SUMMARY1. The actions of the n-alcohols from pentanol to dodecanol on nicotinic acetylcholine receptor (nAChR) channels were investigated by recording single AChactivated channel activity from inside-out membrane patches isolated from cultured rat myotubes. Alcohols were applied to the cytoplasmic side of the membrane; aqueous concentrations ranged from 11 7 mM-pentanol to 0-02 mm-dodecanol.2. The intermediate-chain alcohols (pentanol to octanol) caused channel currents to fluctuate between the fully open and closed state level so that openings occurred in bursts interrupted by brief gaps. Closed time distributions were fitted well with two exponential components, the fast component representing the closures within a burst. The number of gaps within a burst was dependent on alcohol concentration whereas gap duration was independent of concentration but increased with increasing chain length of the alcohol up to octanol.3. Nonanol and decanol reduced the mean duration of bursts of openings but did not cause an increase in the number of short closed intervals within a burst. Beyond decanol there was a decline in the ability of the n-alcohols to affect channel function. A saturated solution of undecanol (0-07 mM) reduced the mean open time by 33+17 %, whereas a saturated solution of dodecanol had no significant effect. Over the range of concentrations tested this describes the effects of all the n-alcohols (C5 to C12) on channel gating reasonably well.6. Blocking rate constants (k+B) for pentanol through to nonanol were calculated to be between 2 8 and 5-7 x 106 M-1 s-1. These values are based on the assumption that the concentration of the alcohols at their site(s) of action was equal to the aqueous concentration applied to the membrane.

Abstract.A method for clamping cytosolic free Ca 2+ ([Ca2+]~) in cultures of rat sympathetic neurons at or below resting levels for several days was devised to determine whether Ca 2+ signals are required for neurite outgrowth from neurons that depend on Nerve Growth Factor (NGF) for their growth and survival. To control [Ca2+]i, normal Ca 2+ influx was eliminated by titration of extracellular Ca 2+ with EGTA and reinstated through voltage-sensitive Ca 2+ channels. The rate of neurite outgrowth and the number of neurites thus became dependent on the extent of depolarization by KC1, and withdrawal of KC1 caused an immediate cessation of growth. Neurite outgrowth was completely blocked by the L type Ca 2+ channel antagonists nifedipine, nitrendipine, D600, or diltiazem at sub-or micromolar concentrations. Measurement of [Ca2+]i in cell bodies using the fluorescent Ca 2+ indicator fura-2 established that optimal growth, similar to that seen in normal medium, was obtained when [Ca2+]i was clamped at resting levels. These levels of [Ca~+]i were set by serum, which elevated [Ca2+]i by ~30 nM, whereas the addition of NGF had no effect on [Ca2+]~. The reduction of [Ca2+]o prevented neurite fasciculation but this had no effect on the rate of neurite elongation or on the number of extending neurites. These results show that neurite outgrowth from NGF-dependent neurons occurs over long periods in the complete absence of Ca 2+ signals, suggesting that Ca 2+ signals are not necessary for operating the basic machinery of neurite outgrowth.

The effects of the homologous series of carboxylic esters, methyl propionate to methyl decanoate, on the steady-state inactivation of the sodium current in squid axons have been studied. The esters moved the relationship between the inactivation parameter, h infinity, and the membrane potential in the hyperpolarizing direction, thus reducing the number of sodium channels available at the resting potential. The concentration dependence of the shift at the mid-point of the curve of h infinity against potential has been measured for all esters except decanoate, which was almost inactive. Two aspects of these concentration dependences suggest that molecular volume is an important determinant of the effectiveness of each ester. Firstly, there is a sharp decline in activity above methyl hexanoate. This cut-off in activity resembles that for hydrocarbons where it has been suggested [e.g., Haydon, D.A., Urban, B.W. 1983, J. Physiol. (London) 341:411-427] to a result from a decrease in uptake with increasing molecular volume. (Further data for the hydrocarbons n-butane to n-heptane are reported here.) Secondly, the smallest compounds, methyl propionate and methyl butyrate, are less effective than would be predicted if equal membrane concentrations of each ester produced the same shift. The aqueous concentration dependences for these esters indicate that below methyl hexanoate, as the series is descended, progressively higher membrane concentrations are required to produce a given shift. This would be expected if the volume of ester in the membrane, rather than the number of molecules, is important.(ABSTRACT TRUNCATED AT 250 WORDS)

We have examined how NGF-dependent rat sympathetic neurons maintain Ca2+ homeostasis when challenged with high K+ or 8-(4-chlorophenylthio)cyclic AMP (CPTcAMP), two survival factors. In the presence of NGF, high K+ (55 mM) caused a stable, 65% reduction in the density of cell soma voltage-sensitive Ca2+ channels within 2 days. Although resting [Ca2+]i was elevated by 1.6-fold, this was 50% less than the rise in [Ca2+]i measured before down-regulation occurred, suggesting that down-regulation may help prevent the toxic effects of persistently elevated [Ca2+]i. Inhibition of protein synthesis by cycloheximide blocked recovery from down-regulation. Moreover, treatment with cycloheximide or actinomycin-D caused a 2-fold rise in the peak Ca2+ current, suggesting that voltage-sensitive Ca2+ channel activity may be tonically attenuated during normal growth. In the absence of NGF, neurons survived for several days in high K+ medium with no significant rise in resting [Ca2+]i, although neurites did not grow. Neither Ca2+ channel density nor resting [Ca2+]i were altered in neurons surviving with CPTcAMP. Moreover, CPTcAMP lowered the dependence on extracellular Ca2+. However, the dihydropyridine antagonist nitrendipine blocked both high K(+)- and CPTcAMP-dependent survival although it had no effect in the presence of NGF. Thus, in the absence of NGF, sympathetic neurons do not require elevation of [Ca2+]i above resting levels to survive with either high K+ or CPTcAMP, but dihydropyridine-sensitive Ca2+ channel activity may be essential for their survival promoting actions.