially sensitive to PKA and PKC. Cotransfecting the Kvβ1.2 subunit resulted in a decrease in the value of the time constant of inactivation of the full-length channel but did not modify its sensitivity to PKA and PKC. The cotransfected Kvβ2 subunit had no effects. Our results indicate that the hKv1.3 lymphocyte channel retains its electrophysiological characteristics when transfected in the Kvβ-negative HEK 293 cell line but its sensitivity to modulation by PKA and PKC is significantly reduced. Received: 18 June 1997/Revised: 7 October 1997     

An Energy-Barrier Model for the Permeation of Monovalent and Divalent Cations Through the Maxi Cation Channel in the Plasma Membrane of Rye Roots

The depolarization-activated, high-conductance ``maxi' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K⁺, Na⁺, Ca²⁺ and Ba²⁺ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model), which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary conductance vs. activity relationships for K⁺, Na⁺ and Ca²⁺, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative cation permeabilities with solution composition, and the complex effects of Ba²⁺ and Ca²⁺ on K⁺ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input to electrocoupling models allowing the relationships between membrane voltage, Ca²⁺ influx and Ca²⁺ signaling to be studied theoretically. Received: 29 April 1998/Revised: 20 November 1998     

Involvement of Protein Tyrosine Kinase in Osmoregulation of Na+ Transport and Membrane Capacitance in Renal A6 Cells

Renal A6 cells have been reported in which hyposmolality stimulates Na⁺ transport by increasing the number of conducting amiloride-sensitive 4-pS Na⁺ channels at the apical membrane. To study a possible role of protein tyrosine kinase (PTK) in the hyposmolality-induced signaling, we investigated effects of PTK inhibitors on the hyposmolality-induced Na⁺ transport in A6 cells. Tyrphostin A23 (a PTK inhibitor) blocked the stimulatory action of hyposmolality on a number of the conducting Na⁺ channels. Tyrphostin A23 also abolished macroscopic Na⁺ currents (amiloride-sensitive short-circuit current, I _Na ) by decreasing the elevating rate of the hyposmolality-increased I _Na . Genistein (another type of PTK inhibitor) also showed an effect similar to tyrphostin A23. Brefeldin A (BFA), which is an inhibitor of intracellular translocation of protein, blocked the action of hyposmolality on I _Na by diminishing the elevating rate of the hyposmolality-increased I _Na , mimicking the inhibitory action of PTK inhibitor. Further, hyposmolality increased the activity of PTK. These observations suggest that hyposmolality would stimulate Na⁺ transport by translocating the Na⁺ channel protein (or regulatory protein) to the apical membrane via a PTK-dependent pathway. Further, hyposmolality also caused an increase in the plasma (apical) membrane capacitance, which was remarkably blocked by treatment with tyrphostin A23 or BFA. These observations also suggest that a PTK-dependent pathway would be involved in the hyposmolality-stimulated membrane fusion in A6 cells. Received: 6 October 1999/Revised: 4 February 2000     

Biased Usages of Arginines and Lysines in Proteins Are Correlated with Local-Scale Fluctuations of the G + C Content of DNA Sequences

Amino acid residues arginine (R) and lysine (K) have similar physicochemical characteristics and are often mutually substituted during evolution without affecting protein function. Statistical examinations on human proteins show that more R than K residues are used in the proximity of R residues, whereas more K than R are used near K residues. This biased use occurs on both a global and a local scale (shorter than ∼100 residues). Even within a given exon, G + C-rich and A + T-rich short DNA segments preferentially encode R and K, respectively. The biased use of R and K on a local scale is also seen in Saccharomyces cerevisiae and Caenorhabdidtis elegans, which lack global-scale mosaic structures with varying GC%, or isochores. Besides R and K, several amino acids are also used with a positive or negative correlation with the local GC% of third codon bases. The local-, or ``within-gene'-, scale heterogeneity of the DNA sequence may influence the sequence of the encoded protein segment. Received: 2 March 1998 / Accepted: 23 April 1998