Inward rectifier K channels in renal epithelioid cells (MDCK) activated by serotonin

Summary The present study has been performed to test for the effect of intracellular calcium and of serotonin on the channel activity in patches from subconfluent MDCK-cells. In inside-out patches, inwardly rectifying potassium-selective channels are observed with open probabilities of 0.01±0.01, 0.24±0.03 and 0.39±0.07, at 100 nmol/liter, 1 mol/liter or 10 mol/liter calcium activity, respectively. The single-channel slope conductance is 34±2 pS, if the potential difference across the patch (V ) is zero, and approaches 59±1 pS, ifV is –50 mV, cell negative. In the cell-attached mode, little channel activity is observed prior to application of serotonin (open probability=0.03±0.03). If 1 mol/liter serotonin is added to the bath perfusate, the open probability increases rapidly to a peak value of 0.34±0.04 within 8 sec. In continued presence of the hormone, the open probability declines to approach 0.06±0.02 within 30 sec. At zero potential difference between pipette and reference in the bath (i.e., the potential difference across the patch is equal to the potential difference across the cell membrane), the single-channel conductance is 59±4 pS. In conclusion, inwardly rectifying potassium channels have been identified in the cell membrane of subconfluent MDCK-cells, which are activated to a similar extent by increase of intracellular calcium activity to 1 mol/liter and by extracellular application of 1 mol/liter serotonin.     

Amiloride-blockable sodium currents in isolated taste receptor cells

Summary Isolated taste receptor cells from the frog tongue were investigated under whole-cell patch-clamp conditions. With the cytosolic potential head at –80 mV, more than 50% of the cells had a stationary inward Na current of 10 to 700 pA in Ringer's solution. This current was in some cells partially, in others completely, blockable by low concentrations of amiloride. With 110mm Na in the external and 10mm Na in the internal solution, the inhibition constant of amiloride was (at –80 mV) near 0.3 m. In some cells the amiloride-sensitive conductance was Na specific; in others it passed both Na and K. The Na/K selectivity (estimated from reversal potentials) varied between 1 and 100. The blockability bysmall concentrations of amiloride resembled that of channels found in some Na-absorbing epithelia, but the channels of taste cells showed a surprisingly large range of ionic specificities. Receptor cells, whichin situ express these channels in their apical membrane, may be competent to detect the taste quality salty. The same cells also express TTX-blockable voltage-gated Na channels.     

Current-voltage relationships for the plasma membrane and its principal electrogenic pump inNeurospora crassa: I. Steady-state conditions

Summary The nonlinear membrane current-voltage relationship (I–V curve) for intact hyphae ofNeurospora crassa has been determined by means of a 3-electrode voltage-clamp technique, plus quasi-linear cable theory. Under normal conditions of growth and respiration, the membraneI–V curve is best described as a parabolic segement convex in the direction of depolarizing current. At the average resting potential of –174 mV, the membrane conductance is 190 mhos/cm²; conductance increases to 240 mhos/cm² at –300 mV, and decreases to 130 mhos/cm² at 0 mV. Irreversible membrane breakdown occurs at potentials beyond this range.Inhibition of the primary electrogenic pump inNeurospora by ATP withdrawal (with 1mm KCN) depolarizes the membrane to the range of –40 to –70 mV and reduces the slope of theI–V curve by a fixed scaling factor of approximately 0.8. For wild-typeNeurospora, compared under control conditions and during steady-state inhibition by cyanide, theI–V difference curve — presumed to define the current-voltage curve for the electrogenic pump — is a saturation function with maximal current of 20 A/cm², a half-saturation potential near –300 mV, and a projected reversal potential of ca. –400 mV. This value is close to the maximal free energy available to the pump from ATP hydrolysis, so that pump stoichiometry must be close to 1 H⁺ extruded:1 ATP split.The time-courses of change in membrane potential and resistance with cyanide are compatible with the steady-stateI–V curves, under the assumption that cyanide has no major effects other than ATP withdrawal. Other inhibitors, uncouplers, and lowered temperature all have more complicated effects.The detailed temporal analysis of voltage-clamp data showed three time-constants in the clamping currents: one of 10 msec, for charging the membrane capacitance (0.9 F/cm²) a second of 50–75 msec; and a third of 20–30 sec, perhaps representing changes of intracellular composition.     

Intracellular gradients of electrical potential in the epithelial cells of theNecturus gallbladder

Summary When single-barrelled electrodes (5–60 M) were advanced under manual control from the mucosal side of the epithelium the mucosal membrane was on average indented by about 40 m before the microelectrode penetrated the cell. Since this dimpling was comparable with the total depth of the cell, which recovered its original shape within 0.5 sec, the steady intracellular potential was recorded only about 14 m from the basal (serosal) membrane. Fast recording of the associated change in potential revealed an abrupt drop to –26 mV at a mean rate of 84 V/sec, followed by a further slow drop to a steady value of about –50 mV at a mean rate of 0.28 V/sec. The initial level of –26 mV may be regarded as the potential difference across the mucosal membrane. This conclusion was confirmed by mounting the microelectrode on a piezoelectric probe, which delivered 3 m jabs in less than 0.5 msec. With this device in operation to prevent dimpling, the mean potential difference across the mucosal membrane was recorded as –29 mV. In all cases the potential across the basal membrane was recorded as –52 mV. Manual advance of the microelectrode tip within the cytoplasm yielded an intracellular potential gradient of 0.6 mV/m. The same potential profile and membrane potentials were demonstrated on penetrating the epithelium from the serosal side, and measurements with multibarrelled electrodes whose tips were staggered in depth gave roughly the same internal potential gradient. The resistivity of the cytoplasm was determined by a triple-barrelled microelectrode, and varied from 10 times that ofNecturus saline at the mucosal end of the cell to 4 times in the middle and 6 times at the serosal end.     

Fluctuation analysis of short-circuit current in a warm-blooded sodium-retaining epithelium: Site current,density, and interaction with triamterene

Summary Density and conductance of the Na-site in hen coprodeum were studied by employing fluctuation analysis of shortcircuit current at sodium concentrations from 26 to 130mm. Fluctuations of current in the frequency range 2–800 Hz were induced by triamterene, a reversible blocker of conducting epithelial Na-sites. At 130mm Na the site density was 5.8±1.0 m^–2 and the site conductance was 4 pS. This conductance is equal to that of the frog skin (W. Van Driessche and B. Lindemann, 1979,Nature (London) 282:519–520). Extrapolation of site density to zero sodium renders a total of 38±28 sites m^–2, which is compared with other estimates for the coprodeum. The site-triamterene association and dissociation constants were 9.5±0.4 rad sec^–1 m ^–1 and 255±20 rad sec^–1 and they were independent of external sodium concentration. An analysis of the affinity constant for triamterene based on the DC-short-circuit current was found to be unrelated to the external sodium concentration and identical to that obtained from fluctuation analysis indicating a noncompetitive interaction between sodium and triamterene. Due to the oxygen demand of the epithelium we have developed an experimental method using short data processing times. A new analytical approach using integration of the power density spectrum proved necessary because of low signal-to-noise ratios.     

Protein interactions with lipid bilayers: The channels of kidney plasma membrane proteolipids

Summary Proteolipids extracted from bovine kidney plasma membrane induce irreversible changes in the electrical properties of lipid bilayers formed from diphytanoyl phosphatidylcholine. The interaction with the proteolipid produces channels which are cation selective. At low protein concentrations (i.e., <0.6 g/ml), the single-channel conductance is approximately 10 pS in 100mm KCl and 3 pS in 100mm NaCl. In the presence of protein concentrations above 1 g/ml, another population of channels appears. These channels have a conductance of about 100 pS in 100mm KCl and 30 pS in 100mm NaCl. Further, these channels are voltage dependent in KCl, closing when the voltage is clamped at values 30 mV. The steady-state membrane conductance, measured at low voltages, was found to increase proportional to a high power (2–3) of the proteolipid concentration present in one of the aqueous phases. In 100mm NaCl, the conductance increases at protein concentrations above 5 g/ml, whereas in 100mm KCl in increases at protein concentrations above 0.6 g/ml. These measurements indicate that the higher steady-state conductance observed in KCl at a given proteolipid concentration in a multi-channel membrane presumably results because more channels incorporate in the presence of KCl than in the presence of NaCl.The two major fractions which comprise the proteolipid complex were also tested on bilayers. It was found that both fractions are required to produce the effects described.     

The cloned cardiac Na channel α-subunit expressed in Xenopus oocytes show gating and blocking properties of native channels

Summary The neonatal rat cardiac Na channel -subunit directed currents in oocytes show characteristic cardiac relative resistance to tetrodotoxin (TTX) block. TTX-sensitive currents obtained by expression in Xenopus oocytes of the -subunits of the rat brain (BrnIIa) and adult skeletal muscle (I) Na channels show abnormally slow decay kinetics. In order to determine if currents directed by the cardiac -subunit (RHI) exhibit kinetics in oocytes like native currents, we compared RHI-directed currents in oocytes to Na currents in freshly isolated neonatal rat myocytes. The decay rate of RHI currents approached that of neonatal myocytes and was faster than BrnIIa and I currents in oocytes. The voltage dependence of availability and activation was the same as that in the rat myocytes except for a 12–19 mV shift in the depolarizing direction. The RHI Na currents were sensitive to Cd²⁺ block, and they showed use dependence of TTX and lidocaine block similar to native currents. The current expressed in oocytes following injection of the cRNA encoding for the -subunit of the cardiac Na channel possesses most of the characteristic kinetic and pharmacological properties of the native cardiac Na current.We are grateful to Dr. Juliet Morgan for providing us with neonatal ventricle cell cultures. We thank Dr. Gail Mandel for providing the pl plasmid and Dr. A. Goldin for rat brain 2a. Aaron Fox kindly provided us with Axobasic 1.0 software and support. We also thank Turi Larsen for oocyte preparation, technical assistance, injections and maintaining the Xenopus colony. Supported by NIH HL 37217, HL 20592, NS 23360-02 and HL 07381, a grant from the International Life Sciences Institute and a grant from the Upjohn Company.     

Cell K activity in frog skin in the presence and absence of cell current

Summary Cell K activity,a _k, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30–60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO₃ Ringer on the apical surface.Under control conditionsa _k ^r was nearly constant among skins (mean±SD=92±8mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 A/cm²). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase ina _k ^r after inhibition of cell current was observed, on the average the change was not significant (98±11mM after amiloride, 101±12mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from –42 to –77 mV, hyperpolarized after inhibition of cell current, initially to –109±5mV, then depolarizing to a stable value (–88±5mV) after 15–25 min. At this time K was above equilibrium (E _k=98±2mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport.The measurement ofa _k ^r permitted the calculation of the passive K current and pump current under control conditions. assuming a constant current source with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.     

The influence of zinc on the modulatory effect of sphingosylphosphorylcholine on Kv1.3 channels in human T lymphocytes

In the present study, the whole-cell patch-clamp technique was applied to investigate the influence of co-application of zinc ions and sphingosylphosphorylcholine (SPC) on the SPC-induced shift of the activation midpoint and slowing of activation kinetics of Kv1.3 channels in human T lymphocytes. The results obtained provided evidence that the effects exerted by SPC and Zn were not additive. The shift was significantly diminished in a concentration-dependent manner upon co-application of 10 M SPC and Zn in the concentration range 10–300 M. However, the shift was not abolished in the presence of 100 and 300 M of Zn co-applied with SPC. It was shown that the extent of the shift upon SPC and Zn co-application was similar to the shift observed for Zn applied without SPC. The slowing of the activation kinetics was also diminished upon SPC and Zn co-application; however, no clear dependence on concentration was observed. Moreover, the slowing was not abolished in the presence of 100 and 300 M of Zn. It was shown that the slowing of the activation kinetics upon Zn and SPC co-application was primarily due to the effect exerted by SPC. The steepness of the voltage dependence of steady-state activation of the channels was not changed upon SPC and Zn co-application. Possible mechanisms underlying the observed phenomena and their possible physiological significance are discussed.Abbreviations 4-AP 4-aminopyridine - SPC sphingosylphosphorylcholine - TL human T lymphocyte     

Single cardiac outwardly rectifying K+ channels modulated by protein kinase A and a G-protein

Elementary K⁺ currents were recorded at 19 °C in cell-attached and in inside-out patches excised from neonatal rat heart myocytes. An outwardly rectifying K⁺ channel which prevented Na⁺ ions from permeating could be detected in about 10% of the patches attaining (at 5 mmol/l external K⁺ and between – 20 mV and + 20 mV) a unitary conductance of 66 +- 3.9 pS. K _{(outw.-rect.)} ⁺ channels have one open and at least two closed states. Open probability and _open rose steeply on shifting the membrane potential in the positive direction, thereby tending to saturate. Open probability (at –7 mV) was as low as 3 ± 1% but increased several-fold on exposing the cytoplasmic surface to Mg-ATP (100 mol/l) without a concomitant change of _open. No channel activation occurred in response to ATP in the absence of cytoplasmic Mg^–+. The cytoplasmic administration of the catalytic subunit of protein kinase A (120–150 /ml) or GTP--S (100 mol/l) caused a similar channel activation. GDP--S (100 mol/l) was also tested and found to be ineffective in this respect. This suggests that cardiac K _{(outw.-rect.)} ⁺ channels are metabolically modulated by both cAMP-dependent phosphorylation and a G-protein. Offprint requests to: M. Kohlhardt     

Voltage dependence of current through the Na,K-exchange pump ofRana oocytes

Summary We have studied current (I _Str) through the Na, K pump in amphibian oocytes under conditions designed to minimize parallel undesired currents. Specifically,I _Str was measured as the strophanthidin-sensitive current in the presence of Ba^2–, Cd²⁺ and gluconate (in place of external Cl^–). In addition,I _Str was studied only after the difference currents from successive applications and washouts of strophanthidin (Str) were reproducible. The dose-response relationship to Str in four oocytes displayed a meanK _0.5 of 0.4 m, with 2–5 m producing 84–93% pump' block. From baseline data with 12 Na⁺-preloaded oocytes, voltage clamped in the range [–170, +50 mV] with and without 2–5 m Str, the averageI _Str depended directly onV _m up to a plateau at 0 mV with interpolated zero current at –165 mV. In three oocytes, lowering the external [Na⁺] markedly decreased the voltage sensitivity ofI _p , while producing only a small change in the maximal outwardI _Str. In contrast, decreasing the external [K⁺] from 25 to 2.5mm reducedI _Str at 0 mV without substantially affecting its voltage dependence. At K⁺ concentrations of 1mm, both the absolute value ofI _Str at 0 mV and the slope conductance were reduced. In eight oocytes, the activation of the averagedI _Str by [K⁺] _o over the voltage interval [–30, +30 mV] was well fit by the Hill equation, with K=1.7±0.4mm andnH (the minimum number of K⁺ binding sites) =1.7±0.4. The results unequivocally establish that the cardiotonic-sensitive current ofRana oocytes displays only a positive slope conductance for [K⁺] _o >1mm. There is therefore no need to postulate more than one voltage-sensitive step in the cycling of the Na, K pump under physiologic conditions. The effects of varying external Na⁺ and K⁺ are consistent with results obtained in other tissues and may reflect an ion-well effect.     

Arsenic concentrations in water and fish from Chautauqua Lake,New York

Synopsis Arsenic persists in Chautauqua Lake, New York waters 13 years after cessation of herbicide (sodium arsenite) application and continues to cycle within the lake. Arsenic concentrations in lake water ranged from 22.4–114.81 g l^–1, = 49.0 ag l^–1. Well water samples generally contained less than 10 g l^–1 arsenic. Arsenic concentrations in lake water exceeded U.S. Public Health Service recommended maximum concentrations (10 g l^–1) and many samples exceeded the maximum permissible limit (50 g l^–1). Fish accumulated arsenic from water but did not magnify it. Fish to water arsenic ratios ranged from 0.4–41.6. Black crappie (Pomoxis nigromaculatus) contained the highest arsenic concentrations (0.14–2.04 g g^–1 ), X = 0.7 g g^–1) while perch (Perca flavescens), muskellunge (Esox masquinongy) and largemouth bass (Micropterus salmoides) contained the lowest concentrations (0.02–0.13 g g^–1). Arsenic concentrations in fish do not appear to pose a health hazard for human consumers.     

Procedures for the axenic isolation of conchocelis and monospores from the red seaweed Porphyra yezoensis

In order to maintain axenic seedstock cultures axenically of thecommercially important red seaweed, Porphyra yezoensis, aprocedure was developed for axenic isolation and culture of conchocelis andmonospores. For axenic isolation of the conchocelis, contaminated microalgaewere most effectively removed by filtering contaminated samples through a100-m mesh after sonication. Removal of bacteria and otheralgaewas accomplished using a mixture of 5 agents (0.02% chitosan, 100 gml^–1 GeO₂, 10 gml^–1 ampicillin, 40 gml^–1 kanamycin and 200 gml^–1 streptomycin). Axenic single colonies wereisolatedfrom a semi-solid medium prepared from 1% transfer gel. After collectingmonospores from the 40–50% density layer on a percoll-gradient, removalofbacteria and fungi from the monospores was accomplished using a mixture of 5antibiotics (3.5 g ml^–1 nystatin, 2 mgml^–1 ampicillin, 400 gml^–1 kanamycin, 50 gml^–1 neomycin and 800 gml^–1 streptomycin). Axenic single juvenile blades wereisolated from a semi-solid medium prepared from 0.5% transfer gel.     

KATP channels of mouse skeletal muscle: mechanism of channel blockage by AMP-PNP

Single ATP-sensitive potassium channels (K _ATP channels) were studied in inside-out membrane patches excised from mouse skeletal muscle. Channel blockage by the non-hydrolysable ATP analogue AMP-PNP was investigated in the absence or presence of 1 mM MgCl₂ with K⁺-rich solutions bathing the internal membrane surface. Currents through single. K _ATP channels were recorded at –40 and +40 mV AMP-PNP (5 to 500 M; Li salt) reduced the open-probability p_o of K _ATP channels and decreased the single-channel currents at high nucleotide concentrations by approximately 10%. Half maximal reduction of p_o at –40 mV was observed at nucleotide concentrations of 29 M in the absence and of 39 M in the presence of Mg²⁺. The steepness of the AMP-PNP concentration-response curves was strongly affected by Mg²⁺, the Hill coefficients of the curves were 0.6 in the absence and 1.6 in the presence of 1 mM MgCl₂. The efficacies of channel blockage by AMP-PNP at –40 and ⁺40 mV were not significantly different. The results indicate that a K _ATP channel can bind more divalent Mg²⁺-complexes of AMP-PNP than trivalent protonated forms of the nucleotide and that channel blockage is hardly affected by the membrane electric field. To estimate the contribution of lithium ions to the observed results, we studied the effects of LiCl (0.8 to 10 mM) in the Mg²⁺-free solution on the single channel current i. At a Li⁺ concentration of 10 mM, i was hardly affected at –40 mV but reduced by a factor of 0.75 at +40 mV. The results are interpreted by a fast, voltage-dependent blockage of K _ATP channels by internal Li⁺ ions. Correspondence to: B. Neumcke     

The contribution of size-fractionated plankton to biomass and primary production of phytoplankton in saline–alkaline ponds

Primary productivity, biomass and chlorophyll-a of size fractionated phytoplankton (<0.22 m, <3 m, <8 m, <10 m, <40 m, <64 m, <112 m and <200 m) were estimated in 6 ponds and 5 experimental enclosures. The results showed that the planktonic algae less than 10 m are important in the biomass and production of phytoplankton in saline–alkaline ponds. The production of size fractionated phytoplankton corresponding to <112 m, <10 m and <3 m in saline–alkaline ponds were 10.5 ± 6.6 , 8.6 ± 5.4 and 0.33 ± 0.1 mgC l^–1 d^–1, respectively. Mean community respiration rate was 1.80 ± 0.73, 1.69 ± 0.90 and 1.38 ± 1.12 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) were 1.61, 8.30 and 0.33 mgC l^–1 d^–1, respectively. The ratio of those to the total phytoplankton production was 15%, 79% and 3%, respectively. The mean respiration rate of the different size groups was 0.11, 0.31 and 1.38 mgC l^–1 d^–1; the ratio of those to total respiration of phytoplankton was 6%, 17% and 77%, respectively. The production of size-fractionated phytoplankton corresponding to <200 m, <10 m and <3 m in enclosures was 2.19 ± 1.63, 2.08 ± 1.75 and 0.22 ± 0.08 mgC l^–1 d^-1, respectively. Mean community respiration rates were 1.25 ± 1.55, 1.17 ± 1.42 and 0.47 ± 0.32 mgC l^–1 d^–1, respectively. The average production of phytoplankton corresponding to micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton was 0.11, 1.86 and 0.22 mgC l^–1 d^–1, respectively. The ratio of those to the total production of phytoplankton was 5%, 85% and 10%, respectively. The mean respiration rate of different size groups were 0.08, 0.72 and 0.46 mgC l^–1 d^–1, the ratio of those to total respiration of phytoplankton was 6%, 57% and 37%, respectively. The concentrations of chlorophyll-a of the phytoplankton in the corresponding size of micro- (10–112 m), nano- (3–10 m) and pico- (<3 m) plankton in the experimental ponds were 19.3, 98.2 and 11. 9 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 15%, 76% and 9%, respectively. The concentrations of chlorophyll-a of phytoplankton micro- (10–200 m), nano- (3–10 m) and pico- (<3 m) plankton in enclosures were 1.7, 34.3 and 3.0 g l^–1, respectively. The ratio of those to the total chlorophyll-a was 4%, 88% and 8%, respectively.     

The kinetics of the degradation of chloroform and benzene in anaerobic sediment from the River Rhine

In anaerobic methanogenic sediment microcosms¹⁴C labelled chloroform was degraded mainly to carbon dioxide. At a concentration of 4 g.l^–1 the mineralization followed first order kinetics with a half life of 12 days at 10°C and 2.6 days at 20°C. At a concentration of 400 g.l^–1 the mineralization rate increased with time and followed logarithmic kinetics with a _max of 0.02.d^–1 at 10°C. The logarithmic kinetics can be explained by growth of the bacteria on the higher concentration of chloroform with a generation time of 35 days. Shaking and oxygenation did not inhibit the mineralization of chloroform, probably because of bacterial consumption of the dissolved oxygen. ¹⁴C labelled benzene was mineralized only for a small percentage to¹⁴C labelled carbon dioxide while other, not acid extractable, degradation products were formed. Under anaerobic conditions after one day when 5% of the benzene was degraded to carbon dioxide, the mineralization ceased, while the disappearance of benzene proceeded. With air in the headspace of the incubation bottles 25% of the benzene was mineralized to carbon dioxide. The anaerobic degradation of benzene at a concentration of 100 .l^–1 showed similar kinetics as the degradation at 1 g.l^–1. Hence no adaptation of the microflora in the sediment occurred during the 63 days of the experiment at 100 g.l^–1.     

Asymmetric diffusion into the postsynaptic neuron: An extremely efficient mechanism for removing excess GABA from synaptic clefts on the Deiters' neurone plasma membrane

Microdissected Deiters' neuron plasma membranes have been used for studying the passage of GABA through the membrane both in the inward and outward direction. Working with 0.2 mM GABA in the compartment simulating the outside of the neurone and with 2.0 mM GABA in the one simulating the inside we found a net transport of GABA towards the inside. This mechanism does not require a Na⁺ ion gradient across the membrane. The nature of the transport process involved was studied by determining the rate of [³H]-GABA inward passage as a function of GABA concentration (1 nM–800 M) on the outward side of the membrane. The results have shown that until 50 M a diffusion process (v=D₁×C, where D₁=3.1×10^–11 1/m²×sec) is the sole mechanism involved. Above 50 M a second diffusion process is activated v=D₂×(C–50×10^–6), where D₂=2.8×10^–11 1/m²×sec. Taking in account both inward and outward directed diffusion, one can calculate 16 M as the equilibrium concentration of GABA on the outward side of the membrane. From a kinetic point of view, these diffusion processes are able to reduce GABA concentration in a synaptic cleft from 3 mM to 20 M within 3 sec. These diffusion systems are discussed as extremely efficient in removing the excess of released GABA in the synaptic cleft.     

Voltage dependent sodium currents in cultured rat cerebellar granule cells

Sodium currents were studied in granule cells dissociated from rat cerebellum. Macroscopic currents were recorded using the patch-clamp technique. Sudium currents, which are TTX sensitive, reached a maximum peak value of 0.42±0.08 pA/m² at 18.4±2.2 mV (n=6). Activation and inactivation kinetics and steady-state properties were described in terms of Hodgkin and Huxley, parameters. The properties of sodium channels in cultured rat cerebellar granule cells are very similar to those reported for various neural preparations.     

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. (Apicomplexa: Eimeriidae) from the black-capped bulbul,Pycnonotus xanthopygos

Oocysts of Isospora ernsti n. sp. and Isospora blagburni n. sp. are described from the black-capped bulbul Pycnonotus xanthopygos from Lincoln Park Zoo, Chicago, Illinois. The bird came from southwestern Africa seven years earlier. I. ernsti oocysts are ellipsoidal to bluntly ovoid, 28–38 × 23–31m (mean 34 × 28 m) and have a single-layered oocyst wall. Micropyle, oocyst residuum and polar granules are absent. Sporocysts are elongate ovoid, 24–30 × 11–16 m (mean 27×13 m). Stieda and substiedal bodies and sporocyst residuum are present. I. blagburni oocysts are spherical to subspherical. 21–28 × 19–26 m (mean 25 × 23 m) and have a single oocyst wall. Sporocysts are ovoid and 17–23 × 10–13 m (mean 20 × 12 m). Stieda and substiedal bodies and sporocyst residuum are present.     

Diversity of amyloid beta protein fragment [1-40]-formed channels

1. The lipid bilayer technique was used to characterize the biophysical and pharmacological properties of several ion channels formed by incorporating amyloid beta protein fragment (AP) 1–40 into lipid membranes. Based on the conductance, kinetics, selectivity, and pharmacological properties, the following AP[1–40]-formed ion channels have been identified: (i) The AP[1–40]-formed bursting fast cation channel was characterized by (a) a single channel conductance of 63 pS (250/50 mM KCl cis/trans) at +140 mV, 17 pS (250/50 mM KCl cis/trans) at –160 mV, and the nonlinear current–voltage relationship drawn to a third-order polynomial, (b) selectivity sequence P _K > P _Na > P _Li = 1.0:0.60:0.47, (c) P_o of 0.22 at 0 mV and 0.55 at +120 mV, and (d) Zn²⁺-induced reduction in current amplitude, a typical property of a slow block mechanism. (ii) The AP[1–40]-formed spiky fast cation channel was characterized by (a) a similar kinetics to the bursting fast channel with exception for the absence of the long intraburst closures, (b) single channel conductance of 63 pS (250/50 KCl) at +140 mV 17 pS (250/50 KCl) at –160 mV, the current–voltage relationship nonlinear drawn to a third-order polynomial fit, and (c) selectivity sequence P _Rb > P _K > P _Cs > P _Na > P _Li = 1.3:1.0:0.46:0.40:0.27. (iii) The AP[1–40]-formed medium conductance channel was charcterized by (a) 275 pS (250/50 mM KCl cis/trans) at +140 mV and 19 pS (250/50 mM KCl cis/trans) at –160 mV and (b) inactivation at V_ms more negative than –120 and more positive than +120 mV. (iv) The AP[1–40]-formed inactivating large conductance channel was characterized by (a) fast and slow modes of opening to seven multilevel conductances ranging between 0–589 pS (in 250/50 mM KCl) at +140 mV and 0–704 pS (in 250/50 mM KCl) at –160 mV, (b) The fast mode which had a conductance of <250 pS was voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.2 s at +36 mV. The slow mode which had a conductance of >250 pS was also voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.0 s at –76 mV, (c) the value of P _K/P _choline for the fast mode was 3.9 and selectivity sequence P _K > P _Cs > P _Na > P _Li = 1.0:0.94:0.87:0.59. The value of P _K/P _choline for the slow mode was 2.7 and selectivity sequence P _K > P _Na > P _Li > P _Cs = 1.0:0.59:0.49:0.21, and (d) asymmetric blockade with 10 mM Zn²⁺-induced reduction in the large conductance state of the slow mode mediated via slow block mechanism. The fast mode of the large conductance channel was not affected by 10 mM Zn²⁺.2. It has been suggested that, although the bursting fast channel, the spiky fast channel and the inactivating medium conductance channel are distinct, it is possible that they are intermediate configurations of yet another configuration underlying the inactivating large conductance channel. It is proposed that this heterogeneity is one of the most common features of these positively-charged cytotoxic amyloid-formed channels reflecting these channels ability to modify multiple cellular functions.3. Furthermore, the formation of -sheet based oligomers could be an important common step in the formation of cytotoxic amyloid channels.