Sidedness of carbamazepine accessibility to voltage-gated sodium channels

Abstract

Voltage-gated sodium channels are inhibited by many local anesthetics, antiarrhythmics, and antiepileptic drugs. The local anesthetic lidocaine appears to be able to access its binding site in the sodium channel only from the membrane phase or from the internal face of the channel. In contrast, the antiepileptic drug carbamazepine was found to inhibit voltage-gated sodium channels only with external, but not internal, application, implying a major difference. We investigated this point using both whole-cell and inside-out patch recordings from human Na(v)1.7 channels in a stable cell line. In the whole-cell configuration, carbamazepine inhibited sodium current within seconds when applied externally, but had little or no effect when applied internally for up to 15 minutes, confirming previous results. However, carbamazepine inhibited sodium channels effectively and rapidly when applied to the internal face of the membrane using inside-out patch recording. We found that lidocaine also has little or no effect when applied intracellularly in whole-cell recording, but blocks effectively and rapidly when applied to the internal surface using inside-out patches. In contrast, the cationic lidocaine derivative QX-314 (N-ethyl-lidocaine) blocks effectively when applied internally with whole-cell dialysis, as well as when applied to inside-out patches. We conclude that carbamazepine and lidocaine access the sodium channel in similar ways and hypothesize that their lack of effect with internal dialysis in whole-cell recording reflects rapid exit through membrane near the pipette recording site. This effect likely limits the ability of any compound with significant membrane permeability to be applied intracellularly by whole-cell dialysis.

Fig. 1.

External but not internal carbamazepine blocks sodium channels in whole-cell recordings. (A) Effect of externally applied 300 μM carbamazepine on sodium current elicited in a whole-cell recording by 30-millisecond steps from −75 to −20 mV delivered every 3 seconds. The left panel shows currents before, during, and after wash-out of 300 μM carbamazepine, whereas the right panel shows the time course of block. (B) Lack of effect of internally applied 300 μM carbamazepine in whole-cell recording (same pulsing protocol as in A). (C) Experimental protocol as in B (with a different cell) but with subsequent application of 300 μM carbamazepine externally. (D) Collected results for inhibition by 300 μM carbamazepine applied externally, internally, or both in whole-cell recording. Data points and error bars are mean ± S.E.M. for measurements in three to five cells. CBZ, carbamazepine; ext, external; int, internal.

Fig. 2.

Shift of voltage dependence of inactivation with inside-out patch recording of sodium Nav1.7 sodium channels. (A) The left panel shows currents evoked by a test pulse to −20 mV from holding potentials of −140, −90, and −70 mV in a whole-cell recording. The right panel shows currents evoked by a test pulse to −20 mV from holding potentials of −140, −90, and −70 mV in a recording from an inside-out patch. (B) Collected results for sodium channel availability in recordings from whole-cell (n = 4) and inside-out patches (n = 5). Data from each cell were fit individually by the Boltzmann function 1/(1 + exp((V − V_h)/k)), where V is holding potential, V_h is voltage of half-maximal inactivation, and k is the slope factor. Solid lines show fits with mean parameters. Whole-cell (open circles): V_h = −70 ± 1 mV, k = 5.7 ± 0.1 mV (n = 4). Inside-out (filled circles): V_h = −94 ± 3 mV, k = 5.5 ± 0.3 (n = 5). Holding potentials were established for 1 second.

Fig. 3.

Carbamazepine rapidly blocks sodium channels when applied to the internal surface in inside-out patch recordings. (A) Effect of 300 μM carbamazepine applied to the internal surface on sodium current elicited in an inside-out patch recording. Currents were evoked by 30-millisecond steps from −100 to −20 mV delivered every 3 seconds. Currents averaged from 10 traces before (black trace) and after (red trace) application of 300 μM carbamazepine are shown. (B) Time course of block and recovery with application of carbamazepine to the internal surface in inside-out recording (same cell as A). (C) Collected results (mean ± S.E.M.) for block by carbamazepine in inside-out patches from four different cells compared with the collected results from Figure 1 for effects of carbamazepine in whole-cell recording. CBZ, carbamazepine; Con, control.

Fig. 4.

Comparison of lidocaine and QX-314 effects with whole-cell and inside-out patch recordings. (A) Effect of externally applied 300 μM lidocaine (30-millisecond steps from −75 to −20 mV delivered every 3 seconds). (B) Lack of effect of internally applied 300 μM lidocaine in whole-cell recording (same pulsing protocol as in A). (C) Experimental protocol as in B (with a different cell) but with subsequent additional application of 300 μM lidocaine externally. (D) Effective block by lidocaine applied to the internal surface in an inside-out patch recording. (Currents before and after lidocaine averaged over 10 sweeps.) (E) Time course of block and partial recovery by lidocaine applied to an inside-out patch. (F) Collected results for inhibition by 300 μM lidocaine applied externally, internally, or both in whole-cell recording and internally in inside-out patch recordings. Data points and error bars are mean ± S.E.M. for measurements in three to five cells with each configuration. (G) Effect of externally applied 1 mM QX-314. (H) Effect of internally applied 300 μM QX-314 in whole-cell recording. (I) Block by QX-314 applied to the internal surface in an inside-out patch recording. (Currents before and after QX-314 averaged over 10 sweeps.) (J) Time course of block by QX-314 applied to an inside-out patch. Con, control; ext, external; int, internal; Lido/lido, lidocaine; QX, QX-314.

Fig. 5.

Illustration of hypothesized mechanism by which internally applied carbamazepine fails to have a significant blocking effect. Hydrophobic carbamazepine molecules are hypothesized to diffuse into and across the membrane near the point of attachment of the pipette, thus failing to accumulate inside the cell. CBZ, carbamazepine.