Abstract
Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary β subunits. Detailed information on the structure–function relationships of Nav1 subunits has been obtained through heterologous expression experiments and analyses of protein structures. The basic properties of Nav1s, including their gating and ion permeation, were classically described in the squid giant axon and other invertebrates. However, heterologous functional expression of Nav1s from marine invertebrates has been unsuccessful. Ascidians belong to the Urochordata, a sister group of vertebrates, and the larval central nervous system of ascidians shows a similar plan to that of vertebrates. Here, we report the biophysical properties of ascidian Ciona Nav1 (CiNav1a) heterologously expressed in Xenopus oocytes. CiNav1a exhibited tetrodotoxin-insensitive sodium currents with rapid gating kinetics of activation and inactivation. Furthermore, consistent with the fact that the Ciona genome lacks orthologous genes to vertebrate β subunits, the human β1 subunit did not influence the gating properties when coexpressed with CiNav1a. Interestingly, CiNav1a contains an ankyrin-binding motif in the II–III linker, which can be targeted to the axon initial segment of mammalian cortical neurons. Our findings provide a platform to gain insight into the evolutionary and biophysical properties of Nav1s, which are important for the development of targeted therapeutics.
Highlights
Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells
ascidian Ciona Nav1 (CiNav1a)–II–III linker– YFP signal overlapped with ankyrin-G signal (Fig. 8), which suggest that the II–III linker of CiNav1a can be targeted to axon initial segment (AIS) of rat cortical neurons
We provide the first functional characterization of marine invertebrate Nav1, the ascidian Nav1 channel (CiNav1a) in a heterologous expression system
Summary
Previous homology search from the Ciona genome identified four putative sodium channel genes [24]. Those critical regions include the pore turrets for ion selectivity in the S5–S6 loop from each quadrant of the four repetitive domains (I–IV) with the signature D/E/K/A as found in other typical Nav1s, and the S4 for voltage sensing, ABM in the II–III loop [32, 33], and III–IV linker for fast inactivation (Fig. 1B) [27, 34] Those critical regions were conserved in TuNa1 (BAA04133) of Halocynthia and mostly in a putative ortholog (CBY22707) of Oikopleura (which belong to another tunicate group of ascidians). Ascidian Nav, CiNav1a, showed TTX-insensitive sodium current with rapid gating in Xenopus oocyte. CiNav1a is grouped in the same clade as the previously characterized ascidian sodium channel, TuNa1 from Halocynthia roretzi (Fig. 1A), which was reported to be TTX insensitive in native neural cells [12]. Coexpression with TipE did not lead to any significant change of the I–V curve, activation speed, and decay kinetics of CiNav1a (Fig. 5, B–D)
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