Figure 1.
1-butanol but not the phospholipase D inhibitor VU0155056 inhibits catecholamine secretion and calcium entry.
A) Secretion was evoked by application of 30 mM KCl for five-minutes. Epinephrine secretion from cells treated with 1-butanol (0.4%; ∼44 mM) or tert-butanol (0.4%; ∼42 mM) was normalized to that from controls. Secretion was significantly inhibited by 1-butanol and to a lesser extent by tert-butanol (one-way ANOVA, F = 20.98, p = 0.0007: * p<0.05, *** p<0.001 for pairwise comparisons using Tukey's post-hoc test). B) KCl-evoked epinephrine secretion was not altered by the phospholipase D inhibitor VU0155056 (1 µM) (p = 0.49, paired t-test). C) A representative experiment showing the intracellular calcium transients evoked by 30 mM KCl (60 s applications) applied before, during, and after washout of 0.4% 1-butanol. D) Mean data from several experiments like that shown in panel C. The peak increase in intracellular [Ca2+] evoked by KCl in the presence (during) of 1-butanol (0.4%; ∼42 mM), tert-butanol (0.4%; ∼42 mM), or VU0155056 (1 µM) was normalized to the first control response in each cell. 1-butanol significantly inhibited Ca2+ entry compared to both tert-butanol and VU0155056 (one-way ANOVA, F = 54.8, p<0.0001: *** p<0.001 for pairwise comparisons using Tukey's post-hoc test). After washout of the drugs (washout) Ca2+ entry was not significantly different in any of the three treatment groups (one-way ANOVA, F = 1.03, p = 0.36).
Figure 2.
Concentration-dependent inhibition of ICa by 1-butanol.
A) Representative time-course showing reversible inhibition of peak ICa amplitude by 1-butanol (0.4%; ∼44 mM). Currents were evoked by 10 ms duration steps from −90 mV to +10 mV. B) Concentration-response curve showing the percent inhibition of ICa amplitude by 1-butanol. Points are mean ± s.e.m. of 4–17 cells (error bars are within the data points in some cases). The solid line shows the fit to a standard dose-response relationship which yielded an estimated EC50 of 52 mM and Hill slope of −1.73. C) Percent inhibition of peak ICa amplitude (elicited by a step from −90 mV to +10 mV) by 1-butanol (n = 17), 2-butanol (n = 5), or tert-butanol (n = 18) (all butanol isomers at 0.4%) (one-way ANOVA, F = 60.7, p<0.0001: *** p<0.001, ns = “not significant” for pairwise comparisons using Tukey's post-hoc test). D) Left panel: Representative experiment showing peak ICa amplitude plotted against time (elicited by a step from −70 mV to +10 mV). Nitrendipine (3 µM) was present throughout the experiment to block any L-type calcium channels. The bath was continually perfused with fresh solution (wash) and 1-butanol applied as indicated by the red bars. At the time indicated by the black bar, the solution flow was stopped and a bolus of ω-conotoxin GVIA was added to the bath (stop + CgTx) to irreversibly block N-type calcium channels. Right panel: The percent inhibition of pharmacologically isolated P/Q-type and N-type ICa by 1-butanol. “P/Q-type” ICa was defined as the CgTx resistant current (i.e. the current remaining after washout of CgTx). “N-type” ICa was defined as the CgTx sensitive component (i.e. the data after Cgtx subtracted from data before CgTx).
Figure 3.
Differential effects of butanol isomers on the current-voltage relationship of ICa.
Cells were stimulated by a series of steps, from a holding potential of −90 mV to test potentials ranging from −60 mV to +60 mV, first in control conditions and then in the presence of either 0.4% (∼44 mM) 1-butanol (panel A, n = 6), 2-butanol (panel B, n = 5), or tert-butanol (panel C, n = 5). The mean current amplitude elicited by each step is plotted. Tert-butanol elicited a clear hyperpolarizing shift in the I-V relationship.
Figure 4.
Butanol isomers differentially modulate the voltage-dependence of ICa activation.
A) The upper panel shows the voltage command protocol and the lower panel shows a family of representative ICa tail current recordings. Cells were stimulated with depolarizing steps from a holding potential of −90 mV to test potentials ranging from −20 to +50 mV and tail currents were recorded upon repolarization to −40 mV. B, C) In each cell, tail currents were recorded first under control conditions and then in the presence of either 1-butanol (panel B, n = 6) or tert-butanol (panel C, n = 6). Tail current amplitude was normalized to that evoked by the +40 mV step and plotted against the potential of the step depolarization (mean ± s.e.m.). The solid line shows the fit with a Boltzmann curve. The inset bar graphs show the mean slope and V50 (potential at half maximal activation) derived from the Boltzmann fits to individual cells. 1-butanol had no effect on V50 but the slope of the curve was significantly shallower. In contrast, tert-butanol had no effect on the slope, but produced a strong hyperpolarizing shift in the V50 (* p<0.05; ** P<0.01; ns = not significant; paired t-test).
Figure 5.
Differential effects of butanol isomers on the deactivation kinetics of ICa.
A) Representative tail currents activated by a step depolarization from −90 mV to +40 mV. In each cell, tail currents were recorded first in control conditions and then in the presence of either 1-butanol (left) or tert-butanol (right). B) The same traces as in panel A, but the time-base was expanded and the currents in the presence of butanol were scaled to the same amplitude as control to more clearly illustrate changes in the decay kinetics. C) The decay of the tail currents was fit with a single exponential function. Bar charts plot the mean time constant of the fit (τ) in control conditions and in the presence of 1-butanol (n = 6) or tert-butanol (n = 6). (* p<0.05; *** p<0.001 compared to matched controls; paired t-test).
Figure 6.
1-butanol and tert-butanol produce a similar hyperpolarization of closed-state inactivation.
A) Schematic depiction of the voltage-stimulus protocol. ICa were elicited by steps to +10 mV. The holding potential was increased in 10 mV increments from −90 mV to −20 mV and maintained at each potential for 30 s. In each cell this was done first in control conditions and then in the presence of either 1-butanol (panel B; n = 5) or tert-butanol (panel C; n = 6). B, C) ICa amplitude was normalized to that evoked from a holding potential of −90 mV and plotted against the holding potential. The graphs show the mean data fit using a Boltzmann function with three free parameters (upper plateau, slope, and V50) and the lower plateau was constrained to ≥0. The inset bar graphs show the hyperpolarizing shift in mean V50 (voltage at half maximal inactivation) derived from the Boltzmann fits to individual cells (** p<0.01; *** p<0.001 compared to matched controls; paired t-test).
Figure 7.
Butanol accelerates voltage-dependent inactivation and reduces total calcium entry during sustained depolarization.
A) Representative recordings of ICa elicited by a 500 ms step depolarization under control conditions (ctl) and in the presence of either 0.4% (∼44 mM) 1-butanol (1-but) or 0.4% (∼42 mM) tert-butanol (tert-but). The patch pipette solution contained 10 mM EGTA to block calcium-dependent inactivation. Thus, the decay in ICa is due to voltage-dependent inactivation. 1-butanol inhibited the peak amplitude and accelerated the the inactivation of ICa. Tert-butanol also accelerated inactivation, but had minimal effects of the peak amplitude of ICa. B) Pooled data (mean ± sem) from multiple experiments like that shown in panel A. To facilitate comparison of inactivation, the amplitude of ICa in the presence of butanol was normalized to control conditions. For clarity, the displayed traces begin at the peak amplitude and error bars are only shown for some data points. 1-butanol and tert-butanol both significantly increased the extent of inactivation. (*** p<0.001 for the extent of inactivation at 490 ms in the presence of butanol compared to control conditions; paired t-test). C) The charge (integral) of ICa is directly proportional to the amount of calcium entry during the depolarizing step. 1-butanol produced a significantly greater inhibition than tert-butanol (*** p<0.001; paired t-test).