Identification of R(-)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels

Abstract

Efonidipine, a derivative of dihydropyridine Ca(2+) antagonist, is known to block both L- and T-type Ca(2+) channels. It remains to be clarified, however, whether efonidipine affects other voltage-dependent Ca(2+) channel subtypes such as N-, P/Q- and R-types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca(2+) channels, including L- and T-types. To address these issues, the effects of efonidipine and its R(-)- and S(+)-isomers on these Ca(2+) channel subtypes were examined electrophysiologically in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk-ts13). Efonidipine, a mixture of R(-)- and S(+)-isomers, exerted blocking actions on L- and T-types, but no effects on N-, P/Q- and R-type Ca(2+) channels. The selective blocking actions on L- and T-type channels were reproduced by the S(+)-efonidipine isomer. By contrast, the R(-)-efonidipine isomer preferentially blocked T-type channels. The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. These findings indicate that the R(-)-isomer of efonidipine is a specific blocker of the T-type Ca(2+) channel.

Figure 1

Effects of efonidipine on five subtypes of Ca²⁺ channels (L-, N-, P/Q-, R- and T-types) expressed in Xenopus oocytes. Membrane currents in response to step depolarizations of a 300- or 150-ms duration from a holding potential of −80 to +10 mV (L, N-, P/Q- and R-types), or −20 mV (T-type) before (Control) and after a 5-min perfusion of 10 μM efonidipine are presented with current traces (top). The decrease in the amplitude of the peak inward currents after a 5-min perfusion of 10 μM efonidipine is expressed as a channel blockade in percent (bottom).

Figure 2

Effects of efonidipine on T-type α_1G channels expressed in Xenopus oocytes (a) and BHK cells (b). Membrane currents in response to step depolarizations of a 100-ms duration from a holding potential of −80 to −20 mV before (Control) and after a 5-min perfusion of 1 and 10 μM efonidipine are presented with current traces (top). Concentration–blockade relationships (bottom) for efonidipine at various holding potentials were obtained in oocytes and BHK cells. The block amounts were obtained after a 5-min perfusion of efonidipine. Each data point is an average of more than eight observations.

Figure 3

Effects of efonidipine on the voltage dependence of α_1G channel availability in oocytes and BHK cells. The membrane current amplitudes after a 5-s conditioning pulse to various potentials were measured by test pulse in the control and 5 min after exposure to efonidipine. The concentration of efonidipine was 10 μM for oocytes (a) and 1 μM for BHK cells (b). The membrane voltage of the test pulse was chosen to elicit the maximal inward current (−30 or −20 mV). The raw value of the current amplitude was averaged in Xenopus oocytes (n=8). The current amplitude was normalized to membrane capacitance in BHK cells, and the values (current density) were averaged (n=8). The data was summarized by the following equation: where I_m is membrane current amplitude, I_max is maximal amplitude of current availability, V_c is the voltage of conditioning pulse, V_h is the conditioning pulse voltage at which a half of the channel is inactivated and s is the slope factor of channel inactivation. The values were the following: oocytes in the control, I_m=0.46± 0.06 μA, V_h=−56.8±2.8 mV, s=4.8±1.8 mV; oocytes in 10 μM efonidipine, I_m=0.31±0.07 μA, V_h=−59.6±5.6 mV, s=5.4±1.6 mV; BHK cells in the control, I_m=0.51±0.07 nA/pF, V_h=−67.2± 4.7 mV, s=5.8±2.4 mV; BHK cells in 1 μM efonidpine, I_m=0.35±0.06 nA/pF, V_h=−74.9±5.9 mV, s=6.3±1.9 mV.

Figure 4

Effects of the optical isomers of efonidipine, S(+)- and R(−)-efonidipine on L- and T-type Ca²⁺ channels expressed in Xenopus oocytes and BHK cells. (a and b) Membrane currents in response to the step depolarization of a 200- (L-type)- or 100-ms duration (T-type) from a holding potential of −80 to +10 mV before (Control) and after a 5-min perfusion of S(+)- (top) or R(−)-efonidipine (bottom) are presented with current traces. L-type α_1Cα₂/δβ_2a and T-type α_1G Ca²⁺ channels were expressed in Xenopus oocytes (a) or BHK cells (b). In BHK cells, β_2a subunit was substituted with β_1a subunit. Note that the S(+)-isomer kept the blocking ability of efonidipine for both L- and T-type channels (see Figure 1), but the R(−)-isomer scarcely affected L-type channels.

Figure 5

Concentration-blockade curves for S(+)- (top) and R(−)-efonidipine (bottom) at three holding potentials (−60, −80 and −100 mV). Blockades of the peak inward currents through T- (left and middle) and L-type channels (right) expressed in Xenopus oocytes (a) and BHK cells (b) were measured after a 5-min perfusion of various concentrations of the isomers.

Figure 6

The IC₅₀ values for efonidipine and its enantiomers on L- and T-type Ca²⁺ channels in oocytes (open symbols) and BHK cells (filled symbols) are summarized. The IC₅₀ at each holding potential was estimated from the data in Figures 2 and 5. The values for oocytes are denoted with open symbols, and those for BHK cells with filled symbols. Asterisks denote that the IC₅₀ for BHK cells was significantly smaller than that for oocytes. The Hill's coefficients were the following: enantiomer-unspecified efonidipine in oocytes, 0.8±0.1 at −100 mV, 0.8±0.2 at −80 mV, 0.6±0.2 at −60 mV; S(+)-efonidipine in oocytes, 0.9±0.1 at −100 mV, 0.8±0.2 at −80 mV, 0.7±0.3 at −60 mV; R(−)-efonidipine in oocytes, 0.8±0.1 at −100 mV, 0.8±0.2 at −80 mV, 0.8±0.2 at −60 mV; enantiomer-unspecified efonidipine in BHK cells, 0.7±0.2 at −100 mV, 0.7±0.3 at −80 mV, 0.6±0.2 at −60 mV; S(+)-efonidipine in BHK cells, 0.9±0.2 at −100 mV, 0.7±0.1 at −80 mV, 0.7±0.2 at −60 mV; R(−)-efonidipine in BHK cells, 1.1±0.2 at −100 mV, 0.9±0.3 at −80 mV, 0.9±0.3 at −60 mV. Each data point is an average of more than eight observations. Significant differences in IC₅₀ values at different holding potentials are denoted in the figure.