Abstract
The Na(v)1.2 and Na(v)1.3 voltage-gated sodium channel isoforms demonstrate distinct differences in their kinetics and voltage dependence of fast inactivation when expressed in Xenopus oocytes. Co-expression of the auxiliary beta1 subunit accelerated inactivation of both the Na(v)1.2 and Na(v)1.3 isoforms, but it did not eliminate the differences, demonstrating that this property is inherent in the alpha subunit. By constructing chimeric channels between Na(v)1.2 and Na(v)1.3, we demonstrate that the carboxyl terminus is responsible for the differences. The Na(v)1.2 carboxyl terminus caused faster inactivation in the Na(v)1.3 backbone, and the Na(v)1.3 carboxyl terminus caused slower inactivation in the Na(v)1.2 channel. Through analysis of truncated channels, we identified a homologous 60-amino acid region within the carboxyl terminus of the Na(v)1.2 and the Na(v)1.3 channels that is responsible for this modulation of fast inactivation. Site-directed replacement of Na(v)1.3 lysine 1826 in this region to its Na(v)1.2 analogue glutamic acid 1880 (K1826E) shifted the voltage dependence of inactivation toward that of Na(v)1.2. The K1826E mutation also accelerated the inactivation kinetics to a level comparable with that of Na(v)1.2. The reverse Na(v)1.2 E1880K mutation exhibited much slower inactivation kinetics and depolarized inactivation voltage dependence. A complementary mutation located within the inactivation linker of Na(v)1.3 (K1453E) caused inactivation changes mirroring those caused by the K1826E mutation in Na(v)1.3. Therefore, we have identified a homologous carboxyl-terminal residue that regulates the kinetics and voltage dependence of fast inactivation in sodium channels, possibly via a charge-dependent interaction with the inactivation linker.
Highlights
There are nine mammalian voltage-gated sodium channel isoforms, all of which have a similar molecular structure
Through analysis of chimeric channels, we showed that the carboxyl terminus is required to transfer the inactivation characteristics of the donor channel
Fast Inactivation Kinetics of Nav1.2 and Nav1.3—We characterized the biophysical differences between the Nav1.2 and Nav1.3 sodium channel isoforms using Xenopus oocytes, a standard and highly controlled heterologous system for studying ion channel function
Summary
Molecular Cloning—The wild-type rat Nav1.3 cDNA coding region was cloned in a pLCT2 low copy vector (pNa3T). Entry into inactivation was analyzed using a two-pulse protocol in which a conditioning pulse to ϩ10 mV was applied from a holding potential of Ϫ100 mV, beginning with a duration of 0 ms and increasing by 2 ms with each consecutive sweep, to elicit inactivation This was immediately followed by a test pulse to Ϫ5 mV to determine the fraction of current that had been inactivated. Data for the Nav1.3, Nav1.3-derived chimeric ␣ subunits alone, Nav1.3 ϩ calmodulin (CaM), and Nav1.3 ϩ CaM1234 were fit from the 2 ms time point instead of the 0 ms time point because a slight potentiation of less than 1% was observed in several oocytes This effect was not observed for Nav1.2 or Nav1.2-derived chimeras or in the presence of the 1 subunit. In the case of ␣ ϩ 1, these data were fit with the double exponential equation,
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