Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel

Summary Eleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (M _r 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion of the channel by the exosialidase, neuraminidase, or partial polysialic acid removal bythe endosialidase, endo-N-acetylneuraminidase, destroy the 3G4 epitope, 3G4 is, therefore, a highly selective probe for the post-translationally attached polysialic acids. Except for this monoclonal antibody, the epitopes recognized by the remaining antibodies were highly resistant to extensive N-linked deglycosylation. Thus, the monoclonal antibodies may be directed against unique post-translationally produced domains of the electroplax sodium channel, presumably sugar groups that are abundant on this protein (Miller, J.A., Agnew, W.S., Levinson, S.R. 1983.Biochemistry 22:462–470). These monoclonal antibodies should prove useful as tools to study discrete post-translational processing events in sodium channel biosynthesis.     

Inner mitochondrial membrane anion channel is present in brown adipocytes but is not identical with the uncoupling protein

Summary Vesicles of inner mitochondrial membrane, mitoplasts, from rat brown adipose tissue were prepared by osmotic swelling and studied using the patch-clamp technique. Current events of a 107.8±8.7 pS (n=16, 21°C) channel were recorded in the mitoplast-attached mode. This channel was selective for anions and its kinetics resembled those of channels previously found in liver and heart mitochondria of mouse and ox. In whole-mitoplast mode each of five purine nucleotides (20 m) blocked the channel. This is the first demonstration of pharmacological blockade of this type of channel. Although a similar anion channel in mouse and ox mitochondria was suggested to be the uncoupling protein (UCP) associated with nonshivering thermogenesis, we present several arguments against this possibility. Thus we describe a high-conductance, purine-nucleotide-binding, anion selective mitochondrial channel, that is not the UCP.     

Prostaglandin E2 Activates Ca2+ Channels in Bovine Adrenal Chromaffin Cells

We have demonstrated that prostaglandin E2 (PGE2) treatment of bovine adrenal chromaffin cells results in a sustained elevation of intracellular Ca2+ concentration ([Ca2+]i) in these cells. Because the continued elevation of [Ca2+]i was dependent on extracellular Ca2+ concentration, it can be assumed that the PGE2-induced [Ca2+]i increase is due, at least in part, to an opening of membrane Ca2+ channels. In this study, we used electrophysiological methods to examine the mechanism of the PGE2-induced [Ca2+]i increase directly. Puff application of PGE2 to the external medium resulted in a prolonged depolarization in about half of the chromaffin cells examined. In whole-cell voltage-clamp recordings, an increase in inward current was observed over a 6-7 min period following bath application of PGE2 (greater than or equal to 10 microM), even in the absence of external Na+. This inward current was abolished when the recordings were made with the cells in a Ca2(+)-free medium, but it was not inhibited by Mn2+, a blocker of voltage-dependent Ca2+ channels. In cell-attached patch-clamp configuration, PGE2 produced an increase in the opening frequency of inward currents. The reversal potential of the PGE2-induced currents was about +40 mV, which is close to the reversal potential of the Ca2+ channel. The opening frequency was not affected by membrane potential changes. In inside-out patch-clamp configuration, inositol 1,4,5-trisphosphate (2 microM) added to the cytoplasmic side activated the Ca2(+)-channel currents, but PGE2 was ineffective when applied to the cytoplasmic side. These results suggest that PGE2 activates voltage-independent Ca2+ channels in chromaffin cells through a diffusible second messenger, possibly inositol 1,4,5-trisphosphate.     

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

A novel potassium-selective channel which is active at membrane potentials between -100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mM-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mM-K and 17 pS with 2.5 mM-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (> 0.2, but often > 0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (tau o = 0.09 msec), closed times with the sum of three exponentials (tau c = 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mM). External Cs ions reduced the apparent unitary conductance for inward currents at Em = -90 mV (IC50 = 1.1 mM). Ba and, more potently, Zn ions lowered not only the apparent single-channel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nM). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.     

Inwardly rectifying potassium current in rabbit osteoclasts: A whole-cell and single-channel study

Summary Ionic conductances of rabbit osteoclasts were investigated using both whole-cell and cell-attached configurations of the patch-clamp recording technique. The predominant conductance found in these cells was an inwardly rectifying K⁺ conductance. Whole-cell currents showed an N-shaped current-voltage (I–13;V) relation with inward current activated at potentials negative to E_K. When external K⁺ was varied, I-V curves shifted 53 mV/10-fold change in [K⁺]_out, as predicted for a K⁺-selective channel. Inward current was blocked by Ba²⁺ and showed a time-dependent decline at negative potentials, which was reduced in Na⁺-free external solution. Inward single-channel currents were recorded in the cell-attached configuration. Single-channel currents were identified as inward-rectifier K⁺ channels based on the following observations: (i) Unitary I-V relations rectified, with only inward current resolved. (ii) Unitary conductance () was 31 pS when recorded in the cell-attached configuration with 140 mm K⁺ in the pipette and was found to be dependent on [K⁺]. (iii) Addition of Ba²⁺ to the pipette solution abolished single-channel events. We conclude that rabbit osteoclasts possess inwardly rectifying K⁺ channels which give rise to the inward current recorded at negative potentials in the whole-cell configuration. This inwardly rectifying K⁺ current may be responsible for setting the resting membrane potential and for dissipating electrical potential differences which arise from electrogenic transport of protons across the osteoclast ruffled border.This work was supported by The Arthritis Society and the Medical Research Council of Canada. M.E.M.K. was supported by a fellowship, S.J.D. a development Grant and S.M.S. a scholarship from the Medical Research Council. We thank Dr. Zu Gang Zheng for help with scanning microscopy.     

Slow voltage inactivation of Ca2+ currents and bursting mechanisms for the mouse pancreatic beta-cell

Summary Recent whole-cell electrophysiological data concerning the properties of the Ca²⁺ currents in mouse -cells are fitted by a two-current model of Ca²⁺ channel kinetics. When the -cell K⁺ currents are added to this model, only large modifications of the measured Ca²⁺ currents will reproduce the bursting pattern normally observed in mouse islets. However, when the measured Ca²⁺ currents are modified only slightly and used in conjunction with a K⁺ conductance that can be modulated dynamically by ATP concentration, reasonable bursting is obtained. Under these conditions it is the K-ATP conductance, rather than the slow voltage inactivation of the Ca²⁺ current, that determines the interburst interval. We find that this latter model can be reconciled with experiments that limit the possible periodic variation of the K-ATP conductance and with recent observations of intracellular Ca²⁺ bursting in isletsThis work was supported in part by NSF grant DIR-90-06104 and the Agricultural Experiment Station of the University of California. P.S. gratefully acknowledges financial support from an NRC Fellowship. We have benefited from numerous conversations with Drs. John Rinzel, Arthur Sherman, Daniel Cook, and Leslie Satin     

Reconstitution of sodium channels in large liposomes formed by the addition of acidic phospholipids and freeze-thaw sonication

Phosphatidylcholine (PC) alone or with phosphatidylethanolamine (PE) are sufficient for the reconstitution of Na+ channels in planar lipid bilayers. However, when Na+ channels were first reconstituted into liposomes using the freeze-thaw-sonication method, addition of acidic phospholipids, such as phosphatidylserine (PS), to the neutral phospholipids was necessary to obtain a significant toxin-modulated 22Na uptake. To further investigate the acidic phospholipid effect on reconstitution into liposomes, Na+ channels purified from Electrophorus electricus electrocytes were reconstituted into liposomes of different composition by freeze-thaw sonication and the effect of batrachotoxin and tetrodotoxin on the 22Na flux was measured. The results revealed that, under our experimental conditions, the presence of an acidic phospholipid was also necessary to obtain a significant neurotoxin-modulated 22Na influx. Though neurotoxin-modulated 22Na fluxes have been reported in proteoliposomes made with purified Na+ channels and PC alone, the 22Na fluxes were smaller than those found using lipid mixtures containing acidic phospholipids. Electron microscopy of negatively stained proteoliposomes prepared with PC, PC/PS (1:1 molar ratio), and PS revealed that the acidic phospholipid increases the size of the reconstituted proteoliposomes. The increment in size caused by the acidic phospholipid, due to the associated increase in internal volume for 22Na uptake and in area for Na+ channel incorporation, appears to be responsible for the large neurotoxin-modulated 22Na fluxes observed.     

Factors affecting chloride conductance in apical membrane vesicles from human placenta

Summary Apical membrane vesicles from human term placenta were isolated using a magnesium precipitation technique, and the purity of the vesicles was assessed morphologically using scanning and transmission electron microscopy, and biochemically, using marker enzymes. The vesicles were found to be morphologically intact and significantly enriched in enzymes associated with apical membranes. ³⁶Cl^– uptake into these vesicles was studied in the presence of an outwardly directed Cl^– gradient. This uptake was found to be time dependent, with an initial rapid uptake tending to peak between 10 and 20 min and thereafter decline. Uptake was found to be voltage dependent since 5 m valinomycin caused a decrease in uptake. The effects of N-phenylanthranilic acid (NPA) and 4,4-diisothiocyanostilbene-2,2-disulphonic acid (DIDS) and bumetanide on the initial rate of Cl^– were examined in the presence and absence of 5 m valinomycin. NPA and DIDS inhibited isotope uptake strongly with IC₅₀ values of 0.83±0.35 m and 3.43±0.37 m, respectively, in the absence of valinomycin. Although valinomycin reduced ³⁶Cl^– uptake by about 80% when added before the isotope, DIDS reduced the uptake which remained in a concentration-dependent fashion with an IC₅₀ of 5.6±2.1 m. Under these conditions, NPA was without effect at concentrations below 100 m. Bumetanide was without effect at the concentrations used in the absence of valinomycin. However, following valinomycin pretreatment, bumetanide reduced ³⁶Cl^– uptake significantly at 100 m concentration. Vesicle diameter, as assessed by flow cytometry, did not change under the conditions employed.The effects of some fatty acids were also investigated. Arachidonic acid and linoleic acid inhibited Cl^– uptake with IC₅₀ values of 37.6±14.9 m and 4.59±0.51 m, respectively. Arachidonyl alcohol and elaidic acid were found to be without effect. These studies show that human placental brush border membrane vesicles possess a chloride conductance channel, the activity of which can be measured in the presence of an outwardly directed Cl^– gradient and this channel is sensitive to Cl^– channel inhibitors, especially N-phenylanthranilic acid, and can be inhibited by unsaturated fatty acids such as arachidonic acid and linoleic acid.This work was supported in part by the Cystic Fibrosis Association of Ireland and Eolas, The Irish Science and Technology Agency. The technical assistance of Mr. Cormac O' Connell in the preparation of the electron micrographs and of Mr. Roddy Monks in the flow cytometric analysis is gratefully acknowledged.