Increased Resistance to Extracellular Cation Block by Mutation of the Pore Domain of the Arabidopsis Inward-rectifying K+ Channel KAT1

Inward-rectifying potassium channels in plant cells provide important mechanisms for low-affinity K⁺ uptake and membrane potential control in specific cell types, including guard cells, pulvinus cells, aleurone cells and root hair cells. K⁺ channel blockers are potent tools for studying the physiological functions and structural properties of K⁺ channels. In the present study the structural and biophysical mechanisms of Cs⁺ and TEA⁺ block of a cloned Arabidopsis inward-rectifying K⁺ channel (KAT1) were analyzed. Effects of the channel blockers Cs⁺ and TEA⁺ were characterized both extracellularly and intracellularly. Both external Cs⁺ and TEA⁺ block KAT1 currents. A mutant of KAT1 (``m2KAT1'; H267T, E269V) was produced by site-directed mutagenesis of two amino acid residues in the C-terminal portion of the putative pore (P) domain. This mutant channel was blocked less by external Cs⁺ and TEA⁺ than the wild-type K⁺ channel. Internal TEA⁺ and Cs⁺ did not significantly block either m2KAT1 or KAT1 channels. Other properties, such as cation selectivity, voltage-dependence and proton activation did not show large changes between m2KAT1 and KAT1, demonstrating the specificity of the introduced mutations. These data suggest that the amino acid positions mutated in the inward-rectifying K⁺ channel, KAT1, are accessible to external blockers and may be located on the external side of the membrane, as has been suggested for outward-rectifying K⁺ channels. Received: 31 July 1995/Revised: 5 January 1996     

Mechanosensitive Properties of BK Channels from Embryonic Rat Neuroepithelium

The mechanosensitive properties of large-conductance Ca²⁺-activated K⁺ (BK) channels from embryonic rat neuroepithelium were investigated with the cell-attached and inside-out configurations of the patch-clamp technique. The channels were activated in both recording configurations by negative pressures applied to the patch electrode, but reversal of the effect was total and immediate in inside-out patches whereas it was incomplete and delayed in on-cell patches. This mechanosensitivity was not mediated by Ca²⁺ ions or fatty acids, suggesting that it is an intrinsic property of these channels. Cytochalasin B did not affect mechanosensitivity in on-cell patches but increased it in inside-out patches. Kinetic studies showed that stretch increased the mean open time of the channels and decreased the slowest time constant of their closed-time distributions. The present as well as previous results suggest complex interactions between embryonic BK channels and their membranous and submembranous environment. Received: 1 February 1996/Revised: 25 March 1996     

Functional regulation of reconstituted Na, K-ATPase by protein kinase A phosphorylation

Reconstituted Na⁺,K⁺-ATPase from either pig kidney or shark rectal glands was phosphorylated by cAMP dependent protein kinase, PKA. The stoichiometry was 0.9 mole P_i/mole -subunit in the pig kidney enzyme and 0.2 mol P_i/mol -subunit in the shark enzyme. In shark Na⁺,K⁺-ATPase PKA phosphorylation increased the maximum hydrolytic activity for cytoplasmic Na⁺ activation and extracellular K⁺ activation without affecting the apparent K_m values. In contrast, no significant functional effect after PKA phosphorylation was observed in pig kidney Na⁺,K⁺-ATPase.     

K252a Modulates the Expression of Nerve Growth Factor-Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor-induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor-dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 n M ) of K252a. At higher concentrations (3 µ M ), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor-dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Lipopolysaccharides of Escherichia coli K12 Strains That Express Cloned Genes for the Ogawa and Inaba Antigens of Vibrio cholerae O1: Identification of O-Antigenic Factors

Structural and serological studies were performed with the lipopolysaccharide (LPS) expressed by Escherichia coli K12 strains No. 30 and No. 64, into which cosmid clones derived from Vibrio cholerae O1 NIH 41 (Ogawa) and NIH 35A3 (Inaba) had been introduced, respectively. The two recombinant strains, No. 30 (Ogawa) and No. 64 (Inaba), produced LPS that included, in common, the O-polysaccharide chain composed of an α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine (4-amino-4,6-dideoxy-D -manno-pyranose) homopolymer attached to the core oligosaccharide of the LPS of E. coli K12. Structural analysis revealed the presence of N-(3-deoxy-L -glycero-tetronyl)-2-O-methyl-D -perosamine at the non-reducing terminus of the O-polysaccharide chain of LPS from No. 30 (Ogawa) but not from No. 64 (Inaba). Serological analysis revealed that No. 30 (Ogawa) and No. 64 (Inaba) LPS were found to share the group antigen factor A of V. cholerae O1. They were distinguished by presence of the Ogawa antigen factor B [co-existing with relatively small amounts of the Inaba antigen factor (c)] in the former LPS and the Inaba antigen factor C in the latter LPS. It appears, therefore, that No. 30 (Ogawa) and No. 64 (Inaba) have O-antigenic structures that are fully consistent with the AB(c) structure for the Ogawa and the AC structure for the Inaba O-forms of V. cholerae O1, respectively. Thus, the present study clearly confirmed our previous finding that the Ogawa antigenic factor B is substantially related to the 2-O-methyl group at the non-reducing terminus of the α(1 → 2)-linked N-(3-deoxy-L -glycero-tetronyl)-D -perosamine homopolymer that forms the O-polysaccharide chain of LPS of V. cholerae O1 (Ogawa).     

High-conductance calcium-activated potassium channels; Structure,pharmacology, and function

High-conductance calcium-activated potassium (maxi-K) channels comprise a specialized family of K⁺ channels. They are unique in their dual requirement for depolarization and Ca²⁺ binding for transition to the open, or conducting, state. Ion conduction through maxi-K channels is blocked by a family of venom-derived peptides, such as charybdotoxin and iberiotoxin. These peptides have been used to study function and structure of maxi-K channels, to identify novel channel modulators, and to follow the purification of functional maxi-K channels from smooth muscle. The channel consists of two dissimilar subunits, and . The subunit is a member of theslo Ca²⁺-activated K⁺ channel gene family and forms the ion conduction pore. The subunit is a structurally unique, membrane-spanning protein that contributes to channel gating and pharmacology. Potent, selective maxi-K channel effectors (both agonists and blockers) of low molecular weight have been identified from natural product sources. These agents, together with peptidyl inhibitors and site-directed antibodies raised against and subunit sequences, can be used to anatomically map maxi-K channel expression, and to study the physiologic role of maxi-K channels in various tissues. One goal of such investigations is to determine whether maxi-K channels represent novel therapeutic targets.     

Transmembrane movements of artificial redox mediators in relation to electron transport and ionic currents in chloroplasts

Electrochemical data obtained with TMPD⁺-sensitive electrodes indicate that ammonium-uncoupled chloroplasts retain TMPD (N,N,N',N'-tetramethyl- p -phenylenediamine) mainly in the reduced form during illumination, whereas uncoupled DCMU-treated chloroplasts accumulate TMPD in the oxidized form (TMPD⁺). This observation indicates that the reduced plastoquinol is the preferred electron donor for photosystem I (PSI) and TMPD can only compete efficiently when plastoquinone reduction is blocked. After adding DCMU the formation of a transmembrane gradient for TMPD⁺ is reflected by a slow-down of the electrogenic electron transport and by the emerging of the overshoot of the membrane current in the light-off response. A light-dependent increase in photoelectric current generated by chloroplasts in the presence of NH₄Cl and TMPD is observed and considered to be caused by a reversible release of current limitation in the interfacial conductance barriers in the lumen.