Browsing by Subject "Sodium channels"
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Item Individual variation and hormonal modulation of sodium channel alpha and beta1 subunits in the electric organ correlate with variation in a social signal(2006) Liu, He; Zakon, HaroldThe electric fish Sternopygus macrurus emits an electric organ discharge (EOD) composed of a series of pulses. EOD frequency and duration are individually unique, sexually dimorphic and regulated by steroid hormones. Previous studies have shown the EOD pulse is partially shaped by a sodium current, whose rate and voltage dependence of inactivation correlate with EOD frequency and pulse duration, and are modulated by androgens. In this study I tested whether the gradient in sodium current inactivation across EOD frequency might be due to regulation on sodium channel α and β1 subunits. Full-length sequences of the two sodium channel α subunits in the electric organ of Sternopygus macrurus, smNav1.4a and smNav1.4b were cloned. Furthermore, two smNav1.4b mRNA transcripts (smNav1.4bL and smNav1.4bS), with alternative first exons and translated into proteins with and without an extended N terminus respectively, were identified. Electric organ expresses smNav1.4a and smNav1.4b at comparable levels and preferentially expresses smNav1.4bL. The mRNA level of smNav1.4bL but not smNav1.4a, correlates with EOD frequency. I also cloned the sodium channel β1 subunit in Sternopygus and found two splice forms of this gene (β1L and β1S). They exhibit a distinct pattern of differential expression in different tissues. In the electric organ, the mRNA levels of β1 and the splicing preference for β1S correlate with EOD frequency. An androgen implant lowered EOD frequency. It also lowered the mRNA levels of smNav1.4bL, smNav1.4bS and β1, but did not affect smNav1.4a or the splicing preference of β1. Expression of smNav1.4bL or smNav1.4bS alone, or together with β1L or β1S in Xenopus oocytes revealed the kinetic properties of these subunits. Importantly, smNav1.4bL and β1S, whose expressions correlate with EOD frequency, show faster inactivation rates and negative shifts of voltage dependence, consistent with the natural phenotype of high EOD frequency fish. Furthermore, two mutagenesis studies addressed the functions of the novel regions in smNav1.4bL and β1S. These results suggest multiple levels of mRNA control on sodium channel α and β1 subunits underlie the cellular excitability in the electric organ and correlate with the variation in an important social signal, EOD, in Sternopygus.Item Role of N- and C- termini in inactivation of sodium channel in weakly electric fish(2009-08) Wu, Mingming; Zakon, H. H.The weakly electric fish Sternopygus macrurus emits an electric organ discharge (EOD) composed of a series of pulses. The EOD pulse is mainly shaped by sodium currents. There are two sodium channel α subunits orthologs of the mammalian Nav1.4 expressed in the EO of Sternopygus. Previous studies identified a novel splice variant of the Nav1.4b (Nav1.4bL), in which an extra 51-amino acid occurs in the N terminal end. Nav1.4bL currents inactivate and recover from inactivation significantly faster than Nav1.4bS. The voltage-dependence of steady-state inactivation of smNav1.4bL shifts to hyperpolarized potential. Structural analysis predicts an α-helix in the middle of the extended N terminus. Removal of a proline right after the α-helix significantly slows down current decay but has no effect on channel recovery from inactivation, suggesting inactivation and recovery have independent mechanism. Mutagenesis analysis of the extended N terminus showed that the short helical region, especially the positive charges in the helix, is an important determinant for channel voltage-dependence of steady-state inactivation. However, other residues outside the helical region are required for regulation of fast inactivation and recovery form inactivation. Functional and structural analysis provides evidence for the importance of the C terminus in fish Nav1.4b channel properties. Chimera in which the C terminus of smNav1.4bS was substituted by the human Nav1.4 C terminus, shows an 11 mV positive shift in voltage-dependence of activation and a -16 mV negative shift in inactivation. Deletion of the distal half of smNav1.4bS negatively shifted voltage-dependence of inactivation and significantly accelerated channel recovery from inactivation. In the deletion mutant, the regulation by the N segment is missing. Substitution of the C terminus mutant retains wild type channel inactivation and recovery properties and can be regulated by N segment again. My study provides evidence that the extended N terminus of smNav1.4bL binds the distal part of C terminal tail to modulate channel inactivation properties. This is the first time to show the distal C terminus is involved in channel recovery from inactivation. Studies in the fish sodium channel properties provide useful information to understand function and structure of voltage-gated sodium channels.