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K+ and Cl- channels are involved in regulating the proliferation of a number of cell types. Two main hypotheses have been proposed to explain the mechanism by which these channels influence cell proliferation: regulation of membrane potential and regulation of cell volume. In order to test these hypotheses, we measured, under different experimental conditions, the volume, membrane potential and rate of proliferation of C6 glioma cells. Cells cultured in control medium for 1-4 days were compared with cells cultured for the same period of time in the presence of broad spectrum channel blockers: tetraethylammonium, 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and Cs+, in hypertonic media (29% increased osmolarity with NaCl, KCl or sucrose), in hypotonic medium (23% decreased osmolarity with H2O) or in the presence of the specific channel blockers, i.e. mast cell degranulating peptide, charybdotoxin or chlorotoxin. In all of these conditions, we observed a close correspondence between the rate of proliferation and the mean cell volume. The proliferation decreased when volume increased. Moreover, whereas control cells were flattened, spindle-shaped, bipolar or multipolar, cells cultured in media supplemented with NPPB, KCl or CsCl were round with few processes. Of the agents tested, only KCl and Cs+ depolarized the cells. These results show that alterations of the rate of proliferation by K+ and Cl- channel blockers or anisotonia are closely related with changes in cell volume or form but are not correlated with changes in membrane potential.

The contribution of an electrogenic proton pump to the membrane potential of neuroblastoma x glioma hybrid NG 108-15 cells was determined with whole-cell voltage and current recordings and cell volume measurements with the preparation bathed in symmetrical 140 mM KCl solutions. The effects of the K+ channel blockers tetraethylammonium and 4-aminopyridine and of the H+-ATPase inhibitor bafilomycin A1 on the membrane potential and input resistance revealed that the membrane potential is generated by an outward H+ pump current of 5-15 pA in equilibrium with an inward passive current. This conclusion is supported by both current- and voltage-clamp results obtained when the preparation was bathed in a Na+-containing external solution after K+ channel blockade with Cs+ in the pipette. Additional support was obtained by measurement of the volume of cells incubated in solutions containing 140 mM KCl. Tetraethylammonium induced a bafilomycin-sensitive increase in inward K+ current and an increase in cell volume of 76% which we believe to be a consequence of the K+ influx. Finally, comparison of membrane potentials obtained in experiments using Na+-containing external, and K+-containing pipette solutions and after K+ channel blockade with Cs+ in the pipette also showed that, under normal physiological conditions, the resting membrane potential is essentially determined by an electrogenic H+ pump.

The effects of the flavonoid quercetin on cell proliferation and voltage-dependent K+ current were studied on mouse neuroblastoma x glioma hybrid cells. In the presence of 1% fetal calf serum, quercetin inhibited both cell proliferation and K+ current with effective doses inducing half-maximum effects of 10 microM and 70 microM respectively. Valinomycin (1 nM) antagonized 80% of the growth-inhibitory effects of 10 microM quercetin. The results suggest that at least a part of the anti-proliferative effect of quercetin is mediated by a K+ channel blockade. They further confirm a role for K+ channels in mitogenesis and cell proliferation.

The effects of fatty acids on voltage-dependent potassium (K+) channels in neuroblastoma cells were studied using the whole-cell current recording technique. At a concentration of 5 microM, unsaturated and medium chain length (C10-C14) saturated fatty acids accelerated the apparent inactivation of the K+ current. This effect was reversed by albumin. In the absence of exogenous fatty acids, albumin slowed the inactivation of the K+ current. The acceleration of the K+ current inactivation induced by unsaturated fatty acids was associated with an increase in the sensitivity of K+ channels to 4-amino-pyridine. It is concluded that kinetic and pharmacological properties of K+ channels are, in part, controlled by membrane fatty acids which, in this way, should contribute to an apparent diversity of K+ channels and the modulation of cell excitability.

The steady-state effects and rate of action of 4-aminopyridine (4-AP) on normal and chloramine-T (CL-T)-modified voltage-dependent potassium (K) currents were studied in neuroblastoma cells with the whole-cell voltage-clamp current recording technique. 4-AP apparently slows both the activation and inactivation of the normal current but does not modify the time course of the CL-T-modified current. These differential effects of 4-AP are interpreted as resulting from the existence of two types of K channels with different 4-AP sensitivities under normal conditions and similar 4-AP sensitivities after CL-T, which furthermore slows their inactivation [8, 9]. While the onset of 4-AP action on the normal current is delayed and can be described by the difference of two exponentials, the onset of 4-AP action on CL-T-modified current starts immediately after the external application of the drug and can be described by the sum of two exponentials. The 4-AP-induced block of the normal current exhibits use-dependent features and is relieved by long conditioning depolarizations. In contrast, the block of the CL-T-modified current is not use-dependent. At high 4-AP concentrations (1-10 mM), the steady-state block of the normal current reaches a saturating value of 95%, while the steady-state block of the CL-T-modified current and the "unblocked" normal current only reaches a saturating value of 35%. The results suggest that CL-T inhibits a channel or membrane constituent which contributes to the inactivation of channels and increases their apparent affinity for 4-AP when they are in closed or open states.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of chloramine-T (CL-T) on voltage-dependent potassium channels in neuroblastoma cells were analysed using the whole-cell current recording technique. CL-T irreversibly decreased the peak whole-cell K current, considerably slowed its inactivation and shifted its activation-voltage curve towards positive voltages by 6 mV. Under control conditions, the inactivation of the whole-cell K current could be described by the sum of two exponentials, F and S, whose time constants at +50 mV were tau F = 1.00 +/- 0.15 S and tau S = 5.72 +/- 0.47 S respectively. After CL-T, it could be described by the sum of two (S1 and S2) or three (F, S1 and S2) exponentials whose time constants at +50 mV were: tau F = 0.81 +/- 0.22 S, tau S1 = 6.46 +/- 0.60 S and tau S2 = 48.56 +/- 3.64 S. Under control conditions, F and S inactivating components of the whole-cell K current were blocked by 4-aminopyridine, with a Hill coefficient of 1 and apparent dissociation constants of 0.04 and 0.7 mM respectively. After CL-T, both S1 and S2 components were equally blocked by 4-aminopyridine with a Hill coefficient of 0.25, being reduced to 64% of their control values by 10 mM. CL-T is known to slow the inactivation of sodium channels and to oxidize sulphydryl amino acids and unsaturated lipids. It is concluded that the inactivation gates of voltage-dependent sodium and potassium channels are either constituted of the same amino acid residues or are controlled by unsaturated lipid surrounding or bound to the channel proteins.

1. The blocking action of oenanthotoxin (OETX) and butanol on Na+ channels was studied in voltage clamp experiments on single myelinated nerve fibres treated by batrachotoxin (BTX). 2. OETX (40 microM) blocked Na+ currents through normal channels but did not affect significantly the BTX modified Na+ current. 3. BTX removed the depolarization-induced charge immobilization and slowed down significantly the OFF charge movement. However, the maximum charge displaced, as well as the kinetics of the ON charge movement during a strong membrane depolarization, remained unchanged. 4. OETX blocked the charge movement in normal Na+ channels but did not affect noticeably the charge movement modified by BTX. 5. BTX did not modify the K+ currents and did not protect them from the blocking action of OETX. 6. Butanol (0.01-0.1 M) decreased almost identically and reversibly both normal and BTX-modified Na+ currents. 7. It is concluded that binding of BTX to its receptor protects the Na+ channel from interaction with OETX but left it accessible to butanol.

The steady state effects and rates of action of tetrodotoxin (TTX) on sodium current were studied in the voltage clamped frog node of Ranvier. Inactivation of the sodium current was separated into fast and slow phases. Both phases were assumed to correspond to two different currents (fast and slow) flowing through fast and slow channels (Benoit et al. 1985). The dose-response curve of the steady state effect of tetrodotoxin on the fast current was sigmoid. An analysis of this effect in double logarithmic coordinates gave a Hill coefficient of 1.74. The rates of tetrodotoxin action on the fast current were determined by the reversible reduction of the peak current recorded at a potential (+20 mV) at which the slow current was relatively small. After an initial delay, the onset of TTX effect followed an exponential function of time whose constant decreased with increasing tetrodotoxin concentrations. Expressed as the time corresponding to a reduction of 2% of the current, the delay (delta t2%) increased from about 100 ms with 300 nM-TTX to about 30 s with 1 nM-TTX. When tetrodotoxin was removed, the offset developed quasi-instantaneously and followed an exponential function of time whose constant was independent of the toxin concentration. Both steady state and rates of tetrodotoxin effects could be fitted well if one assumed that the block of one fast channel occurred after binding of two TTX molecules to two cooperative sites.

Voltage clamp experiments carried out on nodes of Ranvier of myelinated fibres of Rana esculenta showed that a small fractoon of sodium channels fail to inactivate. Thus during long lasting depolarizing pulses there is a small Na-current superimposed on the leakage and potassium currents. This late Na-current appears more marked in sensory fibres than in motor ones.

Voltage clamp experiments done on nodes of Ranvier show that external application of Cesium blocks the inward but not the outward potassium currents. Internal application of Cs ions reduces the outward K-current and the inward K-current is not affected. These results support the hypotheses that K ions cross th- K-channel after dehydration at superficial sites where competition may occur with Cs-ions.

1. Voltage clamp experiments were carried out on frog myelinated fibres to study the origin of the transient inward current occuring when the membrane is repolarized after long lasting depolarizing pulses (tail current denominated "Ip" by Frankenhaeuser). 2. The "tail" of inward current measured during repolarization after break of the depolarizing pulse is insensitive to external application of TTX, is abolished by external treatment with TEA or Cs and decreases when the outward K-current during the pulse is diminished. 3. The time course of the "tail" current is exponential. Its direction depends on the duration of the depolarizing pulse and on the membrane potential level at repolarization. 4. It is concluded that the tail of inward current during repolarization is carried by K-ions accumulated in the perinodal space during a depolarizing pulse. The data suggest that the tail reflects the time course of the restoration of the K-concentration to its initial level. The tail current itself contributes to this restoration depending on the Em value at repolarization. 5. It is shown that one of the two phenomenological models proposed by Frankenhaeuser and Hodgkin to account for the external potassium accumulation observed in the squid giant axon may be also applied to the Ranvier node. Assuming that the thickness of the space is 2900 A and that the K-permeability of the barrier is 0.019 cm/sec, it is possible to account for the observed changes in [K]0 during a long lasting depolarizing pulse. 6. The existence of such a barrier would introduce an electrical resistance in series with the nodal membrane of roughly 150 000 omega.