Single Na' channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers formed from neutral phospholipids and were observed in the presence of batrachotoxin . The batrachotoxinmodified channel activates in the voltage range -120 to -80 mV and remains open almost all the time at voltages positive to -60 mV . Low levels of tetrodotoxin (TTX) induce slow fluctuations .of channel current, which represent the binding and dissociation of single TTX molecules to single channels . The rates of association and dissociation of TTX are both voltage dependent, and the association rate is competitively inhibited by Na'. This inhibition is observed only when Na' is increased on the TTX binding side of the channel . The results suggest that the TTX receptor site is located at the channel's outer mouth, and that the Na' competition site is not located deeply within the channel's conduction pathway.
Forskolin is commonly used to stimulate adenylate cyclase in the study of modulation of ion channels and other proteins by adenosine 3',5'-monophosphate (cAMP)-dependent second messenger systems. In addition to its action on adenylate cyclase, forskolin directly alters the gating of a single class of voltage-dependent potassium channels from a clonal pheochromocytoma (PC12) cell line. This alteration occurred in isolated cell-free patches independent of soluble cytoplasmic enzymes. The effect of forskolin was distinct from those of other agents that raise intracellular cAMP levels. The 1,9-dideoxy derivative of forskolin, which is unable to activate the cyclase, was also effective in altering the potassium channel activity. This direct action of forskolin can lead to misinterpretation of results in experiments in which forskolin is assumed to selectively activate adenylate cyclase.
Voltage-sensitive Na' channels from rat skeletal muscle plasma membrane vesicles were inserted into planar lipid bilayers in the presence of either of the alkaloid toxins veratridine (VT) or batrachotoxin (BTX) . Both of these toxins are known to cause persistent activation of Na' channels. With BTX as the channel activator,, single channels remain open nearly all the time. Channels activated with VT open and close on a time scale of 1-10 s . Increasing the VT concentration enhances the probability of channel opening, primarily by increasing the rate constant of opening. The kinetics and voltage dependence of channel block by 21-sulfo-I I-a-hydroxysaxitoxin are identical for VT and BTX, as is the ionic selectivity sequence determined by bi-ionic reversal potential (Na' -Li' > K+ > Rb+ > Cs'') . However, there are striking quantitative differences in open channel conduction for channels in the presence of the two activators . Under symmetrical solution conditions, the single channel conductance for Na' is about twice as high with BTX as with VT. Furthermore, the symmetrical solution single channel conductances show a different selectivity for BTX (Na' > Li' > K+ ) than for VT (Na' > K + > Li+) . Open channel current-voltage curves in symmetrical Na' and Li' are roughly linear, while those in symmetrical K+ are inwardly rectifying . Na' currents are blocked asymmetrically by K' with both BTX and VT, but the voltage dependence of K+ block is stronger with BTX than with VT . The results show that the alkaloid neurotoxins not only alter the gating process of the Na' channel, but also affect the structure of the open channel . We further conclude that the rate-determining step for conduction by Na' does not occur at the channel's "selectivity filter," where poorly permeating ions like K+ are excluded.
Video microscopy and whole-cell patch-clamp recording were used to monitor changes in relative cell volume (V/Vo), chloride conductance (gCl), and membrane capacitance (Cm) during osmotically induced swelling in Jurkat T lymphocytes. Cellular swelling was initiated with hyperosmotic pipette solutions. Simultaneous evaluation of V/Vo and gCl revealed a 59-s delay between the inception of swelling and the activation of outwardly rectifying, ATP-dependent Cl- channels. Following the delay, increases in V/Vo and gCl progressed in parallel. In contrast, Cm, a measure of cell surface area, fell gradually at a rate of approximately 150 fF/min after whole-cell access was achieved. The decline in Cm lasted 200 s and was followed by a rapid rise (approximately 750 fF/min). The rise in Cm coincided with a variable increase in "leak" current, gCl increased at a slower rate and reached lower peak values in experiments performed without ATP; ATP had no effect on the biphasic Cm time course. The temporal separation of conductance and capacitance during swelling suggests that gCl and Cm vary independently, supporting the hypothesis that a large portion, if not all, of the whole-cell Cl- conductance activated during swelling is provided by volume-sensitive Cl- channels preexisting in the plasma membrane.
Growth factors and hormones induce differentiation of clonal pheochromocytoma (PC12) cells, which are derived from rat adrenal medulla chromaffin cells. On application of nerve growth factor (NGF), PC12 cells extend neurites and express properties characteristic of autonomic ganglion cells. In contrast, incubation of PC12 cells with a corticosteroid, dexamethasone (DEX), does not induce neurite formation but causes an increase in tyrosine hydroxylase activity, suggesting that the cells become chromaffin cell-like. The ability of NGF and DEX to regulate ionic currents has been less well studied. Therefore, we examined how long-term NGF and DEX treatments affected voltage-dependent Na, Ca, and K currents in PC12 cells. Voltage-dependent Na currents were observed only in a small fraction of the PC12 cells in the absence of NGF or DEX. Virtually all NGF-treated cells expressed Na currents within 7 d. DEX increased the number of cells expressing voltage-dependent Na current slowly over 3 weeks, but, unlike NGF, DEX did not change Na current density. Both NGF and DEX also affected the expression of voltage-dependent Ca currents. Most of the untreated cells had only sustained, high-threshold voltage-dependent Ca currents. Chronic application of NGF or DEX increased the fractions of the cells that showed transient, low-threshold T-type Ca currents in addition to the high-threshold currents. The T-type Ca current density, however, increased significantly only in NGF-treated cells. Neither DEX nor NGF affected the voltage-dependent K currents. These results suggest that the expression of voltage-dependent Na and Ca currents are differentially regulated by NGF and DEX. The distinction between treated and untreated cells decreased after 3 weeks in culture as older untreated cells showed increases in the fraction of cells expressing both Na and low-threshold Ca currents. A PC12 subline selected for adherence to uncoated plastic also showed increased fraction of cells expressing these currents, suggesting that interactions with substrate may also influence ionic current expression.
The successful establishment of a postcrisis SV-40 T antigen transformed epithelial cell line, 1HAEo-, which retains tight junctions and vectorial ion transport, is described. Immunocytochemical analysis of 1HAEo- cells shows a defined pattern of cytokeratin staining and a characteristic pericellular localization of the adhesion molecule cellCAM 120/80, indicating the presence of junctional complexes. The presence of both tight junctions and desmosomes has been confirmed by electron microscopy. Cell monolayers have good transepithelial resistance measured in Ussing chambers. Cells increase chloride ion transport in response to addition of agents that raise either intracellular cAMP or calcium, measured either by 36Cl- efflux or whole-cell patch clamp. An increase in short-circuit current, in response to these agents, can be measured in Ussing chambers. The presence of a depolarization-induced outwardly rectifying 45 pS chloride channel has been demonstrated in single cell detached membrane patches. In addition, the cells have been found to express mucin mRNA. These cells therefore demonstrate that it is possible to select transformed cell clones with particular morphologic characteristics, i.e. the presence of tight junctions and cell polarity, which also retain useful epithelial cell-specific functions, including vectorial ion transport. They also provide a major resource for the study of the structure and function of human epithelia.
We have examined whether F-actin integrity is involved in activation of a volume-regulated Cl- current (VRChlC) in B-lymphocytes. VRChlC activation was initiated in response to establishing a whole cell recording in the presence of a hyposmotic gradient. Parallel confocal microscopy experiments using Rhodamine-Phalloidin (R-P) as a specific marker of F-actin showed that the submembrane actin ring is reversibly disrupted in response to an hyposmotic gradient. Disruptions of cortical F-actin integrity by 50 microM cytochalasin B (CB) does not trigger activation of VRChlC under isosmotic conditions or potentiate the rate of activation when the osmolarity of the extracellular solution was decreased by 75%. However, incubation with CB increased the rate of VRChlC activation in response to a 90% hyposmotic gradient. Phalloidin, a stabilizer of F-actin, decreases the rate of VRChlC activation in response to a 90% gradient, but has no effect in response to a 75% gradient. These observations suggest that disassembly of cortical F-actin is not critical for VRChlC activation in B-lymphocytes. The integrity of cortical F-actin, however, can exert a modulatory effect on the rate of VRChlC activation in the presence of a hyposmotic gradient.
Na' channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin . Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation . Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge . The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.