Novel molecular determinants in the pore region of sodium channels regulate local anesthetic binding

Abstract

The pore of the Na+ channel is lined by asymmetric loops formed by the linkers between the fifth and sixth transmembrane segments (S5-S6). We investigated the role of the N-terminal portion (SS1) of the S5-S6 linkers in channel gating and local anesthetic (LA) block using site-directed cysteine mutagenesis of the rat skeletal muscle (Na(V)1.4) channel. The mutants examined have variable effects on voltage dependence and kinetics of fast inactivation. Of the cysteine mutants immediately N-terminal to the putative DEKA selectivity filter in four domains, only Q399C in domain I and F1236C in domain III exhibit reduced use-dependent block. These two mutations also markedly accelerated the recovery from use-dependent block. Moreover, F1236C and Q399C significantly decreased the affinity of QX-314 for binding to its channel receptor by 8.5- and 3.3-fold, respectively. Oddly enough, F1236C enhanced stabilization of slow inactivation by both hastening entry into and delaying recovery from slow inactivation states. It is noteworthy that symmetric applications of QX-314 on both external and internal sides of F1236C mutant channels reduced recovery from use-dependent block, indicating an allosteric effect of external QX-314 binding on the recovery of availability of F1236C. These observations suggest that cysteine mutation in the SS1 region, particularly immediate adjacent to the DEKA ring, may lead to a structural rearrangement that alters binding of permanently charged QX-314 to its receptor. The results lend further support for a role for the selectivity filter region as a structural determinant for local anesthetic block.

Fig. 1.

A, transmembrane topology of the Na⁺ channel. Arrows indicated the P segments, the regions between the fifth and sixth membrane spanning repeats. The residues that were mutated are proximal to the selectivity filter and are shown below the topology cartoon. The putative selectivity residues Asp400, Glu755, Lys1237, and A1529 are enclosed in the box. Cysteine mutants at all positions except Asp400 and Lys1237 (both in italic) were examined. Gly754 and Leu752 are labeled with a strikethrough due to nonexpression of cysteine mutants. B and C, normalized activation curves (B) and steady-state inactivation curves (C) for the wild-type and domain I-IV mutants. The solid curves are fits to a Boltzmann function as described under Materials and Methods. The parameters of the fits are summarized in Table 1.

Fig. 2.

Recovery of the wild-type and mutant channels. To examine recovery from fast inactivation, a paired pulse protocol was used. After a 20-ms inactivating prepulse (P1) to −10 mV, cells were recovered at −100 mV for variable test intervals. Fractional recovery (P2/P1) during the test interval was measured as the peak Na⁺ current elicited by a subsequent test pulse to −10 mV (P2) relative to that measured during the prepulse (P1).

Fig. 3.

A, whole-cell sodium currents of the wild-type, L396C, and A1234C mutants. The currents were elicited by depolarizations from a holding potential of −100 mV to test potentials from −50 to +60 mV in 10-mV increments. B, the times to the peak currents for wild-type, L396C, and A1234C are plotted as a function of the step voltage. C, the time from peak to 50% current decay (t_1/2) for wild-type, L396C, and A1234A is plotted as a function of the test voltage. Significant differences (p < 0.05) are indicated by asterisks (*).

Fig. 4.

A, comparison of the development of use-dependent block of Na⁺ channels by 250 μM internal QX-314. Currents are elicited by voltage steps from −100 to −10 mV and normalized to the first pulse in the train for the wild-type (●, ○), Q399C (■), and F1236C (▲). B, representative whole-cell Na⁺ currents in the presence of QX-314. Currents elicited by the first (a), 20th (b), and 60th (c) pulses for the wild-type, Q399C, and F1236C mutants are shown. The current amplitudes are reduced, but the time constant of current decay is not changed by QX-314. There is no significant difference in the time constant of decay between the wild type and any of the mutant channels except for L396C (see Table 2). C, bar plot of the extent of use-dependent block that develops at steady state with 250 μM QX-314 in the pipette at a stimulation frequency of 1 Hz. The height of the bars represents the fraction of current remaining at 1 min. The magnitude of use-dependent block increases with higher concentrations of QX-314. There is significantly less use-dependent block of the Q399C, L396C, and F1236C mutants (p < 0.05).

Fig. 5.

A, recovery from use-dependent block is determined by depolarizing voltage steps of 20 ms delivered 30 s and 1, 5, 10, and 15 min after use-dependent block is elicited by a 60-s, 1-Hz train of depolarizations. The wild type (○), Q399C (■), and F1236C (▲) data are fit to a single exponential curve with time constants of 9.44, 4.55, and 1.41 min, respectively. The current amplitudes before the pulse train were used for normalization. B, fractional recovery of the current. For the wild-type and each of the mutants, the fractional current at 15 min of recovery minus the fraction of current remaining after a 1-Hz train of depolarizing voltage steps to elicit use-dependent block is plotted. L396C, T397C, M398C, Q399C, and F1236C exhibit significantly larger fractional recoveries at 15 min compared with the wild-type.

Fig. 6.

Entry into and recovery from slow inactivated states. A, conditioning pulses of 3 to 1000 ms to −20 mV (P1) were used to promote entry into slow inactivated states. The rate of entry was assessed by a 50-ms test pulse to −20 mV after 20 ms at −100 mV to permit recovery from fast inactivation from a holding potential of −100 mV (P2) after the conditioning pulse. The P2/P1 ratio after a conditioning pulse duration of 400 ms is plotted. The rate of entry into slow-inactivated states was not changed in any of the mutants except F1236C (*, p < 0.05). B, recovery from inactivation after a 500-ms test pulse to −20 mV (P1) was assessed by a second test pulse to −20 mV (P2) after varying intervals at −100 mV. Recovery from inactivation was uniquely slowed in F1236C.

Fig. 7.

External QX-314 does not block the wild-type skeletal muscle Na⁺ channel or P segment mutants. A, Q399C (○) and F1236C (□) currents are elicited by depolarizing steps from −100 to −10 mV at a rate of 0.5 Hz, and 1 mM QX-314 is applied extracellularly. There is no effect on current amplitude, decay kinetics, or voltage-dependence. The I1575C (homologous to residue Ile1760 in the rat brain IIa channel) mutant in the domain IV-S6 membrane spanning repeat is blocked by ∼30% by extracellular QX-314. B, recovery of the wild-type (○, ●) and F1236C (□, ■) from use-dependent block in the presence of 250 μM internal (○, □) and symmetrical (●, ■) QX-314. External QX-314 does not influence recovery of the wild-type channel but recovery of F1236C is slowed. C, symmetrical QX-314 does not significantly affect the recovery of any of the other P-segment mutants. Bar plot of the fractional recoveries with 250 mM internal (□) and symmetrical (■) QX-314.