Pore properties of rat brain II sodium channels mutated in the selectivity filter domain

Abstract

Ion selectivity of voltage-activated sodium channels is determined by amino-acid residues in the pore regions of all four homologous repeats. The major determinants are the residues DEKA (for repeats I-IV) which form a putative ring structure in the pore; the homologous structure in Ca-channels consists of EEEE. By combining site-directed mutagenesis of a non-inactivating form of the rat brain sodium channel II with electrophysiological methods, we attempted to quantify the importance of charge, size, and side-chain position of the amino-acid residues within this ring structure on channel properties such as monovalent cation selectivity, single-channel conductance, permeation and selectivity of divalent cations, and channel block by extracellular Ca2+ and tetrodotoxin (TTX). In all mutant channels tested, even those with the same net charge in the ring structure as the wild type, the selectivity for Na+ and Li+ over K+, Rb+, Cs+, and NH4+ was significantly reduced. The changes in charge did not correlate in a simple fashion with the single-channel conductances. Permeation of divalent ions (Ca2+, Ba2+, Sr2+, Mg2+, Mn2+) was introduced by some of the mutations. The IC50 values for the Ca2- block of Na+ currents decreased exponentially with increasing net negative charge of the selectivity ring. The sensitivity towards channel block by TTX was reduced in all investigated mutants. Mutations in repeat IV are an exception as they caused smaller effects on all investigated channel properties compared with the other repeats.

Evolution of voltage-gated ion channels at the emergence of Metazoa

“…Moreover, replacing the selectivity filter of NvNa V 2.5 (DKEA) with that from Na V 1 (DEKA) resulted in the loss of sodium selectivity (Gur Barzilai et al, 2012), while replacing the selectivity filter of Na V 1 with that from NvNa V 2.5 also resulted in the loss of sodium selectivity (Schlief et al, 1996). These results suggest that sodium selectivity in NvNa V 2.5 is achieved in a different manner from that in Na V 1 channels.…”

“…The Lys at DIII was shown to be crucial for sodium selectivity as replacing this residue increased potassium and calcium conductance (Schlief et al, 1996;Favre et al, 1996). Moreover, the position of the residues is critical for sodium selectivity and interchanging their position in the domains was shown to result in the loss of sodium selectivity (Schlief et al, 1996).…”

“…The pore is formed by the segments S5 and S6, and the selectivity to specific ions is enabled by the selectivity filter, which is composed of conserved residues, specific for the ion conducted by the channel, and these residues are situated at the pore-lining loops (p-loops) connecting S5 to S6 in the four domains ( Fig. 1) (Yool and Schwarz, 1991;Heinemann et al, 1992;Tang et al, 1993;Schlief et al, 1996;Doyle et al, 1998; Jiang et al., 2003;Payandeh et al, 2011). Further, many voltage-gated ion channels include in addition to the α-subunit one or more auxiliary subunits that modify their expression levels, folding efficiency, functional properties and subcellular localization (Yu and Catterall, 2004).…”

“…The Lys at DIII was shown to be crucial for sodium selectivity as replacing this residue increased potassium and calcium conductance (Schlief et al, 1996;Favre et al, 1996). Moreover, the position of the residues is critical for sodium selectivity and interchanging their position in the domains was shown to result in the loss of sodium selectivity (Schlief et al, 1996).The genomic revolution of the passing decade revealed that homologs of Na V s are present in the unicellular eukaryotes Thecamonas trahens of the Apusozoa (Cai, 2012) as well as in M. brevicollis (Liebeskind et al, 2011). These findings imply that Na Vlike channels emerged before nervous systems or even multicellularity evolved, more than a billion years ago.…”

Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

“…Mutations of these key residues exhibit pronounced influence on the Na + selectivity and tetrodotoxin/saxitoxin binding affinity. Among the four key residues, Lys is indispensable for exclusion of Ca 2+ permeation and for discrimination between Na + and K + [23][24][25][26][27]. Mutating DEKA to EEEE transforms the Na v channel into a Ca 2+ -selective channel [28].…”