Calcium-sensitivity of the plasmalemmal delayed rectifier potassium current suggests that calcium influx in pulvinar protoplasts from Mimosa pudica L. can be revealed by hyperpolarization

Isolated protoplasts from pulvinar motor cells of Mimosa pudica were studied using conventional whole-cell patch clamp techniques. With internal solutions weakly buffered for Ca²⁺ (0.2 mm EGTA), a run-down of the outward delayed rectifier K⁺ current was induced by hyperpolarizing the holding potential, and this effect was strongly promoted by high external Ca²⁺ concentrations. This rundown could be reversed by coming back to less hyperpolarized holding potentials or by lowering the external [Ca²⁺]. Such rundown was absent when pipette internal solutions strongly buffered (10 mm EGTA) for Ca²⁺ were used. Ionomycin induced run-down of the K⁺ current with internal solutions containing 0.2 mm but not 10 mm EGTA. The hyperpolarization-associated rundown was reversibly blocked by Gd³⁺ and La³⁺.We thank Christophe Untereiner and Denis Wagner for expert technical assistance in facilitating the experiments and data acquisition and analysis.     

The Ca2+-induced leak current in Xenopus oocytes is indeed mediated through a Cl− channel

Defolliculated oocytes of Xenopus laevis responded to removal of external divalent cations with large depolarizations and, when voltage clamped, with huge currents. Single channel analysis revealed a Cl^– channel with a slope conductance of about 90 pS at positive membrane potentials with at least four substates. Single channel amplitudes and mean channel currents had a reversal potential of approximately –15 mV as predicted by the Nernst equation for a channel perfectly selective for Cl^–. Readdition of Ca²⁺ immediately inactivated the channel and restored the former membrane potential or clamp current. The inward currents were mediated by a Ca²⁺ inactivated Cl^– channel (CaIC). The inhibitory potency of Ca²⁺ was a function of the external Ca²⁺ concentration with a half maximal blocker concentration of about 20 m.These channels were inhibited by the Cl^– channel blockers flufenamic acid, niflumic acid and diphenylamine-2-carboxylate (DPC). In contrast, 4,4-acetamido-4-isothiocyanatostilbene-2,2-disulfonicacid (SITS), another Cl^– channel blocker, led to activation of this Cl^– channel. Like other Cl^– channels, the CaIC was activated by cytosolic cAMP. Extracellular ATP inhibited the channel while ADP was without any effect. Injection of phorbol 12-myristate 13-acetate (PMA), a protein kinase C activating phorbol ester, stimulated the Cl^– current. Cytochalasin D, an actin filament disrupting compound, reversibly decreased the clamp current demonstrating an influence of the cytoskeleton.The results indicate that removal of divalent cations activates Cl^– channels in Xenopus oocytes which share several features with Cl^– channels of the CLC family. The former so-called leak current of oocytes under divalent cation-free conditions is nothing else than an activation of Cl^– channels.The microelectrode measurements are part of the PhD thesis of K. Liebold; the patch clamp contributions are part of the PhD thesis of F.W. Reifarth. This study was supported by the Deutsche Forschungsgemeinschaft (We1858/2-l) and by Sonderforschungsbereich 249.     

Do neurons have a voltage or a current threshold for action potential initiation?

The majority of neural network models consider the output of single neurons to be a continuous, positive, and saturating firing ratef(t), while a minority treat neuronal output as a series of delta pulses (t — t _i ). We here argue that the issue of the proper output representation relates to the biophysics of the cells in question and, in particular, to whether initiation of somatic action potentials occurs when a certain thresholdvoltage or a thresholdcurrent is exceeded. We approach this issue using numerical simulations of the electrical behavior of a layer 5 pyramidal cell from cat visual cortex. The dendritic tree is passive while the cell body includes eight voltage- and calcium-dependent membrane conductances.We compute both the steady-state (I ^static (V _m )) and the instantaneous (I _o (V_m)) I–V relationships and argue that the amplitude of the local maximum inI ^static (V _m ) corresponds to the current thresholdI _th for sustained inputs, while the location of the middle zero-crossing ofI _o corresponds to a fixed voltage thresholdV _th for rapid inputs. We confirm this using numerical simulations: for rapid synaptic inputs, spikes are initiated if the somatic potential exceedsV _th, while for slowly varying inputI _th must be exceeded. Due to the presence of the large dendritic tree, no charge thresholdQ _th exists for physiological input.Introducing the temporal average of the somatic membrane potential (V _m) while the cell is spiking repetitively, allows us to define a dynamic I-V relationship ^dynamic ((V _m)). We find an exponential relationship between (V _m) and the net current sunk by the somatic membrane during spiking (diode-like behavior). The slope ofI/dynamic((V _m)) allows us to define a dynamic input conductance and a time constant that characterizes how rapidly the cell changes its output firing frequency in response to a change in its input.     

Tay-Sachs disease with atypical chronic course and limited brain storage: Alpha-locus hexosaminidase genetic compound

A 19-year-old Irish-Jewish male had a slow neurologic regression starting at age 4 1/2 years with stuttering. The chronic course resembled that of Spielmeyer-Vogt (juvenile ceroid-lipofuscinosis) disease. The brain was atrophic with neuronal losses and huge compound inclusions in the remaining neurons. Lipid NANA was within normal limits in gray and white matter and G_M2 gangliosides were moderately elevated at 11.5% lipid NANA. Beta-hexosaminidase A activity was lipid composition showed nonspecific abnormalities. Exhaustive tissue extraction ruled out the possibility of tightly bound gangliosides to account for the relatively low G_M2 ganglioside concentration. The extract contained unidentified chromogenic substances interfering with the resorcinol reaction. The similarly affected patient's sister lived to age 26 years and her brain was even more atrophic. No biochemical abnormality to account for progressive neuronal losses and relative lack of G_M2 ganglioside storage was found.Deceased.Special issue dedicated to Dr. Leon S. Wolfe.     

Use of DNA fingerprinting for human population genetic studies

DNA fingerprinting techniques have been used in population genetic studies on many different kinds of organisms. Here, we present new applications for multilocus DNA fingerprint probes in population studies and demonstrate the applicability of DNA fingerprinting to human population genetics, using M13 phage DNA as a probe. The new approach, which is based on a factor method of numerical coding of non-quantitative data (factor correspondence analysis-FCA), shows good agreement between population position, as indicated by the three principal factors, and ethnogenetic proximity.     

Angiotensin and bradykinin metabolism by peptidases identified in cultured human skeletal muscle myocytes and fibroblasts

Angiotensin (ANG) and kinin metabolizing enzymes, angiotensin-converting enzyme (ACE; EC 3.4.15.1), neutral endopeptidase-24.11 (NEP-24.11; EC 3.4.24.11), and aminopeptidase M (AmM; EC 3.4.11.2), have recently been identified in a purified skeletal muscle glycoprotein fraction. We have analyzed the cellular localization of these enzymes. In cultured human skeletal muscle adult myoblasts, myotubes, and fibroblasts, kinins and angiotensins were metabolized by NEP-24.11 and AmM but not by ACE. NEP-24.11 degraded ANG II, ANG III, and bradykinin (BK) and converted ANG I to the active metabolite ANG(1–7). ANG III was converted to the novel ANG IV metabolite [des-Arg¹]ANG III by AmM. These data suggest that, due to their abundance in the body, skeletal muscle myocytes and fibroblasts may play a major role in modulation of the systemic and local effects of angiotensins and kinins. This role could be particularly important in individuals receiving treatment with ACE inhibitors.     

Modelling endplate currents: dependence on quantum secretion probability and decay of miniature current

Quantification of the time course and amplitude of endplate currents (EPC) was made with respect to dispersion of quanta secretion and to changes in the exponential decay of miniature endplate currents (_mepc). The relationship between RPC amplitude and _mepc follows a double-exponential curve with ₁= 0.3 ms and ₂ = 6 ms. If the amplitude of fully synchronised EPC is taken as 100%, then the loss of EPC amplitude is already 42% with physiological parameters of dispersion (the half-rise and decay constant of distribution of secretion probability = 0.5 ms, _mepc =1 ms). This loss is even more substantial if secretion is more dispersed or miniature endplate currents decay faster. Correspondence to: F. Vyskocil