Inward rectifier potassium channels in plants differ from their animal counterparts in response to voltage and channel modulators

We have investigated the electrophysiological basis of potassium inward rectification of the KAT1 gene product from Arabidopsis thaliana expressed in Xenopus oocytes and of functionally related K⁺ channels in the plasma membrane of guard and root cells from Vicia faba and Zea mays. The whole-cell currents passed by these channels activate, following steps to membrane potentials more negative than –100 mV, with half activation times of tens of milliseconds. This voltage dependence was unaffected by the removal of cytoplasmic magnesium. Consequently, unlike inward rectifier channels of animals, inward rectification of plant potassium channels is an intrinsic property of the channel protein itself. We also found that the activation kinetics of KAT1 were modulated by external pH. Decreasing the pH in the range 8.5 to 4.5 hastened activation and shifted the steady state activation curve by 19 mV per pH unit. This indicates that the activity of these K⁺ channels and the activity of the plasma membrane H⁺-ATPase may not only be coordinated by membrane potential but also by pH. The instantaneous current-voltage relationship, on the other hand, did not depend on pH, indicating that H⁺ do not block the channel. In addition to sensitivity towards protons, the channels showed a high affinity voltage dependent block in the presence of cesium, but were less sensitive to barium. Recordings from membrane patches of KAT1 injected oocytes in symmetric, Mg²⁺-free, 100 mM-K⁺, solutions allowed measurements of the current-voltage relation of single open KAT1 channels with a unitary conductance of 5 pS. We conclude that the inward rectification of the currents mediated by the KAT1 gene product, or the related endogenous channels of plant cells, results from voltage-modulated structural changes within the channel proteins. The voltage-sensing or the gating-structures appear to interact with a titratable acidic residue exposed to the extracellular medium. Correspondence to: R. Hedrich     

Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells.

Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells.     

Phosphate uptake and polyphosphate metabolism of mycorrhizal and nonmycorrhizal roots of pine and of Suillus bovinus at varying external pH measured by in vivo 31P-NMR

 Comparative in vivo ³¹P-NMR analyses of mycorrhizal and nonmycorrhizal roots of Pinus sylvestris and the fungus of Suillus bovinus in pure culture were used to investigate alterations in phosphate metabolism due to changes in external pH in the range 3.5–8.5. All control samples maintained a constant pH in both cytoplasm and vacuole. Mycorrhizal roots and pure fungus, but not nonmycorrhizal roots, transformed accumulated inorganic phosphate into mobile polyphosphate with a medium chain length. Phosphate uptake rates and polyphosphate accumulation responded differently to external pH. In all cases, maximal phosphate uptake occurred at an external pH close to 5.5. At an external pH of 8.5, both roots and fungus showed a distinct lag in phosphate uptake, which was abolished when the external pH was lowered to 7.5. An irreversible effect on phosphate uptake as a consequence of variation in external pH was also observed. The central role of the fungus in regulating mycorrhizal phosphate metabolism is discussed. Accepted: 15 April 1997     

Neural and smooth muscle development in the chicken gizzard

The development of the autonomic ganglia of Auerbach's plexus and gizzard smooth muscle was studied in chicken embryos. Nervous system and smooth-muscle-specific antibodies were employed in immunofluorescence stainings on tissue sections to investigate the temporal and spatial frame of neural and muscular differentiation in relation to each other. Subserosal clusters of neural cells were clearly demonstrable at embryonic day 5 (ED5), the earliest stage analysed, with the monoclonal antibody El (SGIII-1). Fine nerve fibres (ED6) and, later, large axon bundles projecting from subserosal neuron clusters towards the lumen were followed and found to reach the luminal border by ED11. Already in early development the area of the future laminar tendons on the ventral and dorsal surface of the gizzard was devoid of neuroblasts, and nerve fibres were not extending to the muscle-tendon borderline until ED16. Double stainings with antibodies to smooth muscle myosin (SMM) and El revealed that SMM expression, taken as an indicator for muscle differentiation, followed neural growth. It was first detectable in close apposition to the differentiating neuroblasts in the caudal and cranial portion of the gizzard at ED6. With further development, myosin expression proceeded inward towards the lumen in a wave which followed the ingrowth of E1-positive nerve fibres from the prospective Auerbach plexus. Neuromuscular differentiation deviated from this pattern in the lateral tendon area where nerve growth was delayed and myosin expression preceeded the arrival of E1-positive nerve fibres. The findings suggest that the gizzard could serve as a model system for the analysis of potential early nervous system imprints on smooth premuscle mesenchyme differentiation.     

Further studies on satellite nucleoli in rat and mouse hepatocytes

To provide more information on satellite nucleoli, these nuclear structures were studied by means of cytochemical and immunofluorescence procedures in rat and mouse hepatocytes without and following experimental inhibition of the RNA synthesis. The immuno-staining specific for nucleoli or B23 as well as C23 proteins demonstrated that satellite nucleoli and characteristic nucleoli exhibit the same fluorescence. The number of satellite nucleoli decreased after inhibition of nucleolar RNA synthesis in a similar way to the number of silver-stained granules (SSGs) of characteristic nucleoli. Inhibition of RNA synthesis also reduced the number of hepatocytes containing satellite nucleoli. Thus, satellite nucleoli seem to be real nucleoli from single NORs which did not fuse in the formation of a characteristic nucleolus.     

Immunofluorescence studies on proteins B23 and C23 in nucleoli of human lymphocytes

Nucleoli of normal and leukemic lymphocytes were studied by cytochemical and immunofluorescence methods to provide more information on the nucleolar presence and distribution of proteins B23 and C23. Annular nucleoli of human lymphocytes represent a very convenient subject for such studies, since they consist of one centrally located large fibrillar center surrounded by RNP components. In such nucleoli, protein C23 was present mainly in the central nucleolar region and protein B23 was found mostly in the periphery. The nucleolar area immunostained for protein B23 was usually larger than that stained for protein C23. The distribution of protein C23 appeared to be similar to that of intensely stained nucleolar argyrophilic components. No substantial differences were found between the distribution of proteins B23 and C23 in nucleoli of normal and leukemic lymphocytes. In lymphocytes of patients treated with chemotherapy, the immunofluorescence was diminished for protein B23 and particularly so for protein C23.     

Interactions of U1 RNP with heterogeneous nuclear RNA in rat Novikoff hepatoma nuclei

Summary The nature of the association of U1 RNA with rapidly sedimenting RNP structures in rat hepatoma nuclei was investigated. The effects of salt and proteinase K treatment on the stability of this bound form of U1 RNA were studied using sucrose density gradient analyses. Quantitation of the amount of U1 RNA remaining associated with large structures after treatment was used to assess the relative contribution of protein-protein(and protein-RNA) versus RNA-RNA interactions. Forty-eight percent of the total nuclear U1 RNA released by sonication was found in a bound form when the sonicate was centrifuged through gradients containing 50 mM NaCl. Fifty percent of this bound U1 RNA remained associated with rapidly sedimenting RNPs when the NaCl concentration was raised to 500 mM. To assess the contribution of protein independent interactions, large RNPs were completely deproteinized and their RNA moieties were then recentrifuged on gradients. By this analysis, 27% of the U1 RNA originally bound to hnRNPs was associated with rapidly sedimenting (>30 S) RNA (at 50 mM NaCl) suggesting their association by RNA-RNA hydrogen bonds. When the concentration of NaCl was 500 mM, 31% of the U1 RNA was associated with large RNA. Hence, approximately 30% of the U1 RNA molecules originally bound (or about 15% of the total nuclear U1 RNA) were found to be associated by RNA-RNA hydrogen bonds while the remainder of the binding of U1 RNP to hnRNP was by protein-protein and/or protein-RNA interactions.     

TRYPTOPHAN TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER DURING ACUTE HEPATIC FAILURE

Abstract— Tryptophan transport across the blood-brain barrier was studied using a single injection dual isotope label technique, in the following three conditions: normal rats, rats with portacaval shunts, and rats with portacaval shunts followed 65 h later by hepatic artery ligation. In both normal rats and those with acute hepatic failure the tryptophan transport system was found to be comprised of two kinetically distinct components. One component was saturable and obeyed Michaelis-Menten kinetics (normal: V_max= 19.5 nmol.min^?1.g^?1. K_m= 113 μM; hepatic failure: V_max, = 33.8 nmol.min^?1.g^?1, K_m= 108 μM), and the second was a high capacity system which transported tryptophan in direct proportion to concentration over the range tested (normal: K= 0.026 ml.min^?1.g^?1; hepatic failure: K= 0.067 ml.min^?1.g^?1). Since the saturable low capacity component transports several neutral amino acids, and their collective plasma concentration is high in relation to the individual K_ms, tryptophan transport by this component is reduced by competitive inhibition under physiological conditions. Thus it was calculated that in normal rats approx 40% of tryptophan influx occurs via the high capacity system. During acute hepatic failure transport via both components was increased substantially, approximately doubling the rate of tryptophan penetration of the blood-brain barrier at all concentrations tested. The contribution by the high capacity component became even more significant than in normal rats, accounting for about 75% of all tryptophan passage from plasma to brain. Brain tryptophan content was 29.9 nmol/g in normal rats and rose to 45.2 nmol/g in rats with portacaval shunts and 50.5 nmol/g in those with acute hepatic failure, correlating with the increased rate of tryptophan transport. In a previous study we found that plasma competing amino acids were greatly increased during acute hepatic failure. Calculations predict that these increased concentrations would cause a reduction in tryptophan transport by the low capacity system. However, because of the increase in the rate of transport by the high capacity component, net tryptophan entry across the blood-brain barrier was actually increased. This increased rate of transport clearly contributes to the increased content of brain tryptophan found during hepatic failure.     

Nucleolar immunofluorescence in human hematological malignancies.

Rabbit antibodies to the nuclear Tris extract of HeLa cells which have been shown by the indirect immunofluorescence technique to localize in nucleoli of a variety of human malignant tumors but not in a number of nontumor tissues also produced bright fluorescence in nucleoli of tumor cells in several hematological malignancies. The tumors studied included Hodgkins malignant lymphoma, non-Hodgkins malignant lymphoma, acute myeloid and acute myelomonocytic leukemia, chronic lymphatic and chronic myeloid leukemia. In contrast, none of the corresponding normal cell lines in the bone marrow exhibited bright nucleolar fluorescence. In addition, neither the cells of patients with acute infectious mononucleosis nor lymphoid hyperplasia exhibited bright nucleolar fluorescence. These studies suggest that antibodies to HeLa cell nucleolar antigens may be useful in immunodiagnosis of human malignancies.