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 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 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.

scholarly journals Allosteric Regulation of Bk Channel Gating by Ca2+ and Mg2+ through a Nonselective, Low Affinity Divalent Cation Site

2001 ◽  
Vol 118 (5) ◽  
pp. 607-636 ◽  
Author(s):  
X. Zhang ◽  
C.R. Solaro ◽  
C.J. Lingle
Keyword(s):  
Divalent Cation ◽  
Binding Sites ◽  
Time Course ◽  
Cytosolic Calcium ◽  
Bk Channels ◽  
Bk Channel ◽  
Cation Binding ◽  
Cation Site ◽  
Current Activation ◽  
Activation Rates

The ability of membrane voltage to activate high conductance, calcium-activated (BK-type) K+ channels is enhanced by cytosolic calcium (Ca2+). Activation is sensitive to a range of [Ca2+] that spans over four orders of magnitude. Here, we examine the activation of BK channels resulting from expression of cloned mouse Slo1 α subunits at [Ca2+] and [Mg2+] up to 100 mM. The half-activation voltage (V0.5) is steeply dependent on [Ca2+] in the micromolar range, but shows a tendency towards saturation over the range of 60–300 μM Ca2+. As [Ca2+] is increased to millimolar levels, the V0.5 is strongly shifted again to more negative potentials. When channels are activated by 300 μM Ca2+, further addition of either mM Ca2+ or mM Mg2+ produces similar negative shifts in steady-state activation. Millimolar Mg2+ also produces shifts of similar magnitude in the complete absence of Ca2+. The ability of millimolar concentrations of divalent cations to shift activation is primarily correlated with a slowing of BK current deactivation. At voltages where millimolar elevations in [Ca2+] increase activation rates, addition of 10 mM Mg2+ to 0 Ca2+ produces little effect on activation time course, while markedly slowing deactivation. This suggests that Mg2+ does not participate in Ca2+-dependent steps that influence current activation rate. We conclude that millimolar Mg2+ and Ca2+ concentrations interact with low affinity, relatively nonselective divalent cation binding sites that are distinct from higher affinity, Ca2+-selective binding sites that increase current activation rates. A symmetrical model with four independent higher affinity Ca2+ binding steps, four voltage sensors, and four independent lower affinity Ca2+/Mg2+ binding steps describes well the behavior of G-V curves over a range of Ca2+ and Mg2+. The ability of a broad range of [Ca2+] to produce shifts in activation of Slo1 conductance can, therefore, be accounted for by multiple types of divalent cation binding sites.

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 High Affinity Divalent Cation Binding to Actin

1989 ◽  
Vol 264 (16) ◽  
pp. 9271-9277 ◽  
Author(s):  
L A Selden ◽  
J E Estes ◽  
L C Gershman
Keyword(s):  
Divalent Cation ◽  
Cation Binding ◽  
High Affinity

scholarly journals Identification of the angiotensin II receptor in rat mesenteric artery

1984 ◽  
Vol 223 (3) ◽  
pp. 659-671 ◽  
Author(s):  
J McQueen ◽  
G D Murray ◽  
P F Semple
Keyword(s):  
Angiotensin Ii ◽  
Binding Sites ◽  
Radioligand Binding ◽  
Specific Binding ◽  
Divalent Cations ◽  
Target Tissue ◽  
Angiotensin Ii Receptor ◽  
High Affinity ◽  
Rat Mesentery ◽  
Rat Mesenteric Artery

Specific binding sites of high affinity and low capacity for 125I-angiotensin II have been identified in a membrane fraction derived from arterial arcades of the rat mesentery. Heterogeneity of binding sites and extensive tracer degradation necessitated the use of nonlinear regression methods for the analysis of radioligand binding data. Forward and reverse rate constants for the high affinity sites obtained by three experimental approaches were in good agreement and gave a dissociation equilibrium constant (Kd) of 19-74 pM (95% confidence interval). Affinities for a number of angiotensin-related peptides calculated from competitive binding curves were in the order 125I-angiotensin II = angiotensin II greater than angiotensin III greater than [Sar1,Ile8]angiotensin II greater than [Sar1,Gly8]angiotensin II. Angiotensin I and biochemically unrelated peptides had virtually no effect on binding of tracer angiotensin II. The divalent cations Mn2+, Mg2+ and Ca2+ stimulated 125I-angiotensin II binding at concentrations of 2-10 mM, as did Na+ at 50-100 mM. In the presence of Na+ or Li+, K+ had a biphasic effect. The chelating agents EDTA and EGTA were inhibitory, as were the thiol reagents dithiothreitol and cysteine. This study defined angiotensin II binding sites in a vascular target tissue of sufficiently high affinity to interact rapidly with plasma angiotensin II at physiological concentrations.

Extracellular Calcium And Platelets

1981 ◽  
Author(s):  
P M Taylor ◽  
S Heptinstall
Keyword(s):  
Divalent Cation ◽  
Binding Sites ◽  
Divalent Cations ◽  
Extracellular Calcium ◽  
Time Dependent ◽  
Aggregation Process ◽  
Platelet Surface ◽  
Intracellular Pool ◽  
Continuous Movement

To gain more information on the role of extracellular Ca in platelet behaviour, the movement of 45Ca between plasma and platelets has been studied. Ttoo experimental procedures have been used: platelets were either studied in plasma that contained near-physiological levels of divalent cations or were studied in divalent cation-depleted plasma.There was a continuous movement of Ca from plasma into platelets when the latter were suspended in plasma that contained near-physiological levels of divalent cations. The iptake was linear with time (2.0 to 2.5 ng ion Ca/109 platelets/60 mins) and was faster at 37°C than at 25°C. The amount of Ca taken up by the platelets increased as the extracellular Ca level was increased and was markedly inhibited by Mg. Sr did not affect the uptake. EGTA displaced only a small amount of the Ca that associated with the plater lets which indicated that Ca was taken up into an intracellular pool rather than sinply bound to the platelet surface. The relevance of this movement of Ca into the cells to platelet behaviour has not been established.Studies using platelets suspended in divalent cation- depleted plasma shewed that extracellular Ca was in equilibrium with Ca bound at or near the platelet surface. The binding of Ca was time-dependent but saturable (0.30 to 0.50 ng ion Ca/109 platelets/30 mins), and the majority was readily displaced by EGTA. The amount of Ca bound to the cells increased as the extracellular Ca level was increased but was little affected by an excess of either Mg or Sr. Mare Ca bound to platelets when they were incubated at 25°C than at 37°C. This was because platelets lost their ability to bind Ca when they were incubated at 37°C in divalent cation-depleted plasma. This phenomenon was time-dependent and irreversible and was paralleled by a loss in the ability of the platelets to aggregate. These Ca binding sites would seem to be relevant to the aggregation process.

scholarly journals Chemical properties of the divalent cation binding site on potassium channels.

1992 ◽  
Vol 100 (2) ◽  
pp. 181-193 ◽  
Author(s):  
S Spires ◽  
T Begenisich
Keyword(s):  
Potassium Channels ◽  
Binding Site ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Chemical Properties ◽  
Cation Binding ◽  
Solution Ph ◽  
Electrophysiological Properties ◽  
Cation Binding Site ◽  
Voltage Gated Ion Channels

The actions of divalent cations on voltage-gated ion channels suggest that these cations bind to specific sites and directly influence gating kinetics. We have examined some chemical properties of the external divalent cation binding sites on neuronal potassium channels. Patch clamp techniques were used to measure the electrophysiological properties of these channels and Zn ions were used to probe the divalent cation binding site. The channel activation kinetics were greatly (three- to fourfold) slowed by low (2-5 mM) concentrations of Zn; deactivation kinetics were only slightly affected. These effects of Zn were inhibited by low solution pH in a manner consistent with competition between Zn and H ions for a single site. The apparent inhibitory pK for this site was near 7.2. Treatment of the neurons with specific amino acid reagents implicated amino, but no histidyl or sulfhydryl, residues in divalent cation binding.

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