A9 Fibroblasts Transfected with the m3 Muscarinic Receptor Clone Express a Ca2+ Channel Activated by Carbachol,GTP and GDP

Muscarinic m3 receptor-mediated changes in cytosolic Ca²⁺ concentration ([Ca²⁺]_l) occur by activation of Ca²⁺ release channels present in the endoplasmic reticulum membrane and Ca²⁺ entry pathways across the plasma membrane. In this report we demonstrate the coupling of m3 muscarinic receptors to the activation of a voltage-insensitive, cation-selective channel of low conductance (3.2 ± 0.6 pS; 25 mm Ca²⁺ as charge carrier) in a fibroblast cell line expressing m3 muscarinic receptor clone (A9m3 cells). Carbachol (CCh)-induced activation of the cation-selective channel occurred both in whole cell and excised membrane patches (CCh on the external side), suggesting that the underlying mechanism involves receptor-channel coupling independent of intracellular messengers. In excised inside-out membrane patches from nonstimulated A9m3 cells GTP (10 μm) and GDP (10 μm) activated cation-selective channels with conductances of approximately 4.3 and 3.3 pS, (25 mm Ca²⁺ as charge carrier) respectively. In contrast, ATP (10 μm), UTP (10 μm) or CTP (10 μm) failed to activate the channel. Taken together, these results suggest that carbachol and guanine nucleotides regulate the activation of a cation channel that conducts calcium. Received: 14 November 1996/Revised: 4 April 1997     

Spatial heterogeneity in the herbaceous layer of a semi-arid savanna ecosystem

Despite increasing recognition of the role spatial pattern can play in ecosystem function, few studies have quantified spatial heterogeneity in savanna ecosystems. The spatial distribution of herbaceous biomass and species composition was measured across three scales in a semi-arid savanna in central Kenya, and patterns were related to environmental variables at different scales. Herbaceous biomass declined across a rainfall gradient and from upper to lower topographic positions, but variation within a site (across 5–50 m) was similar in magnitude to among-site variation associated with rainfall and topography. Geostatistical analyses showed that patchiness at scales of 5–25 m explained 20% of total variation in herbaceous biomass. This pattern arose from the presence of both 5–10-m diameter patches containing high herbaceous biomass (> 170 g m^–2) and 5–10-m diameter patches characterized by nearly bare soil surfaces (< 40 g m^–2). Patch structure was contingent on topography, with larger bare patches at ridgeline and upper hillslope positions. Grass species distributions showed the greatest degree of patch structure and species turnover across distances of 5–45 m. Additional community variation was associated with topography, with minimal variation in species composition across the rainfall gradient. Pattern diversity significantly exceeded levels reported for four other grassland ecosystems, suggesting fundamental differences in local processes generating spatial pattern. It is hypothesized that heterogeneously distributed grazing pressure, interacting with the distribution of shrub canopies, is an important factor generating such high levels of small-scale patch structure in this savanna.     

Hydrodynamics and mass transfer in an airlift loop fermentor

Summary The hydrodynamics and mass transfer behaviour of an airlift fermentor with an external loop (height 10m) has been investigated by measuring gas and liquid velocities, gas hold-up, liquid mixing and oxygen transfer coefficients. Liquid phase properties, i.e., ionic strength, viscosity and surface tension have been varied by altering the fermentation media. Results are compared with those from bubble column experiments performed in the same unit. It is shown, that more uniform two-phase flow in the airlift leads to advantages in scale-up and operation.Nomenclature a Specific interfacial area per volume of dispersion (m²/m³) - c Local concentration of tracer (kmol/m³) - c Concentration of tracer at infinite time (kmol/m³) - C_L Concentration of oxygen in the liquid bulk (kmol/m³) - C_L ^* Concentration of oxygen in the interface (kmol/m³) - D_ax Axial dispersion coefficient (cm²/s) - I Ionic strength (kmol/m³) - i Inhomogeneity [defined in Eq. (2)] - Rate of oxygen transfer (kmol/s) - t_c Circulation time (s) - t_M Mixing time (s) - V_R Volume of gas-liquid dispersion (m³) - V_SG Superficial gas velocity in up-flow column (m/s) Greek letter symbols _L Oxygen transfer coefficient (m/s) - Dynamic viscosity (m Pa s) - Surface tension (m N/m) Presented at the First European Congress on Biotechnology, Interlaken, September 25–29, 1978     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Short Term Bioturbation Activity in the Lagoonal Sediments of Tikehau Atoll (Tuamotu Archipelago,French Polynesia)

To quantify bioturbation activity in Tikehau lagoon, a tracer made of black basaltic sand was poured over the natural white calcareous sediment surface. Three stations respectively located on the inner flat (-3m), the inner slope (-9m), and the lagoon floor (-19m), were studied for short periods of time (48 hours). Bioturbation by macrofauna was quantified by volume of sediment ejected onto the experimental surface and by volume of tracer incorporated into sediment. The results showed a rapid incorporation of sedimented particles at the interface by way of the funnels and burrows of surface deposit feeders and carnivores. Expelled quantities varied with respect to site location: 213 cm³ · m⁻² · 24 h⁻¹ in the inner flat; 98.9 cm³ · m⁻² · 24 h⁻¹ in the inner slope; 7.9 m³ · m⁻² · 24 h⁻¹ in the lagoon floor. Bioturbation by decapod megafauna appeared to be important in the dynamics of the sediments in the deepest areas of the lagoon. In these areas, with almost no hydrodynamical impacts on sediments, bioturbating events were responsible for sediment mixing (despite lower absolute rates than in shallow area). Hydrodynamics controlled the spatial distribution of macroinvertebrate trophic groups by its effects on sedimentation.     

Differential sensitivity of pneumolysin-induced channels to gating by divalent cations

Summary The induction of channels across planar lipid bilayers by purified, recombinant pneumolysin (a hemolytic protein from Streptococcus pneumoniae) has been studied by measuring increases in electrical conductivity. Pneumolysin-induced channels exhibit a wide range of single channel conductances (<50 pS to >1 nS at 0.1 m KCl). Channels can be categorized on the basis of their K⁺:C^– selectivity: the smallest channels are strongly cation selective, with t₊ (the cation transference number) approaching 1.0; the largest channels are unselective (t₊ 0.5). Channels tend to remain open at all voltages (–150 to 150 mV); only the smallest channels exhibit any rectification.In the presence of divalent cations (1–5 mm Zn²⁺; 10–20 mm Ca²⁺), small (<50 pS) and medium-sized (50 pS to 1 nS) channels are closed in a voltage-dependent manner (more closure at higher voltages); at 0 voltage channels reopen. Overall selectivity is reduced by divalent cations, compatible with small, selective channels being closed preferentially to large, nonselective ones.It is concluded that a single molecular species (pneumolysin) induces multiple-sized channels that can be categorized by cation: anion selectivity and by their sensitivity to closure by divalent cations.We are grateful to Dr. G. J. Boulnois and T. J. Mitchell forfruitful discussion and supplies of pneumolysin, and to G. M. Alder for technical assistance. YEK is grateful to Dr. A. A. Lev for leave of absence and to the USSR Academy of Sciences and the Global Network for Molecular and Cell Biology (UNESCO) for support of travel and accommodation, respectively. The work was supported by the Cell Surface Research Fund.     

Mobility of Protozoa through Narrow Channels

Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individual's unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 μm s⁻¹ (or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 μm s⁻¹. Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment.     

Hierarchy theory reveals multiscale predictors of Arkansas darter (Etheostoma cragini) abundance in a Great Plains riverscape

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Sodium-selective channels in membranes of rat macrophages

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na⁺ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na⁺ over K⁺ (as for Cs⁺, NH₄ ⁺, Ca²⁺, Ba²⁺) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li⁺-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722.     

GENOTYPIC VARIATION AND CLONAL STRUCTURE IN CORAL POPULATIONS WITH DIFFERENT DISTURBANCE HISTORIES

Genotypic diversity in six populations of the endemic Hawaiian reef coral, Porites compressa, was directly related to habitat-disturbance history. The highest diversity (lowest amount of clonal proliferation) was found in populations that had been intensely or recently disturbed. In these populations, space was not limited and mean colony size was small (< 500 cm²), suggesting early stages of recolonization. In an undisturbed, protected habitat, lower genotypic diversity was a result of a significant degree of clonal replication of established genotypes. Unoccupied substratum was rare in this habitat, and average colony size was large (> 2500 cm²). Populations in intermediately disturbed habitats showed intermediate levels of diversity and clonal structure as a result of the combined contributions of sexual and asexual reproduction. Individual clones were distributed over small areas (< 4 m²) or distances (< 1 m) in young populations, and more broadly (> 256 m²) and over longer distances (> 90 m) in the older, undisturbed population. Interpretations of life-history parameters and estimates of total genetic variability in species that have the potential to reproduce asexually are dependent upon an assessment of the overall clonal structure of populations. The power of genotypic assays to reliably detect clonal versus unique colonies, as well as the spatial scales over which clonal populations are sampled, are critical to such assessments.     

The Importance of Large-Diameter Trees to Forest Structural Heterogeneity

Large-diameter trees dominate the structure, dynamics and function of many temperate and tropical forests. However, their attendant contributions to forest heterogeneity are rarely addressed. We established the Wind River Forest Dynamics Plot, a 25.6 ha permanent plot within which we tagged and mapped all 30,973 woody stems ≥1 cm dbh, all 1,966 snags ≥10 cm dbh, and all shrub patches ≥2 m². Basal area of the 26 woody species was 62.18 m²/ha, of which 61.60 m²/ha was trees and 0.58 m²/ha was tall shrubs. Large-diameter trees (≥100 cm dbh) comprised 1.5% of stems, 31.8% of basal area, and 17.6% of the heterogeneity of basal area, with basal area dominated by Tsuga heterophylla and Pseudotsuga menziesii. Small-diameter subpopulations of Pseudotsuga menziesii, Tsuga heterophylla and Thuja plicata, as well as all tree species combined, exhibited significant aggregation relative to the null model of complete spatial randomness (CSR) up to 9 m (P≤0.001). Patterns of large-diameter trees were either not different from CSR (Tsuga heterophylla), or exhibited slight aggregation (Pseudotsuga menziesii and Thuja plicata). Significant spatial repulsion between large-diameter and small-diameter Tsuga heterophylla suggests that large-diameter Tsuga heterophylla function as organizers of tree demography over decadal timescales through competitive interactions. Comparison among two forest dynamics plots suggests that forest structural diversity responds to intermediate-scale environmental heterogeneity and disturbances, similar to hypotheses about patterns of species richness, and richness- ecosystem function. Large mapped plots with detailed within-plot environmental spatial covariates will be required to test these hypotheses.     

Plant species diversity and environmental heterogeneity: spatial scale and competing hypotheses

Questions: What is the observed relationship between plant species diversity and spatial environmental heterogeneity? Does the relationship scale predictably with sample plot size? What are the relative contributions to diversity patterns of variables linked to productivity or available energy compared to those corresponding to spatial heterogeneity? Methods: Observational and experimental studies that quantified relationships between plant species richness and within‐sample spatial environmental heterogeneity were reviewed. Effect size in experimental studies was quantified as the standardized mean difference between control (homogeneous) and heterogeneous treatments. For observational studies, effect sizes in individual studies were examined graphically across a gradient of plot size (focal scale). Relative contributions of variables representing spatial heterogeneity were compared to those representing available energy using a response ratio. Results: Forty‐one observational and 11 experimental studies quantified plant species diversity and spatial environmental heterogeneity. Observational studies reported positive species diversity‐spatial heterogeneity correlations at all points across a plot size gradient from ～1.0 × 10^?1 to ～1.0 × 10¹¹ m², although many studies reported spatial heterogeneity variables with no significant relationships to species diversity. The cross‐study effect size in experimental studies was not significantly different from zero. Available energy variables explained consistently more of the variance in species richness than spatial heterogeneity variables, especially at the smallest and largest plot sizes. Main conclusions: Species diversity was not related to spatial heterogeneity in a way predictable by plot size. Positive heterogeneity‐diversity relationships were common, confirming the importance of niche differentiation in species diversity patterns, but future studies examining a range of spatial scales in the same system are required to determine the role of dispersal and available energy in these patterns.     

Use of artificial eggs in studies of washout depth and drift distance for salmonid eggs

Colour-coded artificial trout eggs were used in investigations of washout depth in a natural stream and of drift distance relative to water velocity in an experimental channel and in a section of natural stream.Washout depth was studied in a spawning riffle of a stream whose bankful discharge is 5.6 m³ s^–1. During an experiment when spates never exceeded 6.5 m³ s^–1 egg washout was severe at 5 cm depth within the gravel, variable at 10 cm and negligible at 15 cm. During another experiment when a spate of 9.0 m³ s^–1 (return period 10–20 years) occurred, washout was severe at 5 and 10 cm depth and variable at 15 cm. There was also evidence that some eggs were moved short distances downstream within the gravel without being washed out.Within experimental channels, drift distance could be predicted from multiple regressions relating logarithms of water velocity, percentage of eggs settled and distance from point of release. At a water velocity of 100 cm s^–1 at 0.6 depth, 50% of eggs would settle within 8 m of the point of release. At water velocities of 75 to 100 cm s^–1 drifting eggs would, on average, travel at c. 60% of water velocity and make 1 to 2 bed contacts m^–1 of travel.A similar multiple regression can be applied to data from a natural stream channel. It predicts much larger drift distances (50% settled in 42 m at 100 cm s ^–1 ). However, in the natural channel, settlement appears aggregated and the validity of the concept of permanent settlement is in doubt.     

Transport of coarse particulate organic matter in an Idaho river,USA

I investigated organic matter transport in the Henry's Fork of the Snake River, Idaho, USA, from August 1987 to November 1988. Mean discharge during the study was 15 m³ s^–1. Screens were used to sample very coarse (> 6 mm) transported aquatic macrophyte material (VCTMM). Drift nets were used to sample coarse (1–6 mm) and fine (0.25–1 mm) transported particulate organic matter (CTOM and FTOM). Mean monthly concentration of VCTMM was 0.064 mg AFDWl^–1 and was significantly higher than CTOM (0.024 mg AFDW l^–1) and FTOM (0.036 mg AFDW l^–1). VCTMM concentration was highest in December (0.163 mg AFDW l^–1) and lowest in May (0.018 mg AFDW l^–1). The sample position along a transect across the channel had a significant effect on the amount of transported organic matter collected in many months. The concentration of debris from individual species tracked the standing stock of that species during the growing season. In Fall, a dramatic increase in VCTMM corresponded to a decrease in macrophyte standing stock. FTOM and CTOM concentrations were highest in January (CTOM: 0.048; FTOM: 0.111 mg AFDW l^–1), lowest in November 1988 (<0.006 mg AFDW l^–1), were not correlated with discharge, and were inversely correlated with the standing stocks of macrophytes upstream, probably because macrophyte beds influenced the retentiveness of the channel. Standing stock of aquatic macrophytes was highest in September–October (5.2 kg wet weight m ^–2) and lowest in February (1.7 kg wet weight m^–2). Annual movement of particulate organic matter past the sampling point was about 45 000 kg AFDW, of which 21 000 kg was VCTMM, 8 000 kg was CPOM, and 16 000 kg was FPOM.     

Nonselective Block by La3+ of Arabidopsis Ion Channels Involved in Signal Transduction

Lanthanide ions such as La³⁺ are frequently used as blockers to test the involvement of calcium channels in plant and animal signal transduction pathways. For example, the large rise in cytoplasmic Ca²⁺ concentration triggered by cold shock in Arabidopsis seedlings is effectively blocked by 10 mm La³⁺ and we show here that the simultaneous large membrane depolarization is similarly blocked. However, a pharmacological tool is only as useful as it is selective and the specificity of La³⁺ for calcium channels was brought into question by our finding that it also blocked a blue light (BL)-induced depolarization that results from anion channel activation and believed not to involve calcium channels. This unexpected inhibitory effect of La³⁺ on the BL-induced depolarization is explained by our finding that 10 mm La³⁺ directly and completely blocked the BL-activated anion channel when applied to excised patches. We have investigated the ability of La³⁺ to block noncalcium channels in Arabidopsis. In addition to the BL-activated anion channel, 10 mm La³⁺ blocked a cation channel and a stretch-activated channel in patches of plasma membrane excised from hypocotyl cells. In root cells, 10 mm La³⁺ inhibited the activity of an outward-rectifying potassium channel at the whole cell and single-channel level by 47% and 58%, respectively. We conclude that La³⁺ is a nonspecific blocker of multiple ionic conductances in Arabidopsis and may disrupt signal transduction processes independently of any effect on Ca²⁺ channels. Received: 28 July 1997/Revised: 13 November 1997     

Characterization of ion channels in the plasma membrane of epidermal cells of expanding pea (Pisum sativum arg) leaves

Ion channels in isolated patches of the plasma membrane of pea (Pisum sativum arg) epidermal cells were studied with the patch-clamp technique. One anion and one cation channel were dominantly present in most trials. The anion channel conducts nitrate, halides and malate, with a conductance in symmetrical 100 mm Cl^– of 300 pS and can be blocked by SITS when applied to the cytoplasmic side of the membrane. The cation channel poorly discriminates between potassium, sodium and lithium, is not blocked by either TEA or Ba²⁺, and has a conductance of 35 pS in symmetrical 100 mm K⁺. The open probability of the cation channel increases with increase of the Ca²⁺ concentration on the cytoplasmic side of the membrane from 0.1 to 1 m. The possible role of these two channels in the physiology of epidermal cells is discussed.This work was supported by NSF grant DCB-890 3744 to E.V.     

The Impermeant Ion Methylammonium Blocks K+ and NH+ 4 Currents through KAT1 Channel Differently: Evidence for Ion Interaction in Channel Permeation

The permeation properties of KAT1, an inward rectifying potassium channel from plant cells, were investigated with different ions in the external medium. With either K⁺, NH⁺ ₄ or methylammonium (MA) in the external solution, the channel, expressed in Xenopus oocytes, appeared permeable to K⁺ and, to a lesser extent, to NH⁺ ₄ but not to the slightly bigger, methylated analogue of NH⁺ ₄, MA. Substituting NH⁺ ₄ for K⁺ shifted the voltage dependency of channel activation further negative and hastened activation kinetics. This suggests that channel operation depends on the transported substrate. In mixed solution (50 mm K⁺, 50 mm MA) MA inhibited K⁺ current in a voltage-independent manner. The maximum block did not exceed 50% of the K⁺ current. In contrast, when NH⁺ ₄ was the permeant ion (50 mm NH⁺ ₄, 50 mm MA) MA caused a voltage-dependent, slowly developing open channel block, achieving complete inhibition at very negative voltages. The latter block could be partially overcome by the addition of K⁺ in the external solution. The data support a model in which ions, after entering the channel pore, compete with different affinities for binding sites on their permeation pathway. Received: 6 October 1997/Revised: 28 January 1998     

Multi-scale analysis of plant species richness in Serengeti grasslands

Aim To assess scale dependence between environmental factors and plant species richness. Additionally, we aimed to identify the scales at which niche relations and habitat heterogeneity, as hypothesized by A. Shmida & M.V. Wilson (1985) Journal of Biogeography, 12 , 1–20, operate in the savanna grasslands that were the focus of this study. Location Savanna grassland plant communities of Serengeti National Park, Tanzania. Methods Plant species richness was sampled in 102 modified Whittaker plots and tested for associations with two climate factors, mean annual rainfall (MAP) and potential evapotranspiration (PET), and two landscape variables, plot aspect (ASP) and topographic variation (TOPO), using multiple regressions. Scale dependence was assessed by conducting regressions after altering three aspects of spatial scale: grain, extent and focus. Grain was altered by analysing plant richness at 1, 10, 10² and 10³ m²; extent was investigated by restricting the maximum distance between samples to 75, 100, 125 and 150 km; and focus was manipulated by averaging samples spatially according to geographical land regions. Within the context of our data, we assumed that niche relations were represented by climate factors and habitat heterogeneity by landscape factors. Results Across all 102 plots, plant species richness between 1 and 10² m² had a negative relation to PET and a weak positive relation to MAP. Plant species richness at 10³ m² had a positive association with TOPO and weaker associations with climate factors. ASP stayed in the model between grains of 10 and 10³ m², but had a very weak positive association with richness. When the focus was changed to land regions, associations between plant species richness and explanatory variables strengthened, but were not qualitatively different. At spatial extents of 75 and 100 km, PET was the strongest correlate of plant species richness across all spatial grains. At spatial extents ≥ 125 km, PET explained the majority of the model variance at spatial grains ≤ 10² m², whereas TOPO explained equal amounts or more of the model variance at spatial grains of 10³ m². Main conclusions Both climate and topographic variation explained plant species richness in Serengeti grasslands, but specific patterns depended on grain, extent and, to a lesser degree, focus. Consistent with the ideas of Shmida & Wilson (1985) , determinants of plant species richness shifted from niche relations to habitat heterogeneity between spatial grains of 1 and 10³ m², although this occurred only at relatively large spatial extents (≥ 150 km). Finally, the signs, strength and shape of plant species richness relationships in Serengeti closely match those that describe macro‐scale patterns of woody plant species richness across the entire African continent.     

Calcium-independent K+-selective channel from chromaffin granule membranes

Summary Intact adrenal chromaffin granules and purified granule membrane ghosts were allowed to fuse with acidic phospholipid planar bilayer membranes in the presence of Ca²⁺ (1 mm). From both preparations, we were able to detect a large conductance potassium channel (ca. 160 pS in symmetrical 400 mm K⁺), which was highly selective for K⁺ over Na⁺ (P _k/P _Na = 11) as estimated from the reversal potential of the channel current. Channel activity was unaffected by charybdotoxin, a blocker of the [Ca²⁺] activated K⁺ channel of large conductance. Furthermore, this channel proved quite different from the previously described channels from other types of secretory vesicle preparations, not only in its selectivity and conductance, but also in its insensitivity to both calcium and potential across the bilayer. We conclude that the chromaffin granule membrane contains a K⁺-selective channel with large conductance. We suggest that the role of this channel may include ion movement during granule assembly or recycling, and do not rule out events leading to exocytosis.     

Amphotericin B Kills Unicellular Leishmanias by Forming Aqueous Pores Permeable to Small Cations and Anions

The polyene antibiotic amphotericin B (AmB) is known to form two types of ionic channels across sterol-containing liposomes, depending on its concentration and time after mixing (Cohen, 1992). In the present study, it is shown that AmB only kills unicellular Leishmania promastigotes (LPs) when aqueous pores permeable to small cations and anions are formed. Changes of membrane potential across ergosterol-containing liposomes and LPs were followed by fluorescence changes of 3,3′ dipropylthiadicarbocyanine (DiSC₃(5)). In KCl-loaded liposomes suspended in an iso-osmotic sucrose solution, low AmB concentrations (≤0.1 μm) induced a polarization potential, indicating K⁺ leakage, but no movement of cations and anions was allowed until AmB concentrations greater than 0.1 μm were added. In agreement with these data, it was found that AmB altered the negative membrane potential held across LPs in a manner consistent with the differential cation/anion selectivity exhibited by the channels formed in liposomes. Thus, LPs suspended in an iso-osmotic sucrose solution did not exhibit any AmB-induced membrane depolarization effect brought about by efflux of anions until 0.1 μm or higher AmB concentrations were added. By contrast, LPs suspended in an iso-osmotic NaCl solution and exposed to 0.05 μm AmB exhibited a nearly total collapse of the negative membrane potential, indicating Na⁺ entry into the cells. The concentration dependence of the AmB-induced permeability to different salts was also measured across vesicles derived from the plasma membrane of leishmanias (LMVs), by using a rapid mixing technique. At concentrations above 0.1 μm, AmB induced the formation of aqueous pores across LMVs with a positive cooperativity, yielding Hill coefficients between 2 to 3. Measured anion selectivity across such aqueous pores followed the sequence: SCN > NO3 > Cl > I > Br > acetate (SO²⁻ ₄ being impermeable). Cell killing by AmB was followed by fluorescence changes of the DNA-binding compound ethidium bromide (EB). At low concentrations (≤0.1 μm), AmB was found to be nonlethal against LPs but, above this concentration, leishmanias were rapidly killed. The rate and extent of such an effect were found to be dependent on the type of cation and anion present in the external aqueous solution. For both NH⁺ ₄ and Na⁺ salts, the measured rank order of AmB cell killing followed the same sequence that was determined for AmB-induced salt permeation across LMVs. Further, replacement of either extracellular Na⁺ by choline or Cl⁻ by SO²⁻ ₄, or its partial substitution by sucrose, in iso-osmotic conditions, led to a complete inhibition of the killing effect exerted by otherwise lethal AmB concentrations. Finally, it was shown that tetraethylammonium (TEA⁺), an organic cation that is known to block AmB-induced salt permeation across LMVs was able to retard the time lag observed for EB incorporation across LPs, indicating that this parameter can be taken to represent the time taken for salt accumulation inside the parasites. The present results thus indicate clearly that low AmB concentrations (≤0.1 μm) were able to form across LPs, cation channels that collapsed the parasite membrane potential but are not lytic. At high concentrations (<≥0.1 μm), a salt influx via the aqueous pores formed by the antibiotic was followed by osmotic changes leading to cell lysis. This last stage is supported by electron microscopy observations of the changes of parasite morphology immediately upon addition of AmB, which indicated that the typical elongated promastigote cell forms became rounded and the flagella swells and round up. The present work is the first demonstration of the in vitro sensitivity of Leishmania promastigotes to osmotic lysis by AmB. Received: 25 September 1995/Revised: 11 March 1996