Novel type of signaling molecules: protein kinases covalently linked to ion channels

Recently we identified a new class of protein kinases with a novel type of catalytic domain structurally and evolutionarily unrelated to the conventional eukaryotic protein kinases. This new class, which we named alpha-kinases, is represented by eukaryotic elongation factor-2 kinase and the Dictyostelium myosin heavy chain kinases. Here we cloned, sequenced and analyzed the tissue distribution of five new putative mammalian alpha-kinases: melanoma alpha-kinase, kidney alpha-kinase, heart alpha-kinase, skeletal muscle alpha-kinase, and lymphocyte alpha-kinase. All five are large proteins of more than 1000 amino acids with an alpha-kinase catalytic domain located at the very carboxyl-terminus. We expressed the catalytic domain of melanoma alpha-kinase in Escherichia coli, and found that it autophosphorylates on threonine residues, demonstrating that it is a genuine protein kinase. Unexpectedly, we found that the long amino-terminal portions of melanoma and kidney alpha-kinases represent new members of the transient receptor potential (TRP) ion channel family, which are implicated in the mediation of capacitative Ca2+ entry in nonexcitable mammalian cells. This suggests that melanoma and kidney alpha-kinases, which represent a novel type of signaling molecule, are involved in the regulation of Ca2+ influx in mammalian cells.     

Single channels and surface potential of linear gramicidins

The single channel data for 4 different linear gramicidins containing either 4 Trp, 4 Phe, 4 Tyr or TyrBzl have been analyzed on the basis of 3 barriers-2 sites model. They form 2 families which differ by their single channel behavior and thus different energy profiles of the channel. A relationship between the surface potential and the entry barrier is proposed.     

Proton mobilities in water and in different stereoisomers of covalently linked gramicidin A channels

Proton conductivities in bulk solution (lambda(H)) and single-channel proton conductances (g(H)) in two different stereoisomers of the dioxolane-linked gramicidin A channel (the SS and RR dimers) were measured in a wide range of bulk proton concentrations ([H], 0.1-8000 mM). Proton mobilities (micro(H)) in water as well as in the SS and RR dimers were calculated from the conductivity data. In the concentration range of 0.1-2000 mM, a straight line with a slope of 0.75 describes the log (g(H))-log ([H]) relationship in the SS dimer. At [H] > 2000 mM, saturation is followed by a decline in g(H). The g(H)-[H] relationship in the SS dimer is qualitatively similar to the [H] dependence of lambda(H). However, the slope of the straight line in the log(lambda(H))-log([H]) plot is 0.96, indicating that the rate-limiting step for proton conduction through the SS dimer is not the diffusion of protons in bulk solution. The significant difference between the slopes of those linear relationships accounts for the faster decline of micro(H) as a function of [H] in the SS dimer in relation to bulk solution. In the high range of [H], saturation and decline of g(H) in the SS dimer can be accounted for by the significant decrease of micro(H) in bulk solution. At any given [H], g(H) in the RR dimer is significantly smaller than in the SS. Moreover, the g(H)-[H] relationship in the RR stereoisomer is qualitatively different from that in the SS. Between 1 and 50 mM [H], g(H) can be fitted with an adsorption isotherm, suggesting the presence of a proton-binding site inside the pore (pK(a) approximately 2), which limits proton exit from the channel. At 100 mM < [H] < 3000 mM, g(H) increases linearly with [H]. The distinctive shape of the g(H)-[H] relationship in the RR dimer suggests that the channel can be occupied simultaneously by more than one proton. At higher [H], the saturation and decline of g(H) in the RR dimer reflect the properties of micro(H) in bulk solution. In the entire range of [H], protons seem to cross the SS and RR channels via a Grotthuss-like mechanism. The rate-limiting step for proton transfer in the SS dimer is probably the membrane-channel/bulk solution interface. It is also proposed that the smaller g(H) in the RR dimer is the consequence of a different organization and dynamics of the H-bonded network of water molecules inside the pore of the channel, resulting in a slower proton transfer and multiple pore occupancy by protons.     

Glial ion channels.

It now appears that most of the ion channels discovered in glia are similar or identical to their neuronal equivalents. Recent studies show that glial cells can sense and respond to neuronal signals and that neurons may influence both the development and maintenance of ion channel expression of certain glial cells. Although they lack excitability, glia are probably active participants in brain function.     

Cadaverine is covalently linked to peptidoglycan in Selenomonas ruminantium.

Cadaverine was found to exist as a component of cell wall peptidoglycan of Selenomonas ruminantium, a strictly anaerobic bacterium. [14C]cadaverine added to the growth medium was incorporated into the cells, and about 70% of the total radioactivity incorporated was found in the peptidoglycan fraction. When the [14C]cadaverine-labeled peptidoglycan preparation was acid hydrolyzed, all of the 14C counts were recovered as cadaverine. The [14C]cadaverine-labeled peptidoglycan preparation was digested with lysozyme into three small fragments which were radioactive and were positive in ninhydrin reaction. One major spot, a compound of the fragments, was composed of alanine, glutamic acid, diaminopimelic acid, cadaverine, muramic acid, and glucosamine. One of the two amino groups of cadaverine was covalently linked to the peptidoglycan, and the other was free. The chemical composition of the peptidoglycan preparation of this strain was determined to be as follows: L-alanine-D-alanine-D-glutamic acid-meso-diaminopimelic acid-cadaverine-muramic acid-glucosamine (1.0:1.0:1.0:1.0:1.1:0.9:1.0).     

Pectic polysaccharide rhamnogalacturonan II is covalently linked to homogalacturonan.

A borate-containing pectin was solubilized from sugar beet (Beta vulgaris L. ) cell walls by treatment with 0.5 M imidazole, pH 7. The molecular weight of the pectin was reduced when the borate ester was hydrolyzed by treatment with 1 N HCl. Treatment of the acid-treated pectin with boric acid in the presence of Pb(2+) gave a product whose molecular weight distribution was similar to the imidazole-soluble pectin. The imidazole-soluble pectin was saponified and then digested with endo- and exo-polygalacturonases. These treatments shifted the boron peak at the high molecular weight region to the low molecular weight (10 kDa), which corresponds to rhamnogalacturonan II-borate ester cross-linked dimer (dRG-II-B). The treatment also generated rhamnogalacturonan I (RG-I), dRG-II-B, monomeric rhamnogalacturonan II and galacturonic acid. These results show that imidazole solubilizes a high molecular weight borate-containing pectic complex composed of homogalacturonan-rhamnogalacturonan II and RG-I. Our data suggest that borate esters formed between rhamnogalacturonan II molecules cross-link the macromolecular pectin.     

p53 is covalently linked to 5.8S rRNA.

We report here the isolation and identification of the RNA specifically immunoprecipitated and covalently linked to the tumor suppressor gene product p53. After treatment with proteinase K, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band of p53 yields a single, discrete 157-nucleotide RNA, which was cloned, sequenced, and identified as 5.8S rRNA. 5.8S rRNA was obtained only after proteolysis of the p53 SDS-PAGE band. Free 5.8S rRNA did not comigrate with p53 in SDS-PAGE. This RNA was only immunoprecipitated from cells containing p53. Protein-free RNA obtained by proteolysis of the p53 band hybridized to the single-stranded DNA vector containing the antisense sequence of 5.8S rRNA. The covalence of the p53-5.8S rRNA linkage was demonstrated by the following findings: (i) p53 and the linked 5.8S rRNA comigrated in SDS-PAGE; (ii) only after treatment of the p53-RNA complex with proteinase K did the 5.8S rRNA migrate differently from p53-linked 5.8S rRNA; and (iii) this isolated RNA was found linked to phosphoserine, presumably at the 5' end. Covalent linkage to the single, specific RNA suggests that p53 may be involved in regulating the expression or function of 5.8S rRNA.     

Repair of DNA covalently linked to protein

A potentially lethal form of DNA/RNA modification, a cleavage complex, occurs when a nucleic acid-processing enzyme that acts via a transient covalent intermediate becomes trapped at its site of action. A number of overlapping pathways act to repair these lesions and many of the enzymes involved are those that catalyze recombinational-repair processes. A protein, Tdp1, has been identified that reverses cleavage-complex formation by specifically hydrolyzing a tyrosyl-DNA phosphodiester bond. The study of these pathways is both interesting and pertinent as they modulate the effectiveness of many antitumor/antibacterial drugs that act by stabilizing cleavage-complexes in vivo.     

Solution structure of oligonucleotides covalently linked to a psoralen derivative.

Psoralen (pso) was attached via its C-5 position to the 5'-phosphate group of an oligodeoxynucleotide d(TAAGCCG) by a hexamethylene linker (m6). Complex formation between pso-m6-d(TAAGCCG) and the complementary strands d(CGGCTTA)[7-7mer] or d(CGGCTTAT)[7-8mer] was investigated by nuclear magnetic resonance in aqueous solution. Structural informations derived from DQF-COSY and NOESY maps, revealed that the mini double helix adopts a B-form conformation and that the deoxyriboses preferentially adopt a C2'-endo conformation. The nOe connectivities observed between the protons of the bases or the sugars in each duplex, and the protons of the psoralen and the hexamethylene chain, led us to propose a model involving an equilibrium between two conformations due to different locations of the psoralen. Upon UV-irradiation, the psoralen moiety cross-linked the two DNA strands at the level of 5'TpA3' sequences. NMR studies of the single major photo-cross-linked duplex pso-m6-d(TAAGCCG) and d(CGGCTTA) were performed. The stereochemistry of the diadduct is indeed cis-syn at both cyclobutane rings. In addition, the effects of this diadduct on the helical structure are analyzed in detail.     

Absence of covalently linked core protein from newly synthesized hyaluronate.

1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.     

Coronary vasoactivity of adenosine covalently linked to polylysine

Adenosine was covalently linke to polylysine nonapeptide by reacting adenosine-5-carboxaldehyde with polylysine at pH 9.5, then reducing the Schiff base with NaBH₄. The product (PLADO), which contained an average of two adenosine residues per molecule, did not penetrate cell membranes. When administered to two conscious instrumented dogs PLADO caused maximum coronary vasodilation at a concentration of 4 μM in coronary plasma water. This coronary vasodilatory effect was antagonized by aminophylline. Thus, after the administration of this methylxanthine coronary conductance increased by only 46% of maximum at a PLADO concentration of 6.9 μM. This result confirms earlier work using adenosine coupled to oxidized oligosaccharides that indicates the possibility of adenosine receptors on the surface of coronary myocytes.     

Preparation of covalently linked DNA-RNA hybrids and arabinocytidine containing DNA fragments.

It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment.     

Electromagnetic gating in ion channels.

There have been many attempts to develop a theoretical explanation of the phenomena of electromagnetic field interactions with biological systems. None of the reported efforts have been entirely successful in accounting for the observed experimental results, in particular with respect to the reports of interactions between extremely low frequency (ELF) magnetic fields and biological systems at ion cyclotron resonance frequencies. The approach used in this paper starts with the Lorentz force equation, but use is made of cylindrical co-ordinates and cylindrical boundary conditions in an attempt to more closely model the walls of an ion channel. The equations of motion of an ion that result from this approach suggest that the inside shape of the channel plus the ELF magnetic fields at specific frequencies and amplitudes could act as a gate to control the movement of the ion across the cell membrane.     

Site-specific DNA cleavage by antisense oligonucleotides covalently linked to phenazine di-N-oxide.

Site-specific degradation of DNA was achieved by the use of DNA oligonucleotides covalently tethered to phenazine 5,10-di-N-oxide. When annealed to a complementary DNA target strand, the antisense oligonucleotide effected alkylation of guanosine residues in proximity to the phenazine di-N-oxide prosthetic group. Admixture of dithiothreitol to the formed duplex resulted in reductive activation of the phenazine di-N-oxide moiety with concomitant generation of diffusible oxygen radicals; the latter effected strand scission of the target DNA oligonucleotide. Several parameters of DNA degradation were studied, including the effect on DNA degradation of chain length in the tether connecting the oligonucleotides and prosthetic group, the relative efficiencies of DNA cleavage when the prosthetic group was in the middle or at the end of the antisense oligonucleotide, and the effect of O2 on DNA degradation. Also studied was the actual chemistry of DNA oligonucleotide degradation and the ability of individual diastereomers of the modified oligonucleotides to mediate degradation of the target DNA.     

Photocleavage of DNA by the p-nitrobenzoyl group covalently linked to proflavine.

We have synthesized two novel DNA photocleaving agents,3,6-diamino-10-[6-(4-nitrobenzoyloxy)hexyl]acridinium chloride and 3,6-diamino-10-[6-(4-nitrobenzamido)-hexyl]acridinium chloride, and studied their DNA binding mode and cleavage properties. These compounds contain the photoactive p-nitrobenzoyl group attached to proflavine via an amide or ester linker group and a polymethylene chain. Spectroscopic and viscometric studies have shown that the compounds bind DNA by an intercalative mode. The presence of covalently-bonded intercalator is essential for the UV (310 nm) induced DNA scission. Above a critical ratio, an increase in the relative concentration of compound to DNA did not induce further cleavage. The cleavage efficiency was dependent on the type of linker group. These results are discussed in regard to possible mechanisms for photoinduced DNA breakage.     

Primer-dependent synthesis of covalently linked dimeric RNA molecules by poliovirus replicase.

Poliovirus-specific RNA-dependent RNA polymerase (replicase, 3Dpol) was purified from HeLa cells infected with poliovirus. The purified enzyme preparation contained two proteins of apparent molecular weights 63,000 and 35,000. The 63,000-Mr polypeptide was virus-specific RNA-dependent RNA polymerase, and the 35,000-Mr polypeptide was of host origin. Both polypeptides copurified through five column chromatographic steps. The purified enzyme preparation catalyzed synthesis of covalently linked dimeric RNA products from a poliovirion RNA template. This reaction was absolutely dependent on added oligo(U) primer, and the dimeric product appeared to be made of both plus- and minus-strand RNA molecules. Experiments with 5' [32P]oligo(U) primer and all four unlabeled nucleotides suggest that the viral replicase elongates the primer, copying the poliovirion RNA template (plus strand), and the newly synthesized minus strand snaps back on itself to generate a template-primer structure which is elongated by the replicase to form covalently linked dimeric RNA molecules. Kinetic studies showed that a partially purified preparation of poliovirus replicase contains a nuclease which can cleave the covalently linked dimeric RNA molecules, generating template-length RNA products.     

Single channel properties of heterotetrameric mutant RyR1 ion channels linked to core myopathies

Skeletal muscle excitation-contraction coupling involves activation of homotetrameric ryanodine receptor ion channels (RyR1s), resulting in the rapid release of Ca(2+) from the sarcoplasmic reticulum. Previous work has shown that Ca(2+) release is impaired by mutations in RyR1 linked to Central Core Disease and Multiple Minicore Disease. We studied the consequences of these mutations on RyR1 function, following their expression in human embryonic kidney 293 cells and incorporation in lipid bilayers. RyR1-G4898E, -G4898R, and -DeltaV4926/I4927 mutants in the C-terminal pore region of RyR1 and N-terminal RyR1-R110W/L486V mutant all showed negligible Ca(2+) permeation and loss of Ca(2+)-dependent channel activity but maintained reduced K(+) conductances. Co-expression of wild type and mutant RyR1s resulted in Ca(2+)-dependent channel activities that exhibited intermediate Ca(2+) selectivities compared with K(+), which suggested the presence of tetrameric RyR1 complexes composed of wild type and mutant subunits. The number of wild-type subunits to maintain a functional heterotetrameric channel differed among the four RyR1 mutants. The results indicate that homozygous RyR1 mutations associated with core myopathies abolish or greatly reduce sarcoplasmic reticulum Ca(2+) release during excitation-contraction coupling. They further suggest that in individuals, expressing wild type and mutant alleles, a substantial portion of RyR1 channels is able to release Ca(2+) from sarcoplasmic reticulum.     

Dipole cooperative model of ion channels of excitable biomembranes. Conductance of ion channels

An analysis of ionic channel conductance is presented in terms of dipole cooperative model. The dependence of conductance on displaced charge is found to be an S-shaped function. Basing on this function and kinetics of gating currents, the kinetic curves for the conductance are calculated. These curves are compared with Hodgkin--Huxley results on sodium channel. A good agreement may be observed for the case of positive jumps of the potential. Less accurate coincidence takes place for negative jumps of the potential.     

Activation of NMDA receptors linked to modulation of voltage-gated ion channels and functional implications

Catfish (Ictaluruspunctatus) cone horizontal cells containN-methyl-D-aspartate (NMDA) receptors, thefunction of which has yet to be determined. In the present study, wehave examined the effect of NMDA receptor activation on voltage-gatedion channel activity. NMDA receptor activation produced a long-termdownregulation of voltage-gated sodium and calcium currents but had noeffect on the delayed rectifying potassium current. NMDA'seffect was eliminated in the presence of AP-7. To determine whetherNMDA receptor activation had functional implications, isolated catfish cone horizontal cells were current clamped to mimic the cell's physiological response. When horizontal cells were depolarized, theyelicited a single depolarizing overshoot and maintained a depolarizedsteady state membrane potential. NMDA reduced the amplitude of thedepolarizing overshoot and increased the depolarized steady-statemembrane potential. Both effects of NMDA were eliminated in thepresence of AP-7. These results support the hypothesis that activationof NMDA receptors in catfish horizontal cells may affect the type ofvisual information conveyed through the distal retina.