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.