K+ channels of stomatal guard cells: Abscisic-acid-evoked control of the outward rectifier mediated by cytoplasmic pH

The activation by abscisic acid (ABA) of current through outward-rectifying K⁺ channels and its dependence on cytoplasmic pH (pH_i) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H⁺-selective microelectrodes to record membrane potentials and pH_i during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pH_i. Following impalements, stable pH_i values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pH_i rose over periods of 5–8 min to values 0.27±0.03 pH units above the pH_i before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K⁺ channel current (I_{K, out}) and, once evoked, both pH_i and I_{K, out} responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in I_{K, out}. Butyrate concentrations of 10 and 30 mM (pH₀ 6.1) caused pH_i to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pH_i and concomitant swell in the steady-state conductance of I_{K, out}. The rise in pH_i and i_{K, out} in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K⁺ channels (I_{K, in}). The pH_i dependence of I_{K, out} was consistent with a cooperative binding of at least 2H⁺ (apparent pK_a = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of I_{K, out} in ABA and, thus, affirm a role for H⁺ in signalling and transport control in plants distinct from its function as a substrate in H⁺-coupled transport. Additional evidence implicates a coordinate control of I_{K, in} by cytoplasmic-free [Ca²⁺] and pH_i.Abbreviations ABA abscisic acid - [Ca²⁺]_i cytoplasmic free [Ca²⁺]_i - E_K K⁺ equilibrium potential - I_{K, out}, I_{K, in} outward-, inward-rectifying K⁺ channel (current) - I-V current-voltage (relation) - Mes 2-(N-morpholino)ethanesulfonic acid - pH_i cytoplasmic pH - Tes 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid - V_m membrane potential We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship.     

The bovine tubouterine junction: general organization and surface morphology

The bovine tubouterine junction is composed of three parts (terminal tubal segment, transition region proper, uterine apex) and follows a sigmoidal course displaying a tubal and an uterine curvature. In the terminal tubal segment, 4–8 primary longitudinal folds and a system of lower secondary folds, ridges and chords project into the centrally located lumen. The transition region proper possesses a slit-like lumen because of the existence of a thick mucosal pad containing the first uterine glands. The longitudinal primary folds of the tube broaden, flatten and start to diverge when they reach the transition region proper. The mucosal pad and broadened folds are heavily vascularized. A system of lateral outpocketings with blind ends pointing in an ampullary direction develops between the primary and secondary folds, the ridges and chords of the terminal tubal segment and transition region proper. From the bottom of these outpocketings, short tubulo-alveolar crypts originate. The mucosa of the uterine apex forms low transversal ridges. The musculature of the bovine tubouterine junction is divided into a continuous circular or spiral intermediate layer, flanked by inner and outer longitudinal layers. The outer longitudinal layer is incomplete in the terminal tubal segment but increases in thickness to form a continuous stratum in the uterine apex. An inner longitudinal layer occurs only in the terminal tubal segment where it is best developed in the bases of the primary longitudinal folds. The simple columnar surface epithelium of the tubouterine junction contains ciliated and non-ciliated cells. The former undergo cyclical changes, and increase during estrus and postestrus. During proestrus, groups of non-ciliated cells display bulbous apical protrusions. During proestrus and estrus, circumscribed epithelial lesions expose the underlying basal lamina.     

Cyclic GMP-activated channels of the chick pineal gland: Effects of divalent cations,pH, and cyclic AMP

Chick pineal cells maintained in dissociated cell culture express an intrinsic photosensitive circadian oscillator, but the mechanisms of phototransduction in avian pinealocytes are not fully understood. In this study, we have used inside-out patches to examine the characteristics of cyclic GMP-activated channels of chick pinealocytes in more detail, concentrating on the effects of factors known to modulate the secretion of melatonin and/or the function of circadian pacemakers. In most patches, the predominant conductance state was 19 pS in symmetrical 145 mM NaCl. But in some patches, a second cyclic GMP-activated channel with a unitary conductance of 29 pS was also present. The current flowing through cyclic GMP-activated channels was not affected by application of salines containing 1 M Ca²⁺ to the cytoplasmic face of the patch membrane. By contrast, application of 1 mM Ca²⁺ caused a partial reduction in cyclic GMP-activated current at all membrane potentials. Application of 1–5 mM Mg²⁺ ions caused a virtually complete blockade of current at positive membrane potentials, but caused only a small decrease in current at negative membrane potentials. No obvious differences in the gating of cyclic GMP-activated channels were observed in pH 8.2, 7.4 or 6.2 salines. Application of salines containing 100 M, 500 M, or 1 mM cyclic AMP did not cause activation of the channels, but 5 mM cyclic AMP evoked a low level of channel activity. Application of 5 mM but not 100 M cyclic AMP decreased the probability of channel activation caused by 20–100 M cyclic GMP and also increased the percentage of openings to an 11 pS subconductance state. Thus, cyclic AMP acts as a weak partial agonist. Nevertheless, the gating of these channels does not seem to be controlled directly by physiologically relevant changes in intracellular Ca²⁺, pH, or cyclic AMP.     

Design of a functional calcium channel protein: inferences about an ion channel-forming motif derived from the primary structure of voltage-gated calcium channels.

To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.     

Phosphorylation states greatly regulate the activity and gating properties of Cav3.1 T-type Ca2+ channels

Ca_v3.1 T-type Ca²⁺ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Ca_v3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Ca_v3.1 channel has been poorly investigated. In this work, we analyzed rat Ca_v3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Ca_v3.1 phosphorylation map which includes the reported mouse Ca_v3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Ca_v3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Ca_v3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Ca_v3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.     

Stereoselectivity in β-cyclodextrin complexation of 1,4-dihydropyridine derivatives

Structure–interaction relationships, stereoselectivity, and solubility enhancement in inclusion compexation of β-cyclodextrins (CDs) with some racemic and enantiomerically pure 1,4-dihydropyridine derivatives (DHPs) were investigated. 1:1 and 1:2 (mole ratio) complexes were prepared and characterized by X-ray powder diffraction, differential scanning calorimetry (DSC), MS-FAB spectrometry, ¹H-NMR spectroscopy, water and phase solubility. The solubility studies have revealed different complexation equilibria for optically pure DHP enantiomers, and corresponding racemic mixtures in water solutions. By means of ¹H-NMR chemical shift measurements, the inclusion of aromatic fragments of racemic and enantiomerically pure DHP molecules within the cavities of different CDs was elucidated. Considerable stereoselectivity in complexation interactions was observed. The results indicate the potential use of cyclodextrins as chiral selectors for enantiomeric resolution of 1,4-DHP calcium antagonists. © 1993 Wiley-Liss, Inc.     

Stream channel experiments on downstream movement of recently emerged trout, Salmo trutta L. and salmon, S. salar L.—I. Effect of four different water velocity treatments upon dispersal rate

Four experimental stream channels were used to study instantaneous downstream dispersal rates of young trout, Salmo trutta L., and salmon, S. salur L ., relative to four different water velocities.
Young salmon showed a high rate of dispersal at a low velocity of 7.5 cm s⁻¹ and lower rates at higher velocities of 25 to 70cm s⁻¹. Trout showed their lowest rate at 25cm s⁻¹ with a slightly higher rate at 7.5 cm s⁻¹ and increasingly higher rates at velocities in excess of 25 cm s⁻¹. These results are consistent with field observations on the velocity preferences of young trout and salmon.     

Inhibition of ATP-regulated K+-channels by a photoactivatable ATP-analogue in mouse pancreatic β-cells

The effects of a photoactivatable (DMNPE-caged) ATP-analogue on ATP-regulated K⁺-channels (K_ATP-channel) in mouse pancreatic β-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a K_i of 17 μM; similar to the 11 μM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the K_ATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced K_ATP-channel inhibition.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Conformational analysis of cyclic angiotensin II analogues

Summary Conformationally restricted cyclic analogues of angiotensin II (ANG II), Asp¹-Arg²-Val³-Tyr⁴-Val⁵-His⁶-Pro⁷-Phe⁸, with a link between positions 3 and 5 have considerable biological activity. It is proposed that the spatial arrangement of the pharmacophore groups of Tyr⁴, His⁶ and Phe⁸ side chains and the C-terminal carboxyl group in ANG II and active analogues is similar. Conformational analysis of ANG II and two cyclic analogues c[Sar¹, Lys³,Glu⁵]ANG II and c[Sar¹,Hcy³,Mpt⁵]ANG II was performed, and a geometrical comparison of the low-energy conformations of these compounds allowed one to propose a model of receptor-bound conformation in terms of the spatial arrangement of the pharmacophore groups. This model is characterised by the close spatial location of the His⁶-Phe⁸ side chains and the Tyr⁴ C-terminal carboxyl group and is stabilised by the electrostatic interaction of Arg² and the C-terminal carboxyl group.Abbreviations ANG II angiotensin II - Hcy homocysteine - Mpt trans-4-mercaptoproline