Selectivity filters and cysteine-rich extracellular loops in voltage-gated sodium, calcium, and NALCN channels

Abstract

How nature discriminates sodium from calcium ions in eukaryotic channels has been difficult to resolve because they contain four homologous, but markedly different repeat domains. We glean clues from analyzing the changing pore region in sodium, calcium and NALCN channels, from single-cell eukaryotes to mammals. Alternative splicing in invertebrate homologs provides insights into different structural features underlying calcium and sodium selectivity. NALCN generates alternative ion selectivity with splicing that changes the high field strength (HFS) site at the narrowest level of the hourglass shaped pore where the selectivity filter is located. Alternative splicing creates NALCN isoforms, in which the HFS site has a ring of glutamates contributed by all four repeat domains (EEEE), or three glutamates and a lysine residue in the third (EEKE) or second (EKEE) position. Alternative splicing provides sodium and/or calcium selectivity in T-type channels with extracellular loops between S5 and P-helices (S5P) of different lengths that contain three or five cysteines. All eukaryotic channels have a set of eight core cysteines in extracellular regions, but the T-type channels have an infusion of 4–12 extra cysteines in extracellular regions. The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in NALCN, Cav, and Nav channels, and pairing of shorter loops in Domains II and IV in T-type channel through disulfide bonds involving T-type specific cysteines. Extracellular turrets of increasing lengths in potassium channels (Kir2.2, hERG, and K2P1) contribute to a changing landscape above the pore selectivity filter that can limit drug access and serve as an ion pre-filter before ions reach the pore selectivity filter below. Pairing of extended loops likely contributes to the large extracellular appendage as seen in single particle electron cryo-microscopy images of the eel Nav1 channel.

Evolutionary insights into T-type Ca2+ channel structure, function, and ion selectivity from the Trichoplax adhaerens homologue

“…(A) Replacing 135 mM impermeant external cation NMDG + with Na + , in the presence of invariant 2 mM Ca 2+ produces a 42% increase in peak inward current through TCa v 3 elicited by a voltage step from −110 to −45 mV (black and red current traces, respectively), indicative of a moderate mixing of inward Ca 2+ and Na + ions in macroscopic currents. (B) Mean percent increase in peak inward current at −45 mV (±SE), upon replacement of 135 mM external NMDG + with equimolar Na + in the presence of 2 mM external Ca 2+ , for TCa v 3 compared with previously published data for other cloned Ca v 3 channels (Senatore et al, 2014; Stephens et al, 2015). (C) Macroscopic currents for TCa v 3 are larger in the presence of 2 mM external Ca 2+ versus Ba 2 (inset), where mono-exponential curves fitted over activation and inactivation phases of the current waveforms (τ inact and τ inact , respectively) reveal statistically indistinguishable kinetics under one-way ANOVA.…”

The chemical basis for electrical signaling

“…Instead of four Glu residues at the high field-strength site, mammalian Na V channels typically have Asp/Glu/Lys/Ala in their four homologous domains 67 , and the positively charged Lys residue in domain III is crucial for Na + selectivity 68 . Evolution of invertebrate Na V and Ca V channels has led to a wide array of combinations of amino acid residues in the selectivity filter, which always include at least two negative charges at this high field-strength site 69 .…”

Phenotypic Variability from Benign Infantile Epilepsy to Ohtahara Syndrome Associated with a Novel Mutation in SCN2A

“…A gain of function of Na v 1.2 has been proposed to underlie hyperexcitability in some epilepsy-associated SCN2A mutations; however, conflicting results of in vitro electrophysiological analyses apply for a more complex dysfunction [Ogiwara et al, 2009]. The mutation p.Q383E results in a conformational change within the selectivity filter with a newly formed ED-motif in the first domain of Na v 1.2, showing resemblance to the selectivity filter residues in the second domain of calcium ion channels [Stephens et al, 2015]. Therefore, we hypothesized that a change from sodium selectivity to calcium selectivity can underlie the channel dysfunction and created a computer model of the mutant channel ( fig.…”

“…The ring is formed by residues between P-loop helices from all 4 channel domains (I-IV). Residues at these positions are evolutionary conserved in all voltage-gated cation channels and determine the K + , Ca 2+ , Na + , or mixed ion selectivity [Stephens et al, 2015].…”

“…1 ). The selectivity for the different cations (Na + , Ca 2+ ) is determined by the selectivity filter within the membrane re-entrant loop (Ploop) between the pore-forming S5 and S6 segments of each domain [Mantegazza and Catterall, 2012;Stephens et al, 2015]. SCN2A encodes the pore-forming sodium channel protein type 2 alpha subunit (Na v 1.2), the major alpha subunit in excitatory neurons [Mantegazza and Catterall, 2012;Howell et al, 2015].…”