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.
Highlights
Reviewed by: Maria Isabel Bahamonde Santos, Pompeu Fabra University, Spain Harold H Zakon, The University of Texas, USA
The pattern of conservation suggests a possible pairing of long loops in Domains I and III, which are bridged with core cysteines in Sodium Leak ChaNnel (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
The replacement of sodium and calcium selectivity in invertebrate NALCN channels by splicing at high field strength (HFS) site confirms this site with a lysine in the second or third domain as a key determinant of the sodium selectivity in four repeat domains (4x6TM) channels
Summary
The HFS site at the selectivity filter of eukaryotic Cav and Cav calcium channel is formed by a ring of negatively charged glutamates (EEEE), which resembles the EEEE ring of bacterial. In the same position where the second aspartate substitution (TLDDWSD) generates the calcium-selective bacterial sodium channel, the aspartate in the second repeat is conserved universally in all eukaryotic calcium channels, including Cav, Cav and Cav channels from single cell coanoflagellates to mammalian channels (Tikhonov and Zhorov, 2011; Payandeh and Minor, 2015) This aspartate, which is next to the HFS site glutamate in Domain II, (e.g., TGEDWNS in Cav1.2, see “Ca” site in Figure 4), is likely required for calcium ion selectivity. TTX sensitive vertebrate nerve and skeletal muscle-specific sodium channels possess an outer ring of negatively charged residues, EEDD, perched above the DEKA HFS site (e.g., the TSAGWD sequence in Domain IV of the Nav1.4 channel, see “TTX” site, Figure 4). There are Domain IV mutations in Nav1.4 sodium channels of pufferfish and other species (Jost et al, 2008), which prevent self-poisoning by TTX generated in their own tissues
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