Microtubule cytoskeleton involvement in muscarinic suppression of voltage-gated calcium channel current in guinea-pig ileal smooth muscle

Abstract

1. Effects of agents, which affect microtubule polymerization-depolymerization cycle, on Ba2+ current (IBa) flowing through voltage-gated Ca2+ channels and carbachol (CCh)-induced sustained suppression of IBa were examined in whole-cell voltage-clamped smooth muscle cells of guinea-pig ileum. 2. offchicine (100 microM) and vinblastine (100 microM), microtubule depolymerizers, increased the ampitude of IBa. Lumicolchicine (100 microM), an inactive analogue of colchicine, had no effect on IBa. 3. Taxol (1 - 100 microM), a microtubule polymerizer, decreased IBa in a concentration-dependent manner and accelerated the rate of inactivation of IBa. Baccatin III (100 microM), an inactive analogue of taxol, had no effect on IBa. 4. Colchicine (100 microM) and vinblastine (100 microM), but not lumicolchicine (100 microM), decreased or abolished the sustained component of CCh (10 microM)-induced IBa suppression. 5. Pretreatment with taxol (10 - 100 microM) resulted in a concentration-dependent decrease in IBa and the action of CCh on IBa. The inhibitory effects of taxol and CCh on IBa were not additive. 6. Colchicine (100 microM) or taxol (100 microM) had no effect on voltage-gated K+ channel current or CCh-induced non-selective cationic channel current. 7. These results suggest that polymerization of microtubules leads to suppression of Ca2+ channel activity, and that muscarinic sustained suppression of Ca2+ channel current is mediated by a signal transduction element which involves microtubule cytoskeleton.

Figure 1

Effects of colchicine and taxol on current-voltage relationships of Ba²⁺ current (I_Ba). I_Ba was elicited by depolarizing pulses (2 s duration) to various potentials ranging from −70 to 80 mV in 10 mV increments from the holding potential of −80 mV. (A, B and C) Superimposed current traces recorded from a drug-untreated cell (control cell), a colchicine (100 μM)-treated cell and a taxol (100 μM)-treated cell, respectively. The attached figures near current traces represent the potentials attained by the depolarizing pulses. Leak currents were subtracted and interrupted lines indicate zero I_Ba levels. (D) current-voltage relationships of I_Ba obtained from control cells (n=7), colchicine-treated cells (n=8) and taxol-treated cells (n=7). The peak currents normalized to cell capacitance were averaged, and the mean current densities were plotted against membrane potentials attained by depolarizing pulses.

Figure 2

Effects of colchicine and taxol on the inactivation of I_Ba. I_Ba was elicited by depolarizing pulses (2 s duration) to various potentials ranging from −20 to 20 mV from the holding potential of −80 mV. (A) Current traces of I_Ba elicited by depolarizing pulses to 0 mV in a drug-untreated cell (control cell), a colchicine (100 μM)-treated cell and a taxol (10 μM)-treated cell. The smooth curves in the inactivation phase of I_Ba are the fitted two-exponential function and the time constants for the fast component (τ fast) and the slow component (τ slow) are shown. Note different calibration for each trace. (B and C) Voltage-dependence of τ fast and τ slow, respectively. Mean time constants averaged from seven control cells, eight colchicine-treated cells and seven taxol-treated cells were plotted against voltages attained by depolarizing pulses. ^*Statistically different (P<0.05) from the corresponding mean value for control. Taxol pretreatment decreased the time constants of inactivation for the fast component.

Figure 3

Effects of colchicine and taxol on K⁺ current (I_K). I_K was elicited by depolarizing pulses (2 s duration) to test potentials of −40 to 80 mV in 20 mV increments from the holding potential of −80 mV. (A) Superimposed current traces recorded from a drug-untreated cell (control cell) and a taxol (100 μM)-treated cell. (B) Current-voltage-relationships of I_K obtained from control cells (n=8), colchicine (100 μM)-treated cells (n=7) and taxol (100 μM)-treated cells (n=7). The mean current densities were plotted against voltages attained by depolarizing pulses.

Figure 4

Effects of colchicine and lumicolchicine on transient and sustained components of carbachol (CCh, 10 μM)-induced suppression of I_Ba. I_Ba was repeatedly elicited by depolarizing pulses (30 ms duration) to 0 mV from a holding potential of −60 mV at 0.25 Hz. (A, B and C) Time-series plots for peak I_Ba amplitude (the beginning of CCh application was taken as zero) in a drug-untreated cell (control cell), a colchicine (100 μM)-treated cell and a lumicolchicine (100 μM)-treated cell, respectively. Examples of actual I_Ba traces for before (a) and during CCh application (b) are inserted in each plot. The downward deflection of holding current reflects the induction of non-selective cationic channel current induced by CCh. Pretreatment with colchicine, but not lumicolchicine, caused inhibition of the CCh-induced sustained suppression of I_Ba.

Figure 5

Effect of taxol on transient and sustained components of CCh (10 μM)-induced suppression of I_Ba. (A, B and C) Time-series plots for peak I_Ba amplitude in a drug-untreated cell (control cell), a 10 μM taxol- and a 100 μM taxol-treated cell, respectively. Taxol pretreatment resulted in concentration-dependent inhibition of CCh-induced sustained suppression of I_Ba.