A lysine residue from an extracellular turret switches the ion preference in a Cav3 T-Type channel from calcium to sodium ions
2022; Elsevier BV; Volume: 298; Issue: 12 Linguagem: Inglês
10.1016/j.jbc.2022.102621
ISSN1083-351X
AutoresWendy Guan, Kaidy G. Orellana, Robert F. Stephens, Boris S. Zhorov, J. David Spafford,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoCav3 T-type calcium channels from great pond snail Lymnaea stagnalis have a selectivity-filter ring of five acidic residues, EE(D)DD. Splice variants with exons 12b or 12a spanning the extracellular loop between the outer helix IIS5 and membrane-descending pore helix IIP1 (IIS5-P1) in Domain II of the pore module possess calcium selectivity or dominant sodium permeability, respectively. Here, we use AlphaFold2 neural network software to predict that a lysine residue in exon 12a is salt-bridged to the aspartate residue immediately C terminal to the second-domain glutamate in the selectivity filter. Exon 12b has a similar folding but with an alanine residue in place of lysine in exon 12a. We express LCav3 channels with mutated exons Ala-12b-Lys and Lys-12a-Ala and demonstrate that they switch the ion preference to high sodium permeability and calcium selectivity, respectively. We propose that in the calcium-selective variants, a calcium ion chelated between Domain II selectivity-filter glutamate and aspartate is knocked-out by the incoming calcium ion in the process of calcium permeation, whereas sodium ions are repelled. The aspartate is neutralized by the lysine residue in the sodium-permeant variants, allowing for sodium permeation through the selectivity-filter ring of four negatively charged residues akin to the prokaryotic sodium channels with four glutamates in the selectivity filter. The evolutionary adaptation in invertebrate LCav3 channels highlight the involvement of a key, ubiquitous aspartate, "a calcium beacon" of sorts in the outer pore of Domain II, as determinative for the calcium ion preference over sodium ions through eukaryotic Cav1, Cav2, and Cav3 channels. Cav3 T-type calcium channels from great pond snail Lymnaea stagnalis have a selectivity-filter ring of five acidic residues, EE(D)DD. Splice variants with exons 12b or 12a spanning the extracellular loop between the outer helix IIS5 and membrane-descending pore helix IIP1 (IIS5-P1) in Domain II of the pore module possess calcium selectivity or dominant sodium permeability, respectively. Here, we use AlphaFold2 neural network software to predict that a lysine residue in exon 12a is salt-bridged to the aspartate residue immediately C terminal to the second-domain glutamate in the selectivity filter. Exon 12b has a similar folding but with an alanine residue in place of lysine in exon 12a. We express LCav3 channels with mutated exons Ala-12b-Lys and Lys-12a-Ala and demonstrate that they switch the ion preference to high sodium permeability and calcium selectivity, respectively. We propose that in the calcium-selective variants, a calcium ion chelated between Domain II selectivity-filter glutamate and aspartate is knocked-out by the incoming calcium ion in the process of calcium permeation, whereas sodium ions are repelled. The aspartate is neutralized by the lysine residue in the sodium-permeant variants, allowing for sodium permeation through the selectivity-filter ring of four negatively charged residues akin to the prokaryotic sodium channels with four glutamates in the selectivity filter. The evolutionary adaptation in invertebrate LCav3 channels highlight the involvement of a key, ubiquitous aspartate, "a calcium beacon" of sorts in the outer pore of Domain II, as determinative for the calcium ion preference over sodium ions through eukaryotic Cav1, Cav2, and Cav3 channels. Ion channel chameleons: Switching ion selectivity by alternative splicingJournal of Biological ChemistryVol. 299Issue 3PreviewVoltage-gated sodium and calcium channels are distinct, evolutionarily related ion channels that achieve remarkable ion selectivity despite sharing an overall similar structure. Classical studies have shown that ion selectivity is determined by specific binding of ions to the channel pore, enabled by signature amino acid sequences within the selectivity filter (SF). By studying ancestral channels in the pond snail (Lymnaea stagnalis), Guan et al. showed in a recent JBC article that this well-established mechanism can be tuned by alternative splicing, allowing a single CaV3 gene to encode both a Ca2+-permeable and an Na+-permeable channel depending on the cellular context. Full-Text PDF Open Access Eukaryotic voltage-gated calcium (Cav) and sodium (Nav) channels belong to a large superfamily of P-loop channels, which play key roles in the physiology of electrically excitable cells (1Catterall W.A. Lenaeus M.J. Gamal El-Din T.M. Structure and pharmacology of voltage-gated sodium and calcium channels.Annu. Rev. Pharmacol. Toxicol. 2020; 60: 133-154Crossref PubMed Scopus (126) Google Scholar, 2Zamponi G.W. Striessnig J. Koschak A. Dolphin A.C. The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential.Pharmacol. Rev. 2015; 67: 821-870Crossref PubMed Scopus (704) Google Scholar, 3Tikhonov D.B. Zhorov B.S. P-loop channels: experimental structures, and physics-based and neural networks-based models.Membranes (Basel). 2022; 12: 229Crossref PubMed Scopus (2) Google Scholar). The pore-forming α-subunit of these ion channels folds from a single polypeptide chain of four homologous repeat domains DI, DII, DIII, and DIV. Each domain contains a voltage-sensing module (VSM) and contributes a quarter to the pore module (PM). A VSM has four transmembrane helices (S1-S4) connected by extracellular loops S1-S2 (L1), S3-S4 (L3), and intracellular loop S2-S3 (L2). The PM contains four transmembrane outer helices (S5), which are connected to four VSMs by linker-helices S4-S5 (L4), and four transmembrane inner helices (S6) whose cytoplasmic halves line the ion permeation pathway (the inner pore) and contribute to the bundle-crossing that occludes the ion channel pore in the closed state. Four membrane-reentering extracellular P-loops between helices S5 and S6 contain membrane-descending helices P1 linked to the S5 helix by loop S5-P1 (L5, also called turret) and membrane-ascending helix linked to helix S6 by loop P2-S6 (L6). Side chains of residues in short segments between the P1 and P2 helices contribute to the selectivity filter that along with other residues in the P-loops line the outer pore, which is open to the extracellular space. The distinct classes of calcium channels and sodium channels are defined by their selective passage of calcium and sodium ions (4Hille B. Associates S. Ion Channels of Excitable Membranes. 3rd Ed. Sinauer Associates, Sunderland, MA2001Google Scholar) through the selectivity filters. In Nav channels, the selectivity filter is composed of residues from the four domains: aspartate, glutamate, lysine, and alanine, i.e., the DEKA ring (5Schlief T. Schonherr R. Imoto K. Heinemann S.H. Pore properties of rat brain II sodium channels mutated in the selectivity filter domain.Eur. Biophys. J. 1996; 25: 75-91Crossref PubMed Scopus (101) Google Scholar). In CaV channels, the selectivity-filter ring has five acidic residues, e.g., EE(D)EE or EE(D)DD (6Tang S. Mikala G. Bahinski A. Yatani A. Varadi G. Schwartz A. Molecular localization of ion selectivity sites within the pore of a human L-type cardiac calcium channel.J. Biol. Chem. 1993; 268: 13026-13029Abstract Full Text PDF PubMed Google Scholar). Many animal groups such as nematodes, parasitic platyhelminth (e.g., schistosomes), hemichordates, and echinoderms completely lack voltage-gated NaV2 or NaV1 channel genes in their genomes (7Fux J.E. Mehta A. Moffat J. Spafford J.D. Eukaryotic voltage-gated sodium channels: on their origins, asymmetries, losses, diversification and adaptations.Front. Physiol. 2018; 9: 1406Crossref PubMed Scopus (20) Google Scholar). It is well established that nematodes generate cardiac-like action potentials in pharyngeal muscles that require a voltage-dependent Na+ current, but the gene responsible for this Na+ current has eluded discovery (8Vinogradova I. Cook A. Holden-Dye L. The ionic dependence of voltage-activated inward currents in the pharyngeal muscle of Caenorhabditis elegans.Invert. Neurosci. 2006; 6: 57-68Crossref PubMed Scopus (11) Google Scholar, 9Franks C.J. Pemberton D. Vinogradova I. Cook A. Walker R.J. Holden-Dye L. Ionic basis of the resting membrane potential and action potential in the pharyngeal muscle of Caenorhabditis elegans.J. Neurophysiol. 2002; 87: 954-961Crossref PubMed Scopus (42) Google Scholar). Similarly, the giant pond snail, Lymnaea stagnalis, possesses cardiac action potentials requiring Na+ and Ca2+, but its only NaV1 sodium channel gene in the nervous system does not express in the cardiovascular system (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). We have demonstrated that the source of the Na+ current in the snail heart is a low voltage-activated Cav3 T-type channel that can be blocked by nickel or drugs, e.g., mibefradil (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The T-type currents in cardiomyocytes possess characteristic biophysical properties that enable their participation in rhythm generation to serve a pacemaking role for the molluscan heart (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Measurement by quantitative PCR reveals that the only isoform of the CaV3 T-type channel that is expressed in the snail heart has an alternatively spliced isoform of the LCav3 T-type channel gene containing exon 12a (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). The high Na+ permeation of T-type currents from the snail heart can be achieved in vitro by expressing the snail LCav3 channel harboring exon 12a. Exon 12b, which does not express in the snail heart, but predominates in skeletal muscle tissue, exhibits the typical phenotype of a mostly Ca2+ currents in the LCav3 T-type channel (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). We demonstrate that the human calcium-selective Cav3.2 channel can be converted into channel with a preference for passage of Na+ over Ca2+, in chimeras, which include exon 12a from the snail LCaV3 channel (11Guan W. Stephens R.F. Mourad O. Mehta A. Fux J. Spafford J.D. Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions.Sci. Rep. 2020; 10: 12404Crossref PubMed Scopus (3) Google Scholar). Exons 12a and 12b code for the L5 extracellular turret in domain II (IIS5-P1) of the Cav3 T-type channel, terminating just upstream of the selectivity filter residues (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 11Guan W. Stephens R.F. Mourad O. Mehta A. Fux J. Spafford J.D. Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions.Sci. Rep. 2020; 10: 12404Crossref PubMed Scopus (3) Google Scholar). What has eluded discovery is an atomic model to explain how a typically fast, Ca2+-selective T-type channel converts into a kinetically slow ion channel with a preference for passage of Na+ over Ca2+ without alterations to the selectivity filter or in any Voltage-Sensor Domain (VSD). Here, we employ multiple sequence alignments, 3D molecular modeling with the AlphaFold2 neural network software, and mutational analysis of expressed Cav3 channels to show that what varies is the presence of a single lysine residue within exon 12a, but not exon 12b to neutralize a critical aspartate residue in the ED motif of Domain II contributing to calcium selectivity. Appearance of short, alternative extracellular loops evolved to infiltrate and adjust the pore's ion permeability, provides an ingenious and economical way for nematodes and mollusks to generate multiple phenotypes resembling various CaV and NaV channels within their only T-type channel in the absence of expression of differing CaV or NaV channel isoforms. Cryo-EM structures reveal a common, asymmetrical pattern of extracellular loops forming a canopy that partially encloses the selectivity filters below, contributed by the four homologous domains of eukaryotic NaV1 (12Pan X. Li Z. Zhou Q. Shen H. Wu K. Huang X. et al.Structure of the human voltage-gated sodium channel Nav1.4 in complex with beta1.Science. 2018; 362: eaau2486Crossref PubMed Scopus (239) Google Scholar), CaV1 (13Wu J. Yan Z. Li Z. Qian X. Lu S. Dong M. et al.Structure of the voltage-gated calcium channel Ca(v)1.1 at 3.6 A resolution.Nature. 2016; 537: 191-196Crossref PubMed Scopus (337) Google Scholar), CaV2 (14Gao S. Yao X. Yan N. Structure of human Cav2.2 channel blocked by the painkiller ziconotide.Nature. 2021; 596: 143-147Crossref PubMed Scopus (64) Google Scholar) and CaV3 (15Zhao Y. Huang G. Wu Q. Wu K. Li R. Lei J. et al.Cryo-EM structures of apo and antagonist-bound human Cav3.1.Nature. 2019; 576: 492-497Crossref PubMed Scopus (85) Google Scholar) channels. The structural conservation of extracellular loops extends outside the animals to the basal, single-cell choanoflagellates, such as Salpingoeca rosetta, which possess animal homologs, SroCaV1, SroCav3, and SroNav2, as illustrated in the multiple alignment in Fig. 1. The four pore domains within different CaV and NaV channels in eukaryotes (Fig. 1A) significantly vary from those in bacteria (Fig. 1B) by the presence of long L5 extracellular loops (light green, Fig. 1). The longest of the L5 extracellular loops in eukaryotes is within Domain I (L5I) followed by Domain III (L5III), and these are stabilized by a conserved set of two and one disulfide bonds, respectively, in known eukaryotic NaV and CaV channel homologs (black color, Fig. 1A). The longest of the L6 extracellular loops in eukaryotes is in Domain IV (L6IV), which also is stabilized by a disulfide bond common within eukaryotic CaV and NaV channels (Fig. 1A). Bacterial homologs do not possess extensive L5 extracellular loops like the animals but have a similar disulfide bond (black color, Fig. 1) in the L6 extracellular loop of extended length in some (16Shimomura T. Yonekawa Y. Nagura H. Tateyama M. Fujiyoshi Y. Irie K. A native prokaryotic voltage-dependent calcium channel with a novel selectivity filter sequence.ELife. 2020; 9: e52828Crossref PubMed Scopus (19) Google Scholar) but lacking in other (17Payandeh J. Scheuer T. Zheng N. Catterall W.A. The crystal structure of a voltage-gated sodium channel.Nature. 2011; 475: 353-358Crossref PubMed Scopus (1129) Google Scholar, 18Zhang X. Ren W. DeCaen P. Yan C. Tao X. Tang L. et al.Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel.Nature. 2012; 486: 130-134Crossref PubMed Scopus (3) Google Scholar) bacterial homologs (Fig. 1B). Known CaV3 channels contain two to six additional disulfide bonds (dark orange color, Fig. 1A) above the core of four disulfide bonds (black color, Fig. 1A) in extracellular loops shared with other eukaryotic CaV and NaV channels. These unique "Cav3 only" disulfide bonds reside, in all but one case, in the shortest of the extracellular loops of Domain II (L5II) and Domain IV (L5IV and L6IV), which lie across from each other in the pore domain, and at a lower profile, closer to the membrane and pore selectivity filter than the larger L5I and L5III extracellular loops (Fig. 1A). Cryo-EM structures of human Cav3.1 (15Zhao Y. Huang G. Wu Q. Wu K. Li R. Lei J. et al.Cryo-EM structures of apo and antagonist-bound human Cav3.1.Nature. 2019; 576: 492-497Crossref PubMed Scopus (85) Google Scholar) and Cav3.3 (19He L. Yu Z. Geng Z. Huang Z. Zhang C. Dong Y. et al.Structure, gating, and pharmacology of human CaV3.3 channel.Nat. Commun. 2022; 13: 2084Crossref PubMed Scopus (13) Google Scholar) channels reveal one of these unique cysteines in the short L5II extracellular loop to form a disulfide bond with a cysteine (yellow color, Fig. 1A) in the extracellular loop S1-S2 of VSM-I (dark yellow color, Fig. 1A). This unique tethering of the voltage-sensor and PMs likely contributes to the reported redox sensitivity and variability in ion channel kinetics of CaV3 T-type channels (20Todorovic S.M. Jevtovic-Todorovic V. Meyenburg A. Mennerick S. Perez-Reyes E. Romano C. et al.Redox modulation of T-type calcium channels in rat peripheral nociceptors.Neuron. 2001; 31: 75-85Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 21Karmazinova M. Beyl S. Stary-Weinzinger A. Suwattanasophon C. Klugbauer N. Hering S. et al.Cysteines in the loop between IS5 and the pore helix of Ca(V)3.1 are essential for channel gating.Pflugers.Arch. 2010; 460: 1015-1028Crossref PubMed Scopus (14) Google Scholar). Exons 12a and exons 12b specifically vary in one residue within all molluscan (e.g., L. stagnalis) (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) and nematode (e.g., Caenorhabditis elegans) (22Steger K.A. Shtonda B.B. Thacker C. Snutch T.P. Avery L. The C. elegans T-type calcium channel CCA-1 boosts neuromuscular transmission.J. Exp. Biol. 2005; 208: 2191-2203Crossref PubMed Scopus (55) Google Scholar) Cav3 channels. This is a lysine residue within exon 12a (K1067, Genbank Acc. # JX292155 and K819, Genbank Acc. # AAP84337, respectively) compared to a neutral alanine or methionine residue at a similar position within exon 12b (A1078, Genbank Acc. # AAO83843 or M826, Genbank Acc. # AAP79881, respectively). Figure 2 shows models of calcium-selective and sodium-permeable variants of Cav3 channel from L. stagnalis with exons 12a and 12b, respectively. Both exons are located in the extracellular loop L5II that descends from the extracellular space to the groove between helices IIP1 and IIP2. In the channel with exon 12a, the long side chain of the lysine residue at the apex of loop L5II is salt bridged to the second-domain aspartate of the selectivity-filter ring EE(D)DD and neutralizes the negative charge at this aspartate, a fingerprint residue of eukaryotic calcium channels. In the channel with exon 12b, loop L5II has a similar folding, but alanine, which is located at position of lysine in exon 12a (Fig. 2), is far from the second-domain aspartate and does not affect the negative charge at the aspartate. AlphaFold2 models of nematode Cav3 channels like C. elegans with exons 12a and 12b predicted folding that overlaps with that of the molluscan LCav3 channels. In the CaV3 channels from both phylogenetic groups, a lysine from exon 12a is salt-bridged to the second-domain aspartate in the selectivity filter. In the Cav3 channel from C. elegans, a methionine in exon 12b is engaged in a hydrophobic contact with valine one turn of helix IIP2 downstream from the aspartate and does not neutralize its negative charge. Despite differing sequences and number of intraturret cysteine bridges, the AlphaFold2 neural network predicted identical positioning of the neutral amino acid in exon 12b and the charged lysine in exon 12a whose ammonium group is ∼ 2.6 Å from a carboxylate oxygen in the aspartate residue found in similar position of the outer pore in cryo-EM structures of calcium channels CaV1 (13Wu J. Yan Z. Li Z. Qian X. Lu S. Dong M. et al.Structure of the voltage-gated calcium channel Ca(v)1.1 at 3.6 A resolution.Nature. 2016; 537: 191-196Crossref PubMed Scopus (337) Google Scholar), CaV2 (14Gao S. Yao X. Yan N. Structure of human Cav2.2 channel blocked by the painkiller ziconotide.Nature. 2021; 596: 143-147Crossref PubMed Scopus (64) Google Scholar) and CaV3 (15Zhao Y. Huang G. Wu Q. Wu K. Li R. Lei J. et al.Cryo-EM structures of apo and antagonist-bound human Cav3.1.Nature. 2019; 576: 492-497Crossref PubMed Scopus (85) Google Scholar). Thus, the AlphaFold2 models suggest that it is the salt-bridge between the lysine in exon 12a and the second-domain aspartate in the selectivity filter ring EE(D)DD that renders sodium permeability to T-type channel isoforms with exon 12a. Protostome invertebrates from flatworms, nematodes, arthropods, and mollusks to annelids contain alternative L5II loops encoded by exons 12a or 12b (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 11Guan W. Stephens R.F. Mourad O. Mehta A. Fux J. Spafford J.D. Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions.Sci. Rep. 2020; 10: 12404Crossref PubMed Scopus (3) Google Scholar). In the AlphaFold2 models, exon 12a in molluscan (e.g., L. stagnalis) and nematode (e.g., C. elegans) CaV3 channels possess a similarly placed cysteine disulfide-bonded with a cysteine in the S1-S2 extracellular loop of VSM-I like in mammalian CaV3 channels (15Zhao Y. Huang G. Wu Q. Wu K. Li R. Lei J. et al.Cryo-EM structures of apo and antagonist-bound human Cav3.1.Nature. 2019; 576: 492-497Crossref PubMed Scopus (85) Google Scholar) (Fig. 3A). The disulfide bonding of VSM-I to exon 12b is contained within a pair of intraturret cysteine bridges in most protostome invertebrates such as mollusks (e.g., L. stagnalis) rather than outside the intraturret cysteine bridge of exon 12a. The exception is in nematode Cav3 channels (e.g., C. elegans) in which the cysteine disulfide-bonded to VSM-I is in the same position for exon 12a and exon 12b outside the one or two pairs of intra-L5II loop cysteine bridges, respectively (10Senatore A. Guan W. Boone A.N. Spafford J.D. T-type channels become highly permeable to sodium ions using an alternative extracellular turret region (S5-P) outside the selectivity filter.J. Biol. Chem. 2014; 289: 11952-11969Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 11Guan W. Stephens R.F. Mourad O. Mehta A. Fux J. Spafford J.D. Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in CaV3 T-type channels alter the passage and block of monovalent and divalent ions.Sci. Rep. 2020; 10: 12404Crossref PubMed Scopus (3) Google Scholar) (Fig. 3). We previously reported that all measured kinetic features (activation, inactivation, deactivation, and recovery from inactivation) to a significant degree are slowed in molluscan LCaV3 channels with exon 12a compared to the faster kinetics of the channels with exon 12b, in which the cysteine bridging to the VSM-I is localized within the double set of intraturret cysteines. To confirm the proposed role of lysine residue in rendering sodium permeability to the channels with exon 12a, we generated and expressed in the human embryonic kidney (HEK)-293T cells mutant channels LCaV3-12a_K1067A and LCaV3-12b_A1078K in which the lysine residue of exon 12a and alanine residue in exon 12b were swapped. The relative sodium and calcium permeation through LCaV3 channels was estimated as the relative fold increase in peak current amplitude when external Na+ ion replaced weakly permeant cation, NMDG+ (N-methyl-D-glucamine) in the presence of external Ca2+. Representative peak current traces (Fig. 4A) and current-voltage relationships (Fig. 4B) illustrate a significant fold decrease in peak current size in presence of external Na+ ions when lysine is replaced by alanine in the LCav3-12a_K1067A mutant, (2.24 ± 0.10, n = 9) compared to wildtype LCaV3-12a (15.62 ± 0.57, n = 21). LCav3-12a_K1067A mutant did not significantly vary from the native LCaV3-12b (2.32 ± 0.03, n = 26), suggesting that the only structural feature that is responsible for the changed Na+ to Ca2+ ion preference between exon 12a and exon 12b splice isoforms is the loss of the lysine residue in the L5II loop. And similarly, exchanging the lysine in position of the alanine within the exon 12b in the LCaV3-12b_A1089K mutant yielded sodium permeant T-type channels (13.49 ± 1.14, n = 14). The latter had a fold increase in peak current amplitude in the presence of external Na+ that was dramatically different from the wildtype LCaV3-12b channel (2.32 ± 0.054, n = 22) but not significantly different from native LCaV3-12a (15.66 ± 0.43, n = 21). A means to estimate an ion channel permeability to divalent ions (Ca2+), compared to monovalent ions (X+), PCa/PX, relies on a measurement of the reversal potential in bi-ionic recording conditions (23Fatt P. Ginsborg B.L. The ionic requirements for the production of action potentials in crustacean muscle fibres.J. Physiol. 1958; 142: 516-543Crossref PubMed Scopus (313) Google Scholar). High Ca2+ in external solutions (4 mM) and a high concentration (100 mM) of monovalent ions (Li+, Na+, K+, Cs+) in internal solutions generate a reversal potential elicited from a series of current-generating voltage steps that is considered to reflect the relative permeability of Ca2+ influx normalized to the permeability for the monovalent ion efflux (23Fatt P. Ginsborg B.L. The ionic requirements for the production of action potentials in crustacean muscle fibres.J. Physiol. 1958; 142: 516-543Crossref PubMed Scopus (313) Google Scholar). Notably, Cs+ was observed as a weakly permeable ion through all Cav3 channels (illustrated by the predominance of inward Ca2+ current elicited by voltage steps near the reversal potential in the bottom row of current traces in Fig. 5). Human Cav3.1 channels are not very permeable to any of the monovalent ions tested above (illustrated by the predominance of inward Ca2+ currents elicited in voltage steps generated near the reversal potential in the column of current traces on the right side in Fig. 5). In contrast, monovalent ions (Li+, Na+, K+) dramatically influence the reversal potential of molluscan LCav3 channels with the lysine residue in the L5II extracellular loop (LCav3-12a and LCav3-12b_A1089K). These channels demonstrate statistically significantly higher monovalent ion permeability for Li+, Na+, and K+ than the LCav3 channels with alanine in similar position of the L5II loop (LCav3-12b and LCav3-12a_K1067A). As illustrated in Figure 4, the markedly higher monovalent ion permeability of LCav3 channels with lysine in the L5II loop (LCav3-12a and LCav3-12b_A1089K) is reflected in the large outward monovalent ion conductances observed for Li+, Na+, and K+ predominating over inward Ca2+ conductances elicited in voltage steps generated near the reversal potential. The higher monovalent ion permeabilities of LCav3-12a and LCav3-12b A1089K channels are also reflected in the observed steeper slope of the outward whole cell conductances of Li+, Na+, and K+ ions above the reversal potential (see full-scale current voltage relationships, Fig. 5), and the statistically significant leftward, hyperpolarizing shift in reversal potential (close-up view near the reversal potential, Fig. 5). The position of reversal potentials (sampled in representative current traces, Figure 4, extrapolated from current-voltage relationships Figure 5, and summarized in bar graph form, Fig. 6 inset) are considered a reflection of the relative contribution of inward divalent cation conductance (Ca2+) compared to the outward monovalent ion conductances (Li+/Na+/K+/Cs+) and used to calculate the relative permeabilities of inward Ca2+ flux to monovalent ion (X+) flux (PCa/PX) (illustrated as bar graph ± s.e.m. overlaid with scatter plot of replicates, Fig. 6) according to a bi-ionic equation provided by Fatt and Ginsborg (1958) (23Fatt P. Ginsborg B.L. The ionic requirements for the production of action potentials in crustacean muscle fibres.J. Physiol. 1958; 142: 516-543Crossref PubMed Scopus (313) Google Scholar). The single lysine residue in the extracellular loop of wildtype LCav3-12a and LCav3-12b A1089K mutant compared to the alanine residue in similar position of wildtype LCav3-12b and LCav3-12a K1067A mutant generate statistically significant increases in the relative permeabilities for monovalent ions, Li+, Na+, and K+, over the relative Ca2+ permeability. Reversal potentials and calculated relative permeability ratios (PCa2+/PX+) are also provided for CaV3.1 and CaV3.2 channels in Figure 6, illustrating the much lower relative permeabilities measured for monovalent ions, especially Li+/Na+/K+ in the highly calcium-selective, human homologs than their invertebrate counterparts, measured using the bi-ionic reversal potential method. Our experiments confirm the molecular-modeling pre
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