THE POTASSIUM EQUILIBRIUM IN MUSCLE

1. Analyses were made of the K and HCO₃ content, the irritability, and weight change of isolated frog sartorius muscles after immersion for 5 hours in Ringer''s solutions modified as to pH and potassium content. 2. At each pH a concentration of potassium in the solution was found which was in diffusion equilibrium with the potassium in the muscle. In greater concentrations potassium moved into the muscle against the concentration gradient and vice versa. 3. The greater the alkalinity of the solution the smaller the concentration of the potassium at equilibrium so that the product of the concentrations of OH and K in the solution at equilibrium tends to remain approximately constant. 4. The pH inside the muscle is approximately equal to that outside when first dissected but it tends to change during immersion so as to follow the changes in the pH of the solution. This finding is in direct conflict with the theory according to which the high potassium concentration inside should be accompanied by an equally high hydrogen ion concentration in relation to that outside. 5. The diffusion of potassium into the muscle makes its contents more alkaline but the increase in alkalinity is not always, nor usually, equivalent to the amount of potassium which has diffused and conversely, the pH inside can change in either direction according to the pH outside without there being any diffusion of potassium. Hence potassium is not the only penetrating ion. 6. The irritability of the muscles is at a maximum in concentrations of potassium which are greater than that in normal Ringer''s solution, or about 20 mg. per cent potassium. This optimum does not seem to be a function of pH and is therefore not dependent upon the direction of movement of the potassium but probably on the ratio of potassium outside to that inside. 7. Swelling of the muscles occurs in solutions which injure the muscle so as to permit both cations and anions to enter without permitting the organic protein anions to escape. Anion impermeability is necessary to prevent this same osmotic swelling under normal conditions. 8. An increase in the CO₂ tension in muscle and solution causes a greater increase in acidity in the solution than in the muscle and leads to a loss of potassium. One expects therefore a potassium shift from tissues to blood comparable to the chlorine shift from plasma to corpuscles.     

NMR assignments of a 48 kDa tetramer of the T1 domain of the mammalian voltage gated potassium channel Kv1.4

The N-terminal cytosolic T1 domain of the mammalian voltage gated potassium channel Kv1.4 is strongly involved in the tetramerization of the Kv1.4 subunit that is required for forming a functional ion channel. The T1 domain forms a stable tetramer of 48 kDa in solution that cannot be dissociated into monomers. In spite of the high molecular mass it was possible to completely assign the backbone and part of the side chain resonances by multidimensional NMR spectroscopy on uniformly ²H, ¹³C, ¹⁵N enriched protein. The secondary structure analysis derived from the chemical shifts is in line with the expectations from X-ray structures of related proteins.     

1H nuclear magnetic resonance titration curves and microenvironments of aromatic residues in bovine pancreatic ribonuclease A

The aromatic region of the NMR spectrum of bovine pancreatic ribonuclease A was analyzed in order to clarify the nature of the microenvironments surrounding the individual histidine, tyrosine, and phenylalanine residues and the interactions with inhibitors. The NMR titration curves of ring protons of six tyrosine and three phenylalanine residues as well as four histidine residues were determined at 37 degrees C between pH 1.5 and pH 11.5 under various conditions. The titration curves were analyzed on the basis of a scheme of a simple proton dissociation sequence and the most probable values were obtained for the macroscopic pK values and intrinsic chemical shifts. The microenvironments surrounding the residues and the effects of inhibitors are discussed on the basis of these results. Based on the titration curves of ring protons, the six tyrosine residues were classified into the following four groups: (1) titratable and different chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (2) titratable but similar chemical shifts for C(delta) and C(epsilon) protons (two tyrosine residues), (3) not titratable and different chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residues), and (4) not titratable and similar chemical shifts for C(delta) and C(epsilon) protons (one tyrosine residue). The resonance signals of ring protons were tentatively assigned to tyrosine and phenylalanine residues. The NMR titration curves of His-48 ring protons were continuous in solution containing 0.2 M sodium acetate but were discontinuous in solution containing 0.3 M NaCl because the NMR signals disappeared at pH values between 5 and 6.5. The effects of addition of formate, acetate, propionate, and ethanol were investigated in order to elucidate the mechanism of the continuity of the titration curves of His-48 in the presence of acetate ion. The NMR signal of His-48 C(2) protons was observed at pH 6 in the presence of acetate and propionate ions but was not observed in the presence of formate ion or ethanol. This indicated that both the alkyl chain and the anionic carboxylate group are necessary for the continuity of the titration curves of His-48 ring protons. Based on the results, the mechanism of the effects of acetate ion is discussed.     

Diverse residues of intracellular loop 5 of the Na+/H+ exchanger modulate proton sensing,expression, activity and targeting

The mammalian Na⁺/H⁺ exchanger isoform 1 (NHE1) is an integral membrane protein that regulates intracellular pH (pH_i) by removing a single intracellular proton in exchange for one extracellular sodium ion. It is involved in cardiac hypertrophy and ischemia reperfusion damage to the heart and elevation of its activity is a trigger for breast cancer metastasis. NHE1 has an extensive 500 amino acid N-terminal membrane domain that mediates transport and consists of 12 transmembrane segments connected by intracellular and extracellular loops. Intracellular loops are hypothesized to modulate the sensitivity to pH_i. In this study, we characterized the structure and function of intracellular loop 5 (IL5), specifically amino acids 431–443. Mutation of eleven residues to alanine caused partial or nearly complete inhibition of transport; notably, mutation of residues L432, T433, I436, N437, R440 and K443 demonstrated these residues had critical roles in NHE1 function independent of effects on targeting or expression. The nuclear magnetic resonance (NMR) solution spectra of the IL5 peptide in a membrane mimetic sodium dodecyl sulfate solution revealed that IL5 has a stable three-dimensional structure with substantial alpha helical character. NMR chemical shifts indicated that K438 was in close proximity with W434. Overall, our results show that IL5 is a critical, intracellular loop with a propensity to form an alpha helix, and many residues of this intracellular loop are critical to proton sensing and ion transport.     

An investigation of biooxidation ability of Acidithiobacillus ferrooxidans using NMR relaxation measurement

NMR relaxation measurements can provide a simple means for understanding biological activity of cells in solution with known composition. It has the advantage that it is an in situ, non-intrusive technique, and the acquisition is fast. The iron oxidation ability of Acidithiobacillus ferrooxidans was investigated using NMR relaxation measurements. The transversal relaxation is characterized by a time constant, T?, which is sensitive to the chemical environment. Fe3? ion has more significant T? shortening than Fe2? ion. In the presence of A. ferrooxidans in solutions containing Fe2? ion, T? shortening was found with increasing time as the bacteria oxidize Fe2? to Fe3? ions. In the optimal growth medium, the bacteria concentration increased 80 times and high iron oxidation rate was found. In 10 mM K?SO? medium, however, bacteria concentration remained almost unchanged and the iron oxidation rate was significantly lower.     

Structure of an elastin-mimetic polypeptide by solid-state NMR chemical shift analysis

The conformation of an elastin-mimetic recombinant protein, [(VPGVG)4(VPGKG)]39, is investigated using solid-state NMR spectroscopy. The protein is extensively labeled with 13C and 15N, and two-dimensional 13C-13C and 15N-13C correlation experiments were carried out to resolve and assign the isotropic chemical shifts of the various sites. The Pro 15N, 13Calpha, and 13Cbeta isotropic shifts, and the Gly-3 Calpha isotropic and anisotropic chemical shifts support the predominance of type-II beta-turn structure at the Pro-Gly pair but reject a type-I beta-turn. The Val-1 preceding Pro adopts mostly beta-sheet torsion angles, while the Val-4 chemical shifts are intermediate between those of helix and sheet. The protein exhibits a significant conformational distribution, shown by the broad line widths of the 15N and 13C spectra. The average chemical shifts of the solid protein are similar to the values in solution, suggesting that the low-hydration polypeptide maintains the same conformation as in solution. The ability to measure these conformational restraints by solid-state NMR opens the possibility of determining the detailed structure of this class of fibrous proteins through torsion angles and distances.     

Identification of mixed di-cation forms of G-quadruplex in solution

Multinuclear NMR study has demonstrated that G-quadruplex adopted by d(G3T4G4) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K+ ions into the solution of d(G3T4G4)2 folded in the presence of 15NH4+ ions uncovered a mixed mono-K+-mono-15NH4+ form that represents intermediate in the conversion of di-15NH4+ into di-K+ form. Analogously, 15NH4+ ions were found to replace Na+ ions inside d(G3T4G4)2 quadruplex. The preference of 15NH4+ over Na+ ions for the two binding sites is considerably smaller than the preference of K+ over 15NH4+ ions. The two cation binding sites within the G-quadruplex core differ to such a degree that 15NH4+ ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K+ ions. The second binding site is not taken up by K+ ion until K+ ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K+ ion concentrations afforded equilibrium binding constants. K+ ion binding to sites U and L within d(G3T4G4)2 is more favorable with respect to 15NH4+ ions by Gibbs free energies of approximately -24 and -18 kJ mol(-1) which includes differences in cation dehydration energies, respectively.     

Solution structure and dynamics of a designed monomeric variant of the lambda Cro repressor.

The solution structure of a monomeric variant of the lambda Cro repressor has been determined by multidimensional NMR. Cro K56[DGEVK] differs from wild-type Cro by the insertion of five amino acids at the center of the dimer interface. 1H and 15N resonances for 70 of the 71 residues have been assigned. Thirty-two structures were calculated by hybrid distance geometry/simulated annealing methods using 463 NOE-distance restraints, 26 hydrogen-bond, and 39 dihedral-angle restraints. The root-mean-square deviation (RMSD) from the average structure for atoms in residues 3-60 is 1.03 +/- 0.44 A for the peptide backbone and 1.6 +/- 0.73 A for all nonhydrogen atoms. The overall structure conforms very well to the original design. Although the five inserted residues form a beta hairpin as expected, this engineered turn as well as other turns in the structure are not well defined by the NMR data. Dynamics studies of backbone amides reveal T1/T2 ratios of residues in the alpha2-alpha3, beta2-beta3, and engineered turn that are reflective of chemical exchange or internal motion. The solution structure and dynamics are discussed in light of the conformational variation that has been observed in other Cro structures, and the importance of flexibility in DNA recognition.     

Theoretical study on the proton chemical shifts of hydrogen bonded nucleic acid bases.

The variation of the proton chemical shifts due to the formation intermolecular hydrogen bonds is computed for a number of complexes which can be formed between the bases of the nucleic acids. The shifts expected for the isolated base pairs, in particular for the G-N1 H, T(or U)-N3H protons and the protons of the amino groups of A, G c, when combined with previous computations on the shifts to be expected upon base stacking, may enable a refined analysis of the high resolution NMR spectra of self complementary polynucleotides or tRNAs. Two examples are presented of a direct computation of proton shits associated with helix-coil transitions, helpful for deducing the helical structure in solution.     

Structural consequences of the natural substitution, E9K, on reactive-site-hydrolyzed squash (Cucurbita maxima) trypsin inhibitor (CMTI), as studied by two-dimensional NMR.

Sequence-specific hydrogen-1 NMR assignments were made to all of the 29 amino acid residues of reactive-site-hydrolyzed Cucurbita maxima trypsin inhibitor I (CMTI-I*) by the application of two-dimensional NMR (2D NMR) techniques, and its secondary structural elements (two tight turns, a 3(10)-helix, and a triple-stranded beta-sheet) were identified on the basis of short-range NOESY cross peaks and deuterium-exchange kinetics. These secondary structural elements are present in the intact inhibitor [Holak, T. A., Gondol, D., Otlewski, J., & Wilusz, T. (1989) J. Mol. Biol. 210, 635-648] and are unaffected by the hydrolysis of the reactive-site peptide bond between Arg5 and Ile6, in accordance with the earlier conclusion reached for CMTI-III* [Krishnamoorthi, R., Gong, Y.-X., Lin, C. S., & VanderVelde, D. (1992) Biochemistry 31, 898-904]. Chemical shifts of backbone hydrogen atoms, peptide NH's, and C alpha H's, of CMTI-I* were compared with those of the intact inhibitor, CMTI-I, and of the reactive-site-hydrolyzed, natural, E9K variant, CMTI-III*. Cleavage of the Arg5-Ile6 peptide bond resulted in changes of chemical shifts of most of the backbone atoms of CMTI-I, in agreement with the earlier results obtained for CMTI-III. Comparison of chemical shifts of backbone hydrogen atoms of CMTI-I* and CMTI-III* revealed no changes, except for residues Glu9 and His25. However, the intact forms of the same two proteins, CMTI-I and CMTI-III, showed small but significant perturbations of chemical shifts of residues that made up the secondary structural elements of the inhibitors.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR Studies of the Tautomerism in Pseudoisocytidine

Abstract

The structure of pseudoisocytidine may have two isomers. We would like to designate them as K1 and K3 for N₁H and N₃H, respectively. The authenticity of these two isomers was judged by ¹H NMR. The chemical shift value of N₁H in K1 is found more upfield than N₃H in K3, whereas the chemical shifts of rest protons remain the same. Theoretical calculations show that K1 is less stable than K3 by ca. 9 Kcal/mol in gas phase while a methyl group replaces the furanose moiety. This energy reduces as low as 2 Kcal/mol in solution depending on the polarity of the solvent. Thus, the equilibrium of two tautomers occurs most likely in solution. The ¹H and ¹³C NMR studies have been carried out in the pH range of 1 to 12. The pKa's of deprotonation of N₁ and N₃ sites are found to be 9.36 and 9.42, for K1 and K3, respectively. On the other hand, the pKa's of protonation of the same sites corresponding to these two isomers are 3.79 and 3.69, respectively. A critical analysis of line broadening of C₂ in K1 and K3 in the pH range of 5 to 7 establishes the proton exchange phenomenon. The exchange rate, catalyzed by both [H⁺] and [OH^?], depends on the pH.     

NMR-based localization of ions involved in salting out of hen egg white lysozyme

NaCl-induced aggregation of hen egg white lysozyme (HEWL) was monitored by NMR spectroscopy. Small, but significant, changes induced by salt addition in TOCSY spectra were attributed to the effect of local reorganization of protein backbone upon ion binding. Salt-induced variations in HN and H alpha chemical shifts were mapped on the HEWL 3D structure which allowed the construction of a scheme of the spatial localization of potential ion binding sites. It was found that in a 0.5 M NaCl solution six chloride anions and at least one sodium cation are bound to preferred sites on the HEWL surface.     

High-resolution solid-state 13C NMR study of free and metal-complexed macrocyclic antibiotic ionophores valinomycin, nonactin, and tetranactin: conformational elucidation in solid and solution by conformation-dependent 13C chemical shifts

We recorded high-resolution 13C NMR spectra of the macrocyclic antibiotic ionophores valinomycin, nonactin, and tetranactin in the solid state by the cross-polarization-magic angle spinning (CP-MAS) method, in order to gain insight into the use of conformation-dependent 13C chemical shifts as a convenient means to delineate a conformational change induced by metal ion complexation. The 13C peak splittings in the solid state are consistent with the symmetry properties of the ionophores as revealed by X-ray diffraction: C2 symmetry in free tetranactin and S4 or S6 symmetry for a variety of metal complexes of nonactin and tetranactin or the K+ complex of valinomycin, respectively. Interestingly, many of the 13C NMR peaks of carbons in the skeletal backbones were significantly displaced (up to 8 ppm). The displacements of the peaks were explained by a conformational change as characterized by variations of torsion angles. Accordingly, we were able to obtain conformational features of Na+ and Cs+ complexes of valinomycin, for which X-ray diffraction data are unavailable, on the basis of the displacements of the 13C NMR peaks. Further, we discuss conformational features of these complexes in chloroform solution, with reference to those observed in the solid state.     

Effect of dihydropyridines on cation transport across the skeletal muscle membrane in the frog

2,6-Dimethyl-3,5-dimethoxycarbonyl-4-(o-difluromethoxyphenyl)- 1,4-dihydropyridine (ryodipine) blocks Ca-channels in phasic muscle fibres from ileofibularis and semitendinosus muscle of Rana esculenta. Ryodipine and some other newly synthesized dihydropyridines (10(-7)-10(-4) M) exerted a slight, if any, effect on the steady-state of potassium chord conductance in isotonic K+-sulfate solution. The effluxes of potassium and rubidium from the sartorius muscle of Rana temporaria also remained unchanged after addition of 2 x 10(-4) M ryodipine. Thus, the nonspecific dihydropyridine effect on ion transport seems to be poorly expressed.     

The complex formation of zinc(II) with GHL and related peptides

The formation constants for complexes of Zn(II) with GHL and related peptides have been determined by means of potentiometric titration and ¹H NMR spectroscopy in aqueous solution. GHL has a high affinity for Zn(II) but this somewhat higher affinity compared to the related peptides AH, LH and HL is not a sufficient explanation for its biological role.¹H NMR spectroscopy allows structural assignment of the relative chemical shifts to complex structures and the method, therefore, is a powerful tool for the determination of complex structures when the metal ion is diamagnetic and the ESR method previously applied to the GHLCu(II) system (see ref. 4) cannot be used.     

Conformational analysis of the tetrapeptide Pro-D-Phe-Pro-Gly in aqueous solution

Conformational investigations of the tetrapeptide Pro-D-Phe-Pro-Gly in water solution were carried out by 1H and 13C NMR spectroscopy. The internal proline residue allows for the possibility of cis/trans isomerization about the D-Phe-Pro peptide bond resulting in two conformational isomers. The major isomer was identified as the trans isomer. The pH-dependence of the cis/trans equilibrium supports an additional stabilisation of the trans isomer by an intramolecular ionic interaction between the amino- and carboxy-terminus in the zwitterionic state. Based on 13C spin-lattice relaxation times (T1), different pyrrolidine ring conformations of Pro1 and Pro3 could be determined. By combination of several NMR data (vicinal coupling constants 3JN alpha, temperature dependence of the NH chemical shifts, differences in the chemical shifts between the beta and gamma carbons of the proline residues) and energy minimization calculations, a type II' beta-turn should contribute considerably to the overall structure of the trans isomer.     

Characterization of low populated peptide helical structures in solution by means of NMR proton conformational shifts

A NOE independent NMR method is proposed to characterize unambiguously residues involved in low populated isolated peptide helices. The method is based on the comparison of amide and H alpha chemical shift changes originated upon the addition of stabilizing or denaturing agents with true helical conformational shifts that have been measured for the first time using an isolated model peptide helix, the one formed by Ac-(Leu-Lys-Lys-Leu)3-NHEt in aqueous solution.     

Sodium-23 and potassium-39 nuclear magnetic resonance relaxation in eye lens. Examples of quadrupole ion magnetic relaxation in a crowded protein environment.

Single and multiple quantum nuclear magnetic resonance (NMR) spectroscopic techniques were used to investigate the motional dynamics of sodium and potassium ions in concentrated protein solution, represented in this study by cortical and nuclear bovine lens tissue homogenates. Both ions displayed homogeneous biexponential magnetic relaxation behavior. Furthermore, the NMR relaxation behavior of these ions in lens homogenates was consistent either with a model that assumed the occurrence of two predominant ionic populations, "free" and "bound," in fast exchange with each other or with a model that assumed an asymmetric Gaussian distribution of correlation times. Regardless of the model employed, both ions were found to occur in a predominantly "free" or "unbound" rapidly reorienting state. The fraction of "bound" 23Na+, assuming a discrete two-site model, was approximately 0.006 and 0.017 for cortical and nuclear homogenates, respectively. Corresponding values for 39K+ were 0.003 and 0.007, respectively. Estimated values for the fraction of "bound" 23Na+ or 39K+ obtained from the distribution model (tau C greater than omega L-1) were less than or equal to 0.05 for all cases examined. The correlation times of the "bound" ions, derived using either a two-site or distribution model, yielded values that were at least one order of magnitude smaller than the reorientational motion of the constituent lens proteins. This observation implies that the apparent correlation time for ion binding is dominated by processes other than protein reorientational motion, most likely fast exchange between "free" and "bound" environments. The results of NMR visibility studies were consistent with the above findings, in agreement with other studies performed by non-NMR methods. These studies, in combination with those presented in the literature, suggest that the most likely role for sodium and potassium ions in the lens appears to be the regulation of cell volume by affecting the intralenticular water chemical potential.