A Key Gating Charge Interaction Required for Slow Inactivation of the Navab Bacterial Sodium Channel

Abstract

Bacterial voltage-gated sodium (Nav) channels are considered an ideal model for structure-function studies. The elucidation of the crystal structure of the bacterial channel NavAb (Payandeh et al., Nature 486, 135-139, 2011) opened an avenue to understand electrical signaling in excitable cells at the structural level. NavAb expressed in Hi5 insect cells as for structural studies has unusually negative voltage-dependent activation (Va ∼ 130 mV). NavAb also has three phases of inactivation, a biphasic early inactivation process (τ1∼103 ms, τ2∼3.8 s, at −180 mV) followed by an unusually strong and use-dependent slow-inactivation process. To search for the molecular basis for negative activation and slow inactivation of NavAb, we mutated the outermost gating charge partner in the S2 segment, Asn49, to Lys. This mutation shifted the activation curve ∼ 75 mV toward more positive potentials. Surprisingly, it also completely abolished use-dependent slow inactivation. We showed previously that the equivalent residue in NaChBac (Asp60) interacts with the R3 gating charge in the S4 segment during activation using the disulfide locking method (DeCaen et al, PNAS 2008 105 (39) 15142-15147). To test whether this interaction between R3 and N49 was critical for slow inactivation, we mutated NavAb R3 to Cys. The resulting mutant NavAb_R3C also had positively shifted channel activation (+75 mV) and no use-dependent slow inactivation. The fact that these reciprocal mutations have the same functional effects suggests that interaction between R3 in the S4 segment and N49 in the S2 segment is an important link that stabilizes the activated state of the voltage sensor and triggers the slow inactivation process.