Identification of a cationic channel in synaptosomal membranes

Synaptosomal membranes were fused with liposomes using the hydration technique to produce giant proteoliposomes amenable to patch clamp recordings. Single channel currents of a cationic channel with particular properties were detected. In a solution of 150 mM NaCl, the channel displayed a unit conductance of 136 pS and a mean open state lifetime of 1.1 ms. The gating of the channel was shown to be voltage as well as calcium dependent. Pharmacological studies revealed that the channel was insensitive to a variety of channel blockers, but was inactivated by ruthenium red. Presumably, this channel may play a role in regulating the evoked release of neurotransmitters. Offprint requests to: H. Breer     

Influence of the Molecular Structure of Steroids on Their Ability to Interrupt Gap Junctional Communication

17β-estradiol propionate was found to reduce the gap junctional communication in a concentration range similar to that of testosterone propionate, in primary cultures of rat Sertoli cells and cardiac myocytes. Uncoupling was reversible on washing out and occurred without concomitant rise in the intracellular calcium concentration. Esterification was a prerequisite for the activity of extracellularly applied steroid compounds (for example, testosterone was ineffective even at external concentrations up to 100 μm, whereas its intracellular application at 1 μm totally interrupted intercellular communication), but their uncoupling efficiency did not depend on the nature of the ester chain nor on its position on the steroid nucleus. The derivatives of two other androgen hormones (derivatives of the androstane nucleus) were also efficient as junctional uncouplers. Among five steroid molecules belonging to the pregnane family, only one (pregnanediol diacetate) interrupted the junctional communication. Neither cholic acid nor cholesteryl acetate or ouabain showed this effect. Altogether, no correlation with the presence or position of double bonds nor with the trans- or cis-fusion of the A and B rings could be recognized. These results suggest that this reversible, nondeleterious uncoupling effect of steroids is independent of the shape of the molecules and is more probably related to their size and liposolubility, that condition their insertion into the lipid bilayer. Their incorporation into the membrane could disturb the activity of the membrane proteins by a physical mechanism. Received: 10 April 1995/Revised: 27 October 1995     

External Protons Enhance the Activity of the Hyperpolarization-activated K Channels in Guard Cell Protoplasts of Vicia faba

Hyperpolarization-activated K channels (K _H channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4–8.1 on the activity of the K _H channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the K _H channels by 30–150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by ∼60% between pH 8.1 and 4.4 with an apparent pK _a of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was ∼30 ? and their apparent dissociation constant was 10^−4.6. The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants. Received: 26 April 1996/Revised: 29 June 1996     

Hydraulic control of stomatal conductance in Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and alder [Alnus rubra (Bong)] seedlings

Experiments were conducted on 1-year-old Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] and 2- to 3-month-old alder [Alnus rubra (Bong)] seedlings growing in drying soils to determine the relative influence of root and leaf water status on stomatal conductance (g_c). The water status of shoots was manipulated independently of that of the roots using a pressure chamber that enclosed the root system. Pressurizing the chamber increases the turgor of cells in the shoot but not in the roots. Seedling shoots were enclosed in a whole-plant cuvette and transpiration and net photosynthesis rates measured continuously. In both species, stomatal closure in response to soil drying was progressively reversed with increasing pressurization. Responses occurred within minutes of pressurization and measurements almost immediately returned to pre-pressurization levels when the pressure was released. Even in wet soils there was a significant increase in g_c with pressurization. In Douglas fir, the stomatal response to pressurization was the same for seedlings grown in dry soils for up to 120 d as for those subjected to drought stress over 40 to 60 d. The stomatal conductance of both Douglas fir and alder seedlings was less sensitive to root chamber pressure at higher vapour pressure deficits (D), and stomatal closure in response to increasing D from 1.04 to 2.06 kPa was only partially reversed by pressurization. Our results are in contrast to those of other studies on herbaceous species, even though we followed the same experimental approach. They suggest that it is not always appropriate to invoke a ‘feedforward’ model of short-term stomatal response to soil drying, whereby chemical messengers from the roots bring about stomatal closure.     

Inheritance of stomatal conductance in cotton (Gossypium barbadense)

Stomatal conductance in improved Pima cotton cultivars (Gossypium barbadense) has been previously shown to be positively associated with heat resistance and yield potential. In the present study we determined the mode of inheritance of stomatal conductance in crosses of six G. barbadense parents varying in origin, degree of agronomic development and stomatal conductance. Parents included a primitive tropical cotton (B368), two obsolete cultivars (St Vincent V135, Pima 32), one modern commercial line (Pima S-6) and two elite genotypes of the Pima germplasm (P70, P73). These lines showed distinct differences in stomatal conductance, under greenhouse and field conditions. The primitive B368 had the lowest conductance, and the elite lines the highest. Generation means analysis was used to quantify genetic effects in the crosses P70 × St Vincent V135, Pima S-6 × B368, Pima S-6 × Pima 32, P73 × Pima 32 and P73 × Pima S-6. Best-fit models of the inheritance of stomatal conductance varied in complexity from a simple additive-dominance model in the cross P70 × St. Vincent V135 to models displaying digenic epistatic interactions in the remaining crosses. Significant additive mean effects for stomatal conductance were detected in all crosses. Dominance mean effects were significant in the crosses P73 × Pima 32 and P73 × Pima S-6. Broadsense heritability estimates of stomatal conductance were relatively low (0.16–0.44) in all crosses except Pima S-6 × B368 (0.74). Results also show that the mode of inheritance of stomatal conductance is multigenic, and may have maternal as well as nuclear components. Recouping higher stomatal conductance levels from genetically wider crosses appears feasible and could proceed at a moderate rate. Fixing higher levels of stomatal conductance in populations from crosses of elite germplasm may be more difficult because of the presence of dominant mean effects and digenic epistatic interactions.     

Gas exchange in leaves of the root hemiparasitemelampyrum arvense L. before and after attachment to the host plant

Gas exchange characteristics of a hemiparasiteMelampyrum arvense L. before and after attachment to the hostCapsella bursa pastoris (L.) Med. were compared. The net photosynthetic rates (P_N) on a leaf area basis were extremely low and in comparison to the value obtained for the host were about 15 % and 23 % for the unattached and attached hemiparasite, respectively. Also the concentration of photosynthetic pigments was low (as compared with the host the content of chlorophylls was about 33 % and 49 % and of carotenoids about 38 % and 36 % in the unattached and attached hemiparasite, respectively). On the other hand the rates of respiration were high (about 1.8 and 2.6 times higher in the unattached and attached hemiparasite, respectively, than in the host). In darkness stomatal conductance (g_S) of the host and the unattached hemiparasite was rapidly reduced to 10 % of the value obtained in light, g_S of the attached hemiparasite was decreased only by about 30%. A total reduction of g_S occurred at relative water content (RWC) of 85 %, 75% and 45 % for the unattached hemiparasite, the host, and the attached hemiparasite, respectively. The transpiration (E) rate in the preparasitic stage was very low, being 2.6 and 4.5 times smaller than in the host and the attached hemiparasite, respectively. In the attached hemiparasite WUE was 7.5 and 3 times poorer than in the host and in the preparasitic stage, respectively.     

Ion channel regulation by calmodulin binding

While many ion channels are modulated by phosphorylation, there is growing evidence that they can also be regulated by Ca²⁺-calmodulin, apparently through direct binding. In some cases, this binding activates channels; in others, it modulates channel activities. These phenomena have been documented in Paramecium, in Drosophila, in vertebrate photoreceptors and olfactory receptors, as well as in ryanodine receptor Ca²⁺-release channels. Furthermore, studies on calmodulin mutants in Paramecium have shown a clear bipartite distribution of two groups of mutations in the calmodulin gene that lead to opposite behavioral and electrophysiological phenotypes. These results indicate that the N-lobe of calmodulin specifically interacts with one class of ion-channel proteins and the C-lobe with another.     

A novel Cl− conductance in cultured chick cardiac myocytes: Role of intracellular Ca2+ and cAMP

Cl^– conductance in cultured embryonic chick cardiac myocytes was characterized using whole-cell patch clamp techniques. Following elimination of cation currents in Na⁺and K⁺-free internal and external solutions, the basal whole-cell current was predominantly a Cl^– current. Cl^–-sensitive current (I _Cl) was defined as the difference between the whole-cell currents recorded in normal and low [Cl^–] _o when measured in the same cell. The whole-cell current in the absence or presence of 10 m cAMP was time independent, displayed outward rectification with the pipette [Cl^–] < 40 mm, and was not saturated with a physiological Cl^– gradient. The Cl^– current was also activated by 1 m forskolin and inhibited by 0.3 mm anthracene-9-carboxylic acid (9-AC). Forskolin was less effective than cAMP (internal dialysis) in activating the Cl^– current. The cAMP- or forskolin-activated and basal Cl^– current were reasonably fit by the Goldman-Hodgkin-Katz equation. The calculated P _Cl in the presence of cAMP was increased by fiveto sixfold over the basal level. In the presence of 5 mm EGTA to decrease free [Ca²⁺] _i , the whole-cell current could not be stimulated by cAMP, forskolin or IBMX (0.1 mm). These data suggest that cultured chick cardiac myocytes have a low basal Cl^– conductance, which, as in some mammalian cardiac ventricular myocytes, can be activated by cAMP. However, this study shows that the activation process requires physiological free [Ca²⁺] _i .This study was supported by grants from the National Institutes of Health (HL-17670, HL-27105 and HL-07107) for M.L. and by Institutional funds of the University of Arkansas for Medical Sciences for S.L.We thank Meei-Yueh Liu, Kathleen Mitchell, and Shirley Revels for their technical assistance.     

Physiological effects of five months exposure to low concentrations of O3 and NH3 on Douglas fir (Pseudotsuga menziesii)

Three years old seedlings of Douglas fir (Pseudotsuga menziesii) were exposed lo filtered air, O₃ (day and night concentrations of 78 and 30 μgm^?3: respectively). NH₃ (54 μg m^?3) and to a mixture of NH₃+O₃ (day and night concentrations of 49 + 83 and 49 + 44 μg m^?3 respectively), for 5 months in fumigation chambers. Both gas exchange and chlorophyll fluorescence were measured on shoots which had sprouted at the beginning of the exposure period. After 4. 8, 10 and 20 weeks of exposure, light response curves of electron transport rate (J) were determined, in which J was deduced from chlorophyll fluorescence. Net CO₂ assimiialion was measured at maximum light intensity of 560) μmol m^?2 S^?1 (P_n.560). After 8 and 10 weeks of exposure also light response curves of CO₂ assimilation were assessed. Shoots exposed to O₃ showed a reduction in net CO₂ assimilation as compared to the control shoots during the entire exposure period. The reduction was related lo a lower chlorophyll content and a lower electron transport rate, whereas no effect on quantum yield efficiency (qy) was observed. In contrast, shoots exposed to NH₃ showed a positive effect on photosynthesis. Shoots exposed to NH₃. + O₃ showed a rapid increase in P_n.560, in the period between 4 and 8 weeks to a level equal of that of the NH₃-treatment. After this period a decline in P_n.560 was observed. After 10 weeks of exposure shoots exposed to O₃ showed an increased transpiration rate in the dark as compared to the control shoots. In addition, water use efficiency (WUE) declined as a result of an increase in leaf conductance. Both observations indicate that the stomatal apparatus was affected by O₃. A high transpiration rate in the dark was also found for shoots esposed to NHX. However, shoots exposed to NH₃+ O₃ showed neither an effect on WUE, nor an effect on transpiration rate in the dark. The possibility that NH₃ delayed the O₃ induced effects on photosynthesis and stomatal conductance is discussed.     

Selection for higher yields and heat resistance in Pima cotton has caused genetically determined changes in stomatal conductances

Gas exchange analysis was used to characterize photosynthetic and stomatal responses to key environmental stimuli in five commercial lines of Pima cotton ( Gossypium barbadense L.) which represent a gradient of selection for higher yields and heat resistance, and a primitive, uncultivated G. barbadense. At constant light and vapor pressure deficit, stomatal conductance increased linearly with air temperature in the 23 to 36$C range in all five commercial lines, and conductance at each temperature increased as a function of selection. In contrast, photosynthetic rates had a low sensitivity to temperature in the 23 to 36$C range, particularly in the advanced lines. In a segregating F₂ population from a cross between the advanced line, Pima S-6, and the primitive cotton, B368, the slope of the stomatal response to temperature in each F₂ plant was positively correlated with the stomatal conductance measured at 40$C. An analysis of the frequency distribution of stomatal conductance in F₁ and F₂ progeny of the cross showed that the differences in stomatal conductance between lines were genetically determined. These data indicate that selection for higher yield and heat resistance in Pima cotton has caused genetically determined changes in stomatal properties. Characterization of the relationship between the altered stomatal properties and the attained increases in heat resistance and yields could make it possible to use these physiological traits as selection criteria in future breeding programs.