T-type Ca(2+) channel modulation by otilonium bromide

Abstract

Antispasmodics are used clinically to treat a variety of gastrointestinal disorders by inhibition of smooth muscle contraction. The main pathway for smooth muscle Ca(2+) entry is through L-type channels; however, there is increasing evidence that T-type Ca(2+) channels also play a role in regulating contractility. Otilonium bromide, an antispasmodic, has previously been shown to inhibit L-type Ca(2+) channels and colonic contractile activity. The objective of this study was to determine whether otilonium bromide also inhibits T-type Ca(2+) channels. Whole cell currents were recorded by patch-clamp technique from HEK293 cells transfected with cDNAs encoding the T-type Ca(2+) channels, Ca(V)3.1 (alpha1G), Ca(V)3.2 (alpha1H), or Ca(V)3.3 (alpha1I) alpha subunits. Extracellular solution was exchanged with otilonium bromide (10(-8) to 10(-5) M). Otilonium bromide reversibly blocked all T-type Ca(2+) channels with a significantly greater affinity for Ca(V)3.3 than Ca(V)3.1 or Ca(V)3.2. Additionally, the drug slowed inactivation in Ca(V)3.1 and Ca(V)3.3. Inhibition of T-type Ca(2+) channels may contribute to inhibition of contractility by otilonium bromide. This may represent a new mechanism of action for antispasmodics and may contribute to the observed increased clinical effectiveness of antispasmodics compared with selective L-type Ca(2+) channel blockers.

Fig. 1.

Otilonium bromide (OB) blocks T-type Ca²⁺ channel subunit Ca_V3.1 (α1G) expressed in HEK293 cells. A: representative whole cell T-type Ca²⁺ channel currents recorded in NaCl Ringer solution with 0, 0.1, 1, or 10 μM OB. Inset: OB molecular structure. B: current-voltage relationships of T-type Ca²⁺ currents at each concentration of OB were normalized to the maximum peak inward current of the control record (0 M OB). C: summary of normalized peak inward Ca²⁺ currents (n = 7 cells exposed to the entire sequence of concentrations, *P < 0.05 by nonparametric repeated-measures ANOVA with Friedman posttest). The IC₅₀ value of OB for Ca_V3.1 was 774 ± 109 nM (dotted line).

Fig. 2.

OB blocks T-type Ca²⁺ channel subunit Ca_V3.2 (α1H) expressed in HEK293 cells. A: representative whole cell T-type Ca²⁺ channel currents recorded in NaCl Ringer solution with 0, 0.1, 1, or 10 μM OB. B: current-voltage relationships of T-type Ca²⁺ currents at each concentration of OB were normalized to the maximum peak inward current of the control record (0 M OB). C: summary of normalized peak inward Ca²⁺ currents (n = 6 cells exposed to the entire sequence of concentrations, *P < 0.05 by nonparametric repeated-measures ANOVA with Friedman posttest). The IC₅₀ value of OB for Ca_V3.2 was 1,070 ± 269 nM (dotted line).

Fig. 3.

OB blocks T-type Ca²⁺ channel subunit Ca_V3.3 (α1I) expressed in HEK293 cells. A: representative whole cell T-type Ca²⁺ channel currents recorded in NaCl Ringer solution with 0, 0.1, 1, or 10 μM OB. B: current-voltage relationships of T-type Ca²⁺ currents at each concentration of OB were normalized to the maximum peak inward current of the control record (0 M OB). C: summary of normalized peak inward Ca²⁺ currents (n = 7 cells exposed to the entire sequence of concentrations, *P < 0.05 by nonparametric repeated-measures ANOVA with Friedman posttest). The IC₅₀ value of OB for Ca_V3.3 was 451 ± 90 nM (dotted line).

Fig. 4.

Block by OB is not use dependent. T-type Ca²⁺ channels Ca_V3.1 (A), Ca_V3.2 (B), or Ca_V3.3 (C) were stepped to −30 mV from a holding potential of −120 mV once every 2 s (0.5 Hz, left) or 40 s (0.025 Hz, middle) in the presence of 3 μM OB. Traces at 0 (solid lines) and 200 s (dotted lines) are shown. Right: percent block of T-type Ca²⁺ currents vs. time. Peak Ca²⁺ currents pulsed to −30 mV at 0.5 Hz (solid symbols, 1 of every 4 data points shown) or 0.025 Hz (open symbols, all data points shown) were normalized to the trace at time = 0 in the absence (circles) or presence (triangles) of OB (3 μM).

Fig. 5.

Block of T-type Ca²⁺ channels by OB is reversible. Left: representative traces of maximum peak inward Ca²⁺ currents from Ca_V3.1 (A), Ca_V3.2 (B), or Ca_V3.3 (C) in the presence of 0 (control), 3, or 0 (washout) μM OB. OB (3 μM) inhibited the Ca_V3.1, Ca_V3.2, and Ca_V3.3 current traces by 83, 67, and 92%, respectively, during steps from −100 to −40 mV. Washout recovered 100, 92, and 94% of their respective control traces. Right: peak Ca²⁺ currents normalized to same-cell pre-OB controls (*P < 0.05 by nonparametric repeated-measures ANOVA with Friedman posttest; Ca_V3.1, n = 6; Ca_V3.2, n = 6; Ca_V3.3, n = 8).

Fig. 6.

OB (3 μM) delays activation and inactivation kinetics. Left: equally scaled traces obtained by stepping from −100 to −50 mV, showing Ca_V3.1 (A–B), Ca_V3.2 (C), or Ca_V3.3 (D) currents in response to sham rinses (A) or exposures up to 3 μM extracellular OB (B–D). Traces are from the same experiments shown in Figs. 1A, 2A, and 3A. Middle: time from start of pulse to point of maximum peak inward current (*P < 0.05, control to 3 μM OB; Ca_V3.1, n = 6; Ca_V3.2, n = 5; Ca_V3.3, n = 6). Right: time constant of inactivation vs. step voltage (*P < 0.05, control to 3 μM OB; Ca_V3.1, n = 6; Ca_V3.2, n = 6; Ca_V3.3, n = 6).

Fig. 7.

Mibefradil (3 μM) decreases the frequency of contractions in human colon. Circular muscle layer contractions recorded from a normal human sigmoid colonic muscle strip before (A) and during (B) infusion of 3 μM mibefradil.