The cohering telomeres of Oxytricha.

We have studied the process by which purified Oxytricha macronuclear DNA associates with itself to form large aggregates. The various macronuclear DNA molecules all have the same terminal or telomeric DNA sequences that are shown below. 5' C4A4C4A4C4--mean length----G4T4G4T4G4T4G4T4G4 G4T4G4T4G4T4G4T4G4-----2.4 kb------C4A4C4A4C4. When incubated at high concentrations, these telomeric sequences cohere with one another to form an unusual structure--one that is quite different from any DNA structure so far described. The evidence for this is the following: 1) These sequences cohere albeit slowly, in the presence of relatively high concentrations of Na+, and no other cation tested. This contrasts with the rapid coherence of complementary single-chain terminals of normal DNA (sticky ends) which occurs in the presence of any cation tested. 2) If the cohered form is transferred into buffers containing a special cation, K+, it becomes much more resistant to dissociation by heating. We estimate that K+ increases the thermal stability by 25 degrees or more. The only precedent known (to us) for a cation-specific stabilization is that seen in the quadruplex structure formed by poly I. The thermal stability of double helical macronuclear DNA depends on the cation concentration, but not the cation type. Limited treatment with specific nucleases show that the 3' and 5'-ended strands are essential for the formation of the cohering structure. Once in the cohered form, the telomeric sequences are protected from the action of nucleases. Coherence is inhibited by specific, but not by non-specific, synthetic oligomers, and by short telomeric fragments with or without their terminal single chains. We conclude that the coherence occurs by the formation of a novel condensed structure that involves the terminal nucleotides in three or four chains.     

Interaction of the dye ethidium bromide with DNA containing guanine repeats.

DNA containing one or more copies of the motifs repeated in telomere sequences has unusual conformational properties. The isolated sequence from the protozoan Oxytricha, dT4G4 has the potential to form tetramers in the presence of sodium or potassium ions. We report here that these tetramers bind ethidium tightly, with an interaction that fulfills several criteria for an intercalative mechanism in the G sequence. By contrast, the 4-fold tandem repeat of this subunit, d(T4G4)4, does not interact specifically with ethidium in the presence of Na+. This difference might have a simple structural basis: the tetramer of dT4G4 forms a stack of four G-quartets in the presence of Na+ or K+, whereas the constraint imposed by the T4 "tethers" in the repeat d(T4G4)4 allows only two layers to form in the presence of Na+. In the presence of sufficient K+, the latter can partially form a four-layer G-quartet structure, which interacts with ethidium. This idea is supported by analysis of a "relaxed" sequence, dT4G4(T7G4)3, which allows formation of four G-quartets and binds ethidium in the presence of Na+ as well as K+. Ethidium (and intercalators generally) should thus be able to retard or inhibit the action of telomerase in the presence of K+.     

Monovalent cation-induced structure of telomeric DNA: the G-quartet model

We have investigated the structures formed by oligonucleotides composed of two or four repeats of the telomeric sequences from Oxytricha and Tetrahymena. The Oxytricha four-repeat molecule (d(T4G4)4 = Oxy-4) forms structures with increased electrophoretic mobility in nondenaturing gels containing Na+, K+, or Cs+, but not in gels containing Li+ or no added salt. Formation of the folded structure results in protection of a set of dG's from methylation by dimethyl sulfate. Efficient UV-induced cross-links are observed in Oxy-4 and the related sequence from Tetrahymena (d(T2G4)4 = Tet-4), and join thymidine residues in different repeats. Models proposed to account for these data involve G-quartets, hydrogen-bonded structures formed from four guanosine residues in a square-planar array. We propose that the G-quartet structure must be dealt with in vivo by the telomere replication machinery.     

Effect of monovalent cation-induced telomeric DNA structure on the binding of Oxytricha telomeric protein.

Oligonucleotides bearing 4 repeats of telomeric deoxyguanosine-rich sequence undergo a monovalent cation-induced transition to a folded conformation with G-G base pairs, modeled as a 'G-quartet' structure. We have now deduced the rates of folding and unfolding of d(TTTTGGGG)4, which has four repeats of the Oxytricha telomeric DNA sequence. The estimated average values of delta G for the folded form at 37 degrees C are -2.2 kcal/mol and -4.7 kcal/mol in 50 mM na+ and K+, respectively. The fully folded DNA is not recognized by the Oxytricha telomere-binding protein; the substrate for protein binding has properties consistent with its being partly or fully unfolded. In confirmation of this conclusion, prevention of DNA folding by methylation enables the protein to bind as rapidly in the presence of monovalent cations as in their absence. The slow unfolding (t1/2 = 4 hr and 18 hr at 37 degrees C in Na+ and K+, respectively) of the DNA suggests that such structures would be long-lived if they formed in vivo, unless they can be actively unfolded. The inability of the telomere-binding protein to bind the stable, folded form of the 4-repeat telomeric sequence is a problem that may be circumvented in vivo by avoiding four single-stranded repeats.     

Thermal stability of the K+ channel tetramer: cation interactions and the conserved threonine residue at the innermost site (S4) of the KcsA selectivity filter

The selectivity filter of most K+ channels contains a highly conserved Thr residue that uniquely forms the S4 binding site for K+ by dual coordination with the backbone carbonyl oxygen and side chain hydroxyl of the same residue. This study examines the effect of mutations of Thr75 in the S4 site of theKcsA K+ channel on the cation dependence of the thermal stability of the tetramer, a phenomenon that reflects the structural role of cations in the filter. Conservative mutations of Thr75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity ofK+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by approximately 10(3)- or approximately 10(4)-fold for Ala and Cys substitution, respectively,consistent with the known preference of the S4 site for Ba2+. In contrast, substitution of Ala or Cys at T75 anomalously enhances the ability of Na+ to stabilize the tetramer, suggesting that the native Thr residue at S4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature orCu2+ cation catalyzes formation of covalent dimers of the T75C mutant of KcsA via formation of disulfide bonds between Cys residues of adjacent subunits. Thiophilic cations such as Hg2+ and Ag+ specifically protect the T75C tetramer against heat-induced dimer formation, demonstrating the contribution of cation interactions to tetramer stability in a channel with a non-native S4 site engineered to bind foreign cations.     

Luminescence study of G-quadruplex formation in the presence of Tb3+ ion

The interactions of Tb3+ with the quadruplex-forming oligonucleotide bearing human telomeric repeat sequence d(G(3)T(2)AG(3)T(2)AG(3)T(2)AG(3)), (htel21), have been studied using luminescence spectroscopy and circular dichroism (CD). Enhanced luminescence of Tb3+, resulting from energy transfer from guanines, indicated encapsulation of Tb3+ ion in the central cavity of quadruplex core. The ability of lanthanide ions (Eu3+ and Tb3+) to mediate formation of quadruplex structure has been further evidenced by the fluorescence energy transfer measurements with the use of oligonucleotide probe labeled with fluorescein and rhodamine FRET partners, FAM-htel21-TAMRA. The CD spectra revealed that Tb3+/htel21 quadruplex possesses antiparallel strand orientation, similarly as sodium quadruplex. Tb3+ binding equilibria have been investigated in the absence and the presence of competing metal cations. At low Tb3+ concentration (8 microM) Tb3+/htel21 quadruplex stability is very high (5 x 10(6) M(-1)) and stoichiometry of 5-7 Tb3+ ions per one quadruplex molecule is observed. Luminescence and CD titration experiments suggested that the cavity of quadruplex accommodates two Tb3+ ions and the remaining Tb3+ ions bind probably to TTA loops of quadruplex. Higher concentration of Tb3+ (above 10 microM) results in the excessive binding of Tb3+ ions that finally destabilizes quadruplex, which undergoes transformation into differently organized assemblies. Such assemblies (probably possessing multiple positive charge) exhibit kinetic stability, which is manifested by a very slow kinetics of displacement of Tb3+ ion by competing cations (Li+, Na+, K+).     

Identification of N-[p-(2-benzimidazolyl)phenyl]maleimide-modified residue participating in dynamic fluorescence changes accompanying Na+,K+-dependent ATP hydrolysis

Na+,K+-ATPase from pig kidney was specifically modified with a sulfhydryl fluorescent reagent, N-[p-(2-benzimidazolyl)phenyl]maleimide (BIPM), by pretreatment of N-ethylmaleimide. The preparation thus obtained retained 100% of initial Na+,K+-ATPase activity and contained 1 BIPM residue/alpha-chain, and it showed almost 2-fold larger fluorescence changes accompanying ATP hydrolysis than the previous preparations which retained 60% of initial activity and contained 3-4 BIPM residues/alpha-chain (Taniguchi, K., Suzuki, K., and Iida, S. (1982) J. Biol. Chem. 257, 10659-10667). Extensive trypsin (Sigma type I) treatment of the new preparation produced mainly two different fluorescent peptide peaks in both ion-exchange and reverse-phase chromatography. Amino acid sequence analysis of both peptides showed that they had the same common sequence, Ser-Tyr-X-Pro-Gly-Met-Gly-Val, except that the larger one contained Ala-Leu next to the Val residue. From the comparison of the amino acid sequence deduced from cDNA from sheep kidney (Shull, G. E., Schwartz, A., and Lingrel, J. B. (1985) Nature 316, 691-695), X was shown to correspond to Cys-964 of the alpha-chain in Na+,K+-ATPase. The data suggest that the microenvironment of the BIPM residue covalently bound to the sulfhydryl group of Cys-964 changes accompanying sequential appearance of reaction intermediates of Na+,K+-ATPase.     

Capacity of the outer membrane of a gram-negative marine bacterium in the presence of cations to prevent lysis by Triton X-100.

Cells of marine pseudomonad B-16 (ATCC 19855) washed with a solution containing 0.3 M NaCl, 50 mM MgCl2, and 10 mM KCl (complete salts) could be protected from lysis in a hypotonic environment if the suspending medium contained either 20 mM Mg2+, 40 mM Na+, or 300 mM K+. When the outer double-track layer (the outer membrane) of the cell envelope was removed to yield mureinoplasts, the Mg2+, Na+ or K+, requirements to prevent lysis were raised to 80, 210, and 400 mM, respectively. In the presence of 0.1% Triton X-100, 220, 320, and 360 mM Mg2+, Na+ or K+, respectively, prevented lysis of the normal cells. Mureinoplasts and protoplasts, however, lysed instantly in the presence of the detergent at all concentrations of Mg2+, Na+, or K+ tested up to 1.2 M. Thus, the structure of the outer membrane appears to be maintained by appropriate concentrations of Mg2+ or Na+ in a form preventing the penetration of Triton X-100 and thereby protecting the cytoplasmic membrane from dissolution by the detergent. K+ was effective in this capacity with cells washed with complete salts solution but not with cells washed with a solution of NaCl, suggesting that bound Mg2+ was required in the cell wall membrane for K+ to be effective in preventing lysis by the detergent. At high concentrations (1 M) K+ and Mg2+, but not Na+, appeared to destabilize the structure of the outer membrane in the presence of Triton X-100.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

Selective inhibition by ionophore A23187 of the enzyme isomerization in the catalytic cycle of Na+, K+ -ATPase

The effect of an ionophore A23187 on the purified Na+,K+-ATPase from the outer medulla of pig kidney was investigated. When the enzyme was pretreated with A23187 in the presence of Na+ and K+, the ATPase activity was inhibited almost completely. When the pretreatment was performed in the presence of Na+ and absence of K+, formation of the phosphoenzyme (EP) from ATP was only slightly retarded. The steady state level of EP thus formed was not altered, but EP decomposition was strongly inhibited. Under these conditions, the accumulated EP was sensitive to ADP and insensitive to K+. On the other hand, when the pretreatment was performed in the absence of Na+ and presence of K+, EP formation following simultaneous addition of Na+ and ATP was extremely slow, but the steady state level of EP was not substantially altered. When the pretreatment was performed in the absence of Na+ and presence of K+, EP formation from Pi was unaffected, and the EP formed was in rapid equilibrium with Pi of the medium. These results demonstrate that A23187 selectively inhibits isomerization of the enzyme between the high Na+ and low K+ affinity form and the low Na+ and high K+ affinity form in the catalytic cycle, whether or not the enzyme is phosphorylated. This inhibition is quite similar to the A23187-induced inhibition of the enzyme isomerization in the catalytic cycle of the Ca2+ -ATPase from sarcoplasmic reticulum (Hara, H., and Kanazawa, T. (1986)J. Biol. Chem.261, 16584-16590). These findings suggest that some common mechanism, which is involved in the enzyme isomerization, between these two transport ATPases is strongly disturbed by A23187.     

Effect of gamma-hydroxypropano deoxyguanosine, the major acrolein-derived adduct, on monomolecular quadruplex structure of telomeric repeat d(TTAGGG)(4)

The three oligodeoxyribonucleotides (ODNs) a-c, having the telomeric repeat d(TTAGGG)(4) sequence and incorporating gamma-hydroxypropano deoxyguanosine at different positions, were synthesized. Gel electrophoresis and CD analyses indicated that the ODNs assume monomolecular quadruplex structures in Na+ and in K+ buffers. The T(m) values, obtained by CD melting experiments, showed that the presence of the acrolein-dG adduct into the ODN b decreases the thermal stability of the monomolecular quadruplex structure in Na+ solution, whereas for a and c no significant effect could be detected in the same experimental conditions. On the contrary, all ODNs a-d show the same behaviour in K+ buffer. These findings are briefly discussed.     

Primary structures of two types of alpha-subunit of rat brain Na+,K+,-ATPase deduced from cDNA sequences

A rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+,K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+,K+-ATPase. The deduced amino acid sequence consists of 1,018 amino acids. The alpha-subunit of the rat kidney-type Na+,K+-ATPase shows 97% homology in amino acid sequence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+,K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+,K+-ATPase.     

Coupling of a loop diuretic-sensitive Na+ influx with the net loop diuretic-sensitive K+ efflux in mouse NIH 3T3 cells

Mouse 3T3 fibroblasts have a loop diuretic sensitive Na+ transport system, responsible for more than 50% of the total Na+ influx. This transport system is dependent on the simultaneous presence of all three ions; Na+, K+, (Rb+) and Cl- in the extracellular medium. The same requirement for these three ions was also found for the loop diuretic-sensitive K+ efflux. In addition, the sensitivities of Na+ influx and Rb+ efflux for the two loop diuretics, furosemide and bumetanide were found to be similar. The similar ionic requirement and sensitivity towards loop diuretics of the two fluxes, support the hypothesis, that this loop diuretic-sensitive Na+ influx in mouse 3T3 cells, is accompanied by the net loop diuretic-sensitive K+ efflux.     

Simultaneous bindings of ATP and vanadate to (Na+ + K+)-ATPase. Implications for the reaction mechanism of the enzyme

Inhibition of (Na+ + K+)-dependent adenosine triphosphatase phosphatase by vanadate is thought to occur through the tight binding of vanadate to the same site from which Pi is released. To see if ATP binds to [48V] vanadate-enzyme complex, just as it does to the phosphoenzyme, the effects of Na+, K+, and ATP on the dissociation rate of the complex at 10 degrees C were studied. The rate constant was increased by Na+, and this increase was blocked by K+, indicating that either Na+ or K+ binds to the complex. ATP alone, or in combination with K+, had no effect on the rate constant. In the presence of Na+, however, ATP caused a further increase in the rate constant. The value of K0.5 of Na+ was the same in the presence or absence of ATP; K0.5 of ATP (0.2 mM) did not seem to change significantly when Na+ concentration was varied, and K0.5 of K+, at a constant Na+ concentration, was the same in the presence or absence of ATP. The data indicate that ATP binds to the enzyme-vanadate complex regardless of the presence or absence of Na+ or K+, but it affects the dissociation rate only when Na+ is bound simultaneously. The value of K0.5 of Na+ decreased as pH was increased in the range of 6.5-7.8, but K0.5 of ATP was independent of pH. Demonstration of ATP binding to the enzyme-vanadate complex provides further support for the suggestion that the oligomeric enzyme contains a low-affinity regulatory site for ATP that is distinct from the interacting high-affinity catalytic sites.     

The new models of the human telomere d[AGGG(TTAGGG)3] in K+ solution

The human telomeric sequence d[AGGG(TTAGGG)(3)] has been found to form different types of G-quadruplex structures. NMR revealed that in Na(+) solution this 22 nucleotide (nt) sequence exhibits an antiparallel structure, whereas crystallographic studies in the presence of K(+) showed a dramatically different parallel structure. The structure of this 22 nt sequence in the presence of K(+) has drawn intense interest as the intracellular K(+) concentration is greater than that of Na(+). However, the question of the type of structure for the 22 nt telomeric sequence in K(+) solution remains open. In this study, we substituted the Gs in the sequence with 8-bromoguanine and examined the resultant structures and thermal stabilities by circular dichroism (CD) spectroscopy. The results suggest that the 22 nt in K(+) solution exists as a mixture of mixed-parallel/antiparallel and chair-type G-quadruplex. To date, the exact structure of human telomeric G-quadruplex in K(+) solution is extremely controversial. The present study provides valuable information for understanding the discrepancies between the crystal and solution studies. We discuss the possible implications of the structure in understanding higher-order telomeric DNA structure and T-loop formation.     

Effect of thyroid hormone receptors in normal and cancerous cells on the ionic conductivity of bilayer phospholipid membranes

The effect of thyroid hormones receptors isolated from normal and cancer cells on bilayer phospholipid membranes (BPhLM) conductivity, has been studied. The receptor isolated from normal cells in complex X with the hormone selectively induces H+-conductivity of BPhLM generating transmembrane potential equal to 42 mV on the membrane at pH gradient equal to 1. In the presence of K+, Na+, Ca+, Mn2+, Sr2+, Mg2+ the changes of BPhLM are not observed. Neither hormones (T3, T4) nor receptor in free position affect the BPhLM conductivity. Thyroid hormone receptor isolated from mamalignantly transformed cells in a complex with T3 or T4 increases the BPhLM permeability for Ca2+. The transmembrane potential measured at 10fold Ca2+ ion concentration is equal to 16 mV. In the presence of H+, K+, Na+, Mn2+, Sr2+, Mg2+, Ba2+, the resistance of BPhLM doesn't change.     

Ionic selectivity of Ih channels of rod photoreceptors in tiger salamanders.

Ionic selectivity of Ih channels of tiger salamander rod photoreceptors was investigated using whole-cell voltage clamp. Measured reversal potentials and the Goldman-Hodgkin-Katz voltage equation were used to calculate permeability ratios with 20 mM K+ as a reference. In the absence of external K+, Ih is small and hard to discern. Hence, we defined Ih as the current blocked by 2 mM external Cs+. Some small amines permeate Ih channels, with the following permeability ratios (PX/PK):NH4+, 0.17; methylammonium, 0.06; and hydrazine, 0.04. Other amines are tially impermeant: dimethylammonium (< 0.02), ethylammonium (< 0.01), and tetramethylammonium (< 0.01). When K+ is the only external permeant ion and its concentration is varied, the reversal potential of Ih follows the Nernst potential for a K+ electrode. Ih channels are also permeable to other alkali metal cations (PX/PK): T1+, > 1.55; K+, 1; Rb+, > 0.55; Na+, 0.33; Li+, 0.02. Except for Na+, the relative slope conductance had a similar sequence (GX/GK): T1+, 1.07; K+, 1; Rb+, 0.37; NH4+, 0.07; Na+, 0.02. Based on permeabilities to organic cations, the narrowest part of the pore has a diameter between 4.0 and 4.6 A. Some permeant cations have large effects on the gating kinetics of Ih channels; however, permeant cations appear to have little effect on the steady-state activation curve of Ih channels. Lowering K+ or replacing K+ with Na+ reduces the maximal conductance of Ih but does not shift or change the steepness of its voltage dependence. With ammonium or methylammonium replacing K+ a similar pattern is seen, except that there is a small positive shift of approximately 10 mV in the voltage dependence.     

Ion specificity of cardiac sarcolemmal Na+/H+ antiporter

In bovine cardiac sarcolemmal vesicles, an outward H+ gradient stimulated the initial rate of amiloride-sensitive uptake of 22Na+, 42K+, or 86Rb+. Release of H+ from the vesicles was stimulated by extravesicular Na+, K+, Rb+, or Li+ but not by choline or N-methylglucamine. Uptakes of Na+ and Rb+ were half-saturated at 3 mM Na+ and 3 mM Rb+, but the maximal velocity of Na+ uptake was 1.5 times that of Rb+ uptake. Na+ uptake was inhibited by extravesicular K+, Rb+, or Li+, and Rb+ uptake was inhibited by extravesicular Na+ or Li+. Amiloride-sensitive uptake of Na+ or Rb+ increased with increase in extravesicular pH and decrease in intravesicular pH. In the absence of pH gradient, there were stimulations of Na+ uptake by intravesicular Na+ and K+ and of Rb+ uptake by intravesicular Rb+ and Na+. Similarly, there were trans stimulations of Na+ and Rb+ efflux by extravesicular alkali cations. The data suggest the existence of a nonselective antiporter catalyzing either alkali cation/H+ exchange or alkali cation/alkali cation exchange. Since increasing Na+ caused complete inhibition of Rb+/H+ exchange, but saturating K+ caused partial inhibitions of Na+/H+ exchange and Na+/Na+ exchange, the presence of a Na(+)-selective antiporter is also indicated. Although both antiporters may be involved in pH homeostasis, a role of the nonselective antiporter may be in the control of Na+/K+ exchange across the cardiac sarcolemma.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Hydrophobic substitution mutations in the S4 sequence alter voltage-dependent gating in Shaker K+ channels

Voltage-activated Na+, Ca2+, and K+ channels contain a common motif, the S4 sequence, characterized by a basic residue at every third position interspersed mainly with hydrophobic residues. The S4 sequence is proposed to function as the voltage sensor and to move in response to membrane depolarization, triggering conformational changes that open the channel. This hypothesis has been tested in previous studies which revealed that mutations of the S4 basic residues often shift the curve of voltage dependence of activation along the voltage axis. We find that comparable or larger shifts are caused by conservative substitutions of hydrophobic residues in the S4 sequence of the Shaker K+ channel. We suggest that the S4 structure plays an essential role in determining the relative stabilities of the closed and open states of the channel.