scholarly journals Divalent cation and chloride ion sites of chicken acid sensing ion channel 1a elucidated by x-ray crystallography

2018 ◽  
Author(s):  
Nate Yoder ◽  
Eric Gouaux
Keyword(s):  
Ion Channels ◽  
Divalent Cation ◽  
Binding Sites ◽  
Divalent Cations ◽  
Cation Binding ◽  
Ion Binding ◽  
X Ray ◽  
High Ph ◽  
X Ray Crystallography ◽  
Ion Binding Sites

AbstractAcid sensing ion channels (ASICs) are proton-gated ion channels that are members of the degenerin/epithelial sodium channel superfamily and are expressed throughout central and peripheral nervous systems. ASICs have been implicated in multiple physiological processes and are subject to numerous forms of endogenous and exogenous regulation that include modulation by Ca2+ and Cl− ions. However, the mapping of ion binding sites as well as a structure-based understanding of the mechanisms underlying ionic modulation of ASICs have remained elusive. Here we present ion binding sites of chicken ASIC1a in resting and desensitized states at high and low pH, respectively, determined by anomalous diffraction x-ray crystallography. The acidic pocket serves as a nexus for divalent cation binding at both low and high pH, while we observe divalent cation binding within the central vestibule on the resting channel at high pH only. Moreover, neutralization of residues positioned to coordinate divalent cations via individual and combined Glu to Gln substitutions reduced, but did not extinguish, modulation of proton-dependent gating by Ca2+. Additionally, we demonstrate that anion binding at the canonical thumb domain site is state-dependent and present a previously undetected anion site at the mouth of the extracellular fenestrations on the resting channel. Our results map anion and cation sites on ASICs across multiple functional states, informing possible mechanisms of modulation and providing a blueprint for the design of therapeutics targeting ASICs.

The Effect Of Metal Ions On Human Factor IX Antigenicity

1981 ◽  
Author(s):  
R M Lewis ◽  
H M Reisner ◽  
B C Abels ◽  
H R Roberts
Keyword(s):  
Divalent Cation ◽  
Binding Sites ◽  
Human Factor ◽  
Antibody Binding ◽  
Factor Ix ◽  
Ion Binding ◽  
Human Factor Ix ◽  
Excess Mg ◽  
Ion Binding Sites ◽  
Two Populations

Affinity chromatography of an inhibitor to human factor IX (F.IX) separated the antibody into two populations. The ion dependent population of antibodies had an absolute divalent cation (Me++) binding requirement. The non-ion dependent population bound F.IX equally in the presence or absence of Me++. The concentration of Me++ required for ½ the maximum ion dependent antibody binding (½ max) was (in nM) Ca++ 0.40, Mn++ 0.05, Sr++ 0.70 and Mg++ 0.65.Ca++ potentiated the binding of antibody in the presence of excess Mg++. In addition, the ½ max for Ca++ was reduced about four fold. These observations are consistent with separate binding sites on the F.IX molecule for Ca++ and Mg++ and potentiation of Ca++ binding by Mg++. Scat- chard analysis of ion dependent antibody binding indicates about a 10 fold greater affinity of antibody in the presence of Ca++ than Mg++. In the presence of both cations, affinity was at least as high as in the presence of Ca++ alone supporting the presence of separate ion binding sites on the F.IX molecule.

Location of ion-binding sites in the gramicidin channel by X-ray diffraction

1991 ◽  
Vol 218 (4) ◽  
pp. 847-858 ◽  
Author(s):  
Glenn A. Olah ◽  
Huey W. Huang ◽  
Wenhan Liu ◽  
Yili Wu
Keyword(s):  
Binding Sites ◽  
Ion Binding ◽  
X Ray Diffraction ◽  
X Ray ◽  
Gramicidin Channel ◽  
Ion Binding Sites

scholarly journals Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction.

1993 ◽  
Vol 101 (2) ◽  
pp. 153-182 ◽  
Author(s):  
D D Doyle ◽  
Y Guo ◽  
S L Lustig ◽  
J Satin ◽  
R B Rogart ◽  
...  
Keyword(s):  
Divalent Cation ◽  
Binding Sites ◽  
Structural Model ◽  
Divalent Cations ◽  
Ph Dependence ◽  
Monovalent Cation ◽  
Cation Binding ◽  
Na Channel ◽  
Na Channels ◽  
Ph Titration

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Accessibility of Cations to the Selectivity Filter of KcsA in the Inactivated State: An Equilibrium Binding Study

2019 ◽  
Vol 20 (3) ◽  
pp. 689 ◽  
Author(s):  
Ana Giudici ◽  
Maria Renart ◽  
Clara Díaz-García ◽  
Andrés Morales ◽  
José Poveda ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Binding Sites ◽  
Cation Binding ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Channel State ◽  
Equilibrium Conditions ◽  
Equilibrium Binding ◽  
Potassium Channel Kcsa ◽  
Ion Binding Sites

Cation binding under equilibrium conditions has been used as a tool to explore the accessibility of permeant and nonpermeant cations to the selectivity filter in three different inactivated models of the potassium channel KcsA. The results show that the stack of ion binding sites (S1 to S4) in the inactivated filter models remain accessible to cations as they are in the resting channel state. The inactivated state of the selectivity filter is therefore “resting-like” under such equilibrium conditions. Nonetheless, quantitative differences in the apparent KD’s of the binding processes reveal that the affinity for the binding of permeant cations to the inactivated channel models, mainly K+, decreases considerably with respect to the resting channel. This is likely to cause a loss of K+ from the inactivated filter and consequently, to promote nonconductive conformations. The most affected site by the affinity loss seems to be S4, which is interesting because S4 is the first site to accommodate K+ coming from the channel vestibule when K+ exits the cell. Moreover, binding of the nonpermeant species, Na+, is not substantially affected by inactivation, meaning that the inactivated channels are also less selective for permeant versus nonpermeant cations under equilibrium conditions.

scholarly journals Mutation of putative divalent cation sites in the alpha 4 subunit of the integrin VLA-4: distinct effects on adhesion to CS1/fibronectin, VCAM-1, and invasin.

1993 ◽  
Vol 123 (1) ◽  
pp. 245-253 ◽  
Author(s):  
A Masumoto ◽  
M E Hemler
Keyword(s):  
Cell Adhesion ◽  
Divalent Cation ◽  
Binding Sites ◽  
Divalent Cations ◽  
K562 Cells ◽  
Cation Binding ◽  
Bacterial Protein ◽  
Wild Type ◽  
Vascular Cell Adhesion ◽  
Cation Sites

To investigate the functional significance of putative integrin divalent cation binding sites, several mutated alpha 4 subunit cDNAs were constructed. Mutants contained the conservative substitution of Glu for Asp or Asn at the third position in each of three putative divalent cation sites. Transfection of wild-type or mutated alpha 4 into K562 cells yielded comparable expression levels and immunoprecipitation profiles. However, for all three alpha 4 mutants, adhesion to CS1/fibronectin was greatly diminished in either the presence or absence of the stimulatory anti-beta 1 mAb TS2/16. Constitutive adhesion to vascular cell adhesion molecule (VCAM) 1 was also diminished but, unlike CS1 adhesion, was restored upon TS2/16 stimulation. In contrast, adhesion to the bacterial protein invasin was minimally affected by any of the three mutations. For each of the mutants, the order of preference for divalent cations was unchanged compared to wild-type alpha 4, on CS1/fibronectin (Mn2+ > Mg2+ > Ca2+), on VCAM-1 (Mn2+ > Mg2+ = Ca2+) and on invasin (Mg2+ = Ca2+). However for the three mutants, the efficiency of divalent cation utilization was decreased. On VCAM-1, 68-108 microM Mn2+ was required to support half-maximal adhesion for the mutants compared with 14-18 microM for wild-type alpha 4. These results indicate (a) that three different ligands for VLA-4 show widely differing sensitivities to mutations within putative divalent cation sites, and (b) each of the three putative divalent cation sites in alpha 4 have comparable functional importance with respect to both divalent cation usage and cell adhesion.

scholarly journals Divalent Cation Sensitivity of BK Channel Activation Supports the Existence of Three Distinct Binding Sites

2005 ◽  
Vol 125 (3) ◽  
pp. 273-286 ◽  
Author(s):  
Xu-Hui Zeng ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Divalent Cation ◽  
Binding Sites ◽  
Divalent Cations ◽  
Bk Channels ◽  
Cation Binding ◽  
Regulatory Mechanisms ◽  
Α Subunit ◽  
Physiological Regulation ◽  
Cation Selectivity ◽  
High Affinity

Mutational analyses have suggested that BK channels are regulated by three distinct divalent cation-dependent regulatory mechanisms arising from the cytosolic COOH terminus of the pore-forming α subunit. Two mechanisms account for physiological regulation of BK channels by μM Ca2+. The third may mediate physiological regulation by mM Mg2+. Mutation of five aspartate residues (5D5N) within the so-called Ca2+ bowl removes a portion of a higher affinity Ca2+ dependence, while mutation of D362A/D367A in the first RCK domain also removes some higher affinity Ca2+ dependence. Together, 5D5N and D362A/D367A remove all effects of Ca2+ up through 1 mM while E399A removes a portion of low affinity regulation by Ca2+/Mg2+. If each proposed regulatory effect involves a distinct divalent cation binding site, the divalent cation selectivity of the actual site that defines each mechanism might differ. By examination of the ability of various divalent cations to activate currents in constructs with mutationally altered regulatory mechanisms, here we show that each putative regulatory mechanism exhibits a unique sensitivity to divalent cations. Regulation mediated by the Ca2+ bowl can be activated by Ca2+ and Sr2+, while regulation defined by D362/D367 can be activated by Ca2+, Sr2+, and Cd2+. Mn2+, Co2+, and Ni2+ produce little observable effect through the high affinity regulatory mechanisms, while all six divalent cations enhance activation through the low affinity mechanism defined by residue E399. Furthermore, each type of mutation affects kinetic properties of BK channels in distinct ways. The Ca2+ bowl mainly accelerates activation of BK channels at low [Ca2+], while the D362/D367-related high affinity site influences both activation and deactivation over the range of 10–300 μM Ca2+. The major kinetic effect of the E399-related low affinity mechanism is to slow deactivation at mM Mg2+ or Ca2+. The results support the view that three distinct divalent-cation binding sites mediate regulation of BK channels.

scholarly journals X-ray crystallography reveals molecular recognition mechanism for sugar binding in a melibiose transporter MelB

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Lan Guan ◽  
Parameswaran Hariharan
Keyword(s):  
Molecular Recognition ◽  
Binding Pocket ◽  
Cation Binding ◽  
Recognition Mechanism ◽  
X Ray ◽  
X Ray Crystallography ◽  
Sugar Binding ◽  
Sugar Specificity ◽  
Major Facilitator ◽  
Mfs Transporters

AbstractMajor facilitator superfamily_2 transporters are widely found from bacteria to mammals. The melibiose transporter MelB, which catalyzes melibiose symport with either Na+, Li+, or H+, is a prototype of the Na+-coupled MFS transporters, but its sugar recognition mechanism has been a long-unsolved puzzle. Two high-resolution X-ray crystal structures of a Salmonella typhimurium MelB mutant with a bound ligand, either nitrophenyl-α-d-galactoside or dodecyl-β-d-melibioside, were refined to a resolution of 3.05 or 3.15 Å, respectively. In the substrate-binding site, the interaction of both galactosyl moieties on the two ligands with MelBSt are virturally same, so the sugar specificity determinant pocket can be recognized, and hence the molecular recognition mechanism for sugar binding in MelB has been deciphered. The conserved cation-binding pocket is also proposed, which directly connects to the sugar specificity pocket. These key structural findings have laid a solid foundation for our understanding of the cooperative binding and symport mechanisms in Na+-coupled MFS transporters, including eukaryotic transporters such as MFSD2A.

scholarly journals Synthesis and Photoregulated Metal Coordination of Azobenzene Polymer Having Ion Binding Sites

1991 ◽  
Vol 23 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Masato Nanasawa ◽  
Takahiro Nishiyama ◽  
Hiroyoshi Kamogawa
Keyword(s):  
Binding Sites ◽  
Metal Coordination ◽  
Ion Binding ◽  
Azobenzene Polymer ◽  
Ion Binding Sites
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