Effects of divalent cations on single-channel conduction properties of XenopusIP3 receptor

1998 ◽  
Vol 275 (1) ◽  
pp. C179-C188 ◽  
Author(s):  
Don-On Daniel Mak ◽  
J. Kevin Foskett
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Channel Current ◽  
Current Voltage ◽  
Conduction Pathway ◽  
Wide Range ◽  
Trisphosphate Receptor ◽  
Channel Properties ◽  
Current Blocking ◽  
Conduction Properties

The effects of Mg2+ and Ba2+ on single-channel properties of the inositol 1,4,5-trisphosphate receptor (IP3R) were studied by patch clamp of isolated nuclei from Xenopusoocytes. In 140 mM K+ the IP3R channel kinetics and presence of conductance substates were similar over a range (0–9.5 mM) of free Mg2+. In 0 mM Mg2+ the channel current-voltage ( I-V) relation was linear with conductance of ∼320 pS. Conductance varied slowly and continuously over a wide range (SD ≈ 60 pS) and sometimes fluctuated during single openings. The presence of Mg2+ on either or both sides of the channel reduced the current (blocking constant ∼0.6 mM in symmetrical Mg2+), as well as the range of conductances observed, and made the I-V relation nonlinear (slope conductance ∼120 pS near 0 mV and ∼360 pS at ±70 mV in symmetrical 2.5 mM Mg2+). Ba2+ exhibited similar effects on channel conductance. Mg2+ and Ba2+ permeated the channel with a ratio of permeability of Ba2+ to Mg2+ to K+ of 3.5:2.6:1. These results indicate that divalent cations induce nonlinearity in the I-V relation and reduce current by a mechanism involving permeation block of the IP3R due to strong binding to site(s) in the conduction pathway. Furthermore, stabilization of conductance by divalent cations reveals a novel interaction between the cations and the IP3R.

Pathways of HERG inactivation

1999 ◽  
Vol 277 (1) ◽  
pp. H199-H210 ◽  
Author(s):  
Johann Kiehn ◽  
Antonio E. Lacerda ◽  
Arthur M. Brown
Keyword(s):  
Xenopus Oocytes ◽  
Single Channel ◽  
Inward Rectification ◽  
Single Channels ◽  
Channel Current ◽  
Cardiac Action ◽  
Current Voltage ◽  
Inwardly Rectifying ◽  
Channel Properties ◽  
Current Voltage Relation

The rapid, repolarizing K+ current in cardiomyocytes ( I Kr) has unique inwardly rectifying properties that contribute importantly to the downstroke of the cardiac action potential. The human ether-à-go-go-related gene ( HERG) expresses a macroscopic current virtually identical to I Kr, but a description of the single-channel properties that cause rectification is lacking. For this reason we measured single-channel and macropatch currents heterologously expressed by HERG in Xenopus oocytes. Our experiments had two main findings. First, the single-channel current-voltage relation showed inward rectification, and conductance was 9.7 pS at −100 mV and 3.9 pS at 100 mV when measured in symmetrical 100 mM K+ solutions. Second, single channels frequently showed no openings during depolarization but nevertheless revealed bursts of openings during repolarization. This type of gating may explain the inward rectification of HERG currents. To test this hypothesis, we used a three-closed state kinetics model and obtained rate constants from fits to macropatch data. Results from the model are consistent with rapid inactivation from closed states as a significant source of HERG rectification.

scholarly journals Modeling Macroscopic Currents of Ion Channels

2021 ◽  
Author(s):  
Di Wu
Keyword(s):  
Open Channel ◽  
Single Channel ◽  
Ion Permeation ◽  
Model Studies ◽  
Proposed Model ◽  
Current Voltage ◽  
Improved Model ◽  
Boltzmann Model ◽  
Channel Properties ◽  
Current Voltage Relation

Ion-channel functions are often studied by the current-voltage relation, which is commonly fitted by the Boltzmann equation, a powerful model widely used nowadays. However, the Boltzmann model is restricted to a two-state ion-permeation process. Here we present an improved model that comprises a flexible number of states and incorporates both the single-channel conductance and the open-channel probability. Employing the channel properties derived from the single-channel recording experiments, the proposed model is able to describe various current-voltage relations, especially the reversal ion-permeation curves showing the inward- and outward-rectifications. We demonstrate the applicability of the proposed model using the published patch-clamp data of BK and MthK potassium channels, and discuss the similarity of the two channels based on the model studies.

scholarly journals How does ryanodine modify ion handling in the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel?

1994 ◽  
Vol 104 (3) ◽  
pp. 425-447 ◽  
Author(s):  
A R Lindsay ◽  
A Tinker ◽  
A J Williams
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Relative Permeability ◽  
Single Channel ◽  
Divalent Cations ◽  
Monovalent Cations ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Release Channel ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Conduction Pathway

Under appropriate conditions, the interaction of the plant alkaloid ryanodine with a single cardiac sarcoplasmic reticulum Ca(2+)-release channel results in a profound modification of both channel gating and conduction. On modification, the channel undergoes a dramatic increase in open probability and a change in single-channel conductance. In this paper we aim to provide a mechanistic framework for the interpretation of the altered conductance seen after ryanodine binding to the channel protein. To do this we have characterized single-channel conductance with representative members of three classes of permeant cation; group 1a monovalent cations, alkaline earth divalent cations, and organic monovalent cations. We have quantified the change in single-channel conductance induced by ryanodine and have expressed this as a fraction of conductance in the absence of ryanodine. Fractional conductance seen in symmetrical 210 mM solutions is not fixed but varies with the nature of the permeant cation. The group 1a monovalent cations (K+, Na+, Cs+, Li+) have values of fractional conductance in a narrow range (0.60-0.66). With divalent cations fractional conductance is considerably lower (Ba2+, 0.22 and Sr2+, 0.28), whereas values of fractional conductance vary considerably with the organic monovalent cations (ammonia 0.66, ethylamine 0.76, propanolamine 0.65, diethanolamine 0.92, diethylamine 1.2). To establish the mechanisms governing these differences, we have monitored the affinity of the conduction pathway for, and the relative permeability of, representative cations in the ryanodine-modified channel. These parameters have been compared with those obtained in previous studies from this laboratory using the channel in the absence of ryanodine and have been modeled by modifying our existing single-ion, four-barrier three-well rate theory model of conduction in the unmodified channel. Our findings indicate that the high affinity, essentially irreversible, interaction of ryanodine with the cardiac sarcoplasmic reticulum Ca(2+)-release channel produces a conformational alteration of the protein which results in modified ion handling. We suggest that, on modification, the affinity of the channel for the group 1a monovalent cations is increased while the relative permeability of this class of cations remains essentially unaltered. The affinity of the conduction pathway for the alkaline earth divalent cations is also increased, however the relative permeability of this class of cations is reduced compared to the unmodified channel. The influence of modification on the handling by the channel of the organic monovalent cations is determined by both the size and the nature of the cation.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Endogenous Protease Activation of ENaC

2005 ◽  
Vol 126 (4) ◽  
pp. 339-352 ◽  
Author(s):  
Adedotun Adebamiro ◽  
Yi Cheng ◽  
John P. Johnson ◽  
Robert J. Bridges
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Open Channels ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Protease Inhibition ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Channel Properties

Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain INa at some minimal threshold value.

scholarly journals Ionic interactions of Ba2+ blockades in the MthK K+ channel

2014 ◽  
Vol 144 (2) ◽  
pp. 193-200 ◽  
Author(s):  
Rui Guo ◽  
Weizhong Zeng ◽  
Hengjun Cui ◽  
Liping Chen ◽  
Sheng Ye
Keyword(s):  
Single Channel ◽  
K Channel ◽  
K Channels ◽  
X Ray ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Single File ◽  
Conduction Pathway ◽  
Internal Side ◽  
Channel Properties

The movement and interaction of multiple ions passing through in single file underlie various fundamental K+ channel properties, from the effective conduction of K+ ions to channel blockade by Ba2+ ions. In this study, we used single-channel electrophysiology and x-ray crystallography to probe the interactions of Ba2+ with permeant ions within the ion conduction pathway of the MthK K+ channel. We found that, as typical of K+ channels, the MthK channel was blocked by Ba2+ at the internal side, and the Ba2+-blocking effect was enhanced by external K+. We also obtained crystal structures of the MthK K+ channel pore in both Ba2+–Na+ and Ba2+–K+ environments. In the Ba2+–Na+ environment, we found that a single Ba2+ ion remained bound in the selectivity filter, preferably at site 2, whereas in the Ba2+–K+ environment, Ba2+ ions were predominantly distributed between sites 3 and 4. These ionic configurations are remarkably consistent with the functional studies and identify a molecular basis for Ba2+ blockade of K+ channels.

scholarly journals Single Channel Properties of P2X2 Purinoceptors

1999 ◽  
Vol 113 (5) ◽  
pp. 695-720 ◽  
Author(s):  
Shinghua Ding ◽  
Frederick Sachs
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
Excess Noise ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Channel Current ◽  
Single Channel Current ◽  
The Mean ◽  
Single Channel Currents ◽  
Channel Properties

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.

scholarly journals The effects of free [Ca2+] on the cytosolic face of the inositol (1,4,5)-trisphosphate receptor at the single channel level

1998 ◽  
Vol 330 (1) ◽  
pp. 559-564 ◽  
Author(s):  
C. Edwin THROWER ◽  
J. A. Edward LEA ◽  
P. Alan DAWSON
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Planar Lipid Bilayers ◽  
Channel Activity ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Inositol 1,4,5 Trisphosphate ◽  
Trisphosphate Receptor ◽  
Artificial Bilayers

Cytosolic free Ca2+ has been shown to have both activating and inhibitory effects upon the inositol (1,4,5) trisphosphate receptor (InsP3R) during intracellular Ca2+ release. The effects of cytosolic free Ca2+ on the InsP3R have already been monitored using cerebellar microsomes (containing InsP3R) incorporated into planar lipid bilayers [Bezprozvanny, Watras and Ehrlich (1991) Nature (London) 351, 751-754]. In these experiments the open probability of the channel exhibited a ‘bell-shaped Ca2+ dependence’. However, this has only been seen when the receptor is in the presence of its native membrane (e.g. microsomal vesicles). Using solubilized, purified InsP3R incorporated into planar lipid bilayers using the ‘tip-dip’ technique, investigations were carried out to see if the same effect was seen in the absence of the native membrane. Channel activity was observed in the presence of 4 μM InsP3 and 200 nM free Ca2+. Mean single channel current was 2.69 pA and more than one population of lifetimes was observed. Two populations had mean open times of approx. 9 and 97 ms. Upon increasing the free [Ca2+] to 2 μM, the mean single channel current decreased slightly to 2.39 pA, and the lifetimes increased to 30 and 230 ms. Elevation of free [Ca2+] to 4 μM resulted in a further decrease in mean single channel current to 1.97 pA as well as a decrease in lifetime to approx. 8 and 194 ms. At 10 μM free [Ca2+] no channel activity was observed. Thus, with purified receptor in artificial bilayers, free [Ca2+] on the cytosolic face of the receptor has major effects on channel behaviour, particularly on channel closure, although inhibition of channel activity is not seen until very high free [Ca2+] is reached.

scholarly journals Block by calcium of ATP-activated channels in pheochromocytoma cells.

1993 ◽  
Vol 101 (3) ◽  
pp. 377-392 ◽  
Author(s):  
K Nakazawa ◽  
P Hess
Keyword(s):  
Patch Clamp ◽  
Single Channel ◽  
Divalent Cations ◽  
Mean Amplitude ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Similar Reduction ◽  
Site Model ◽  
Channel Current ◽  
Voltage Dependent

We have investigated the effects of Ca2+ on Na+ influx through ATP-activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1-21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides.

scholarly journals Inositol Trisphosphate Receptor Ca2+ Release Channels

2007 ◽  
Vol 87 (2) ◽  
pp. 593-658 ◽  
Author(s):  
J. Kevin Foskett ◽  
Carl White ◽  
King-Ho Cheung ◽  
Don-On Daniel Mak
Keyword(s):  
Single Channel ◽  
Cell Types ◽  
Inositol Trisphosphate Receptor ◽  
Channel Regulation ◽  
Physiological Processes ◽  
Quantitative Studies ◽  
Numerous Cell ◽  
Trisphosphate Receptor ◽  
Channel Properties ◽  
Structural Aspects

The inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are a family of Ca2+ release channels localized predominately in the endoplasmic reticulum of all cell types. They function to release Ca2+ into the cytoplasm in response to InsP3 produced by diverse stimuli, generating complex local and global Ca2+ signals that regulate numerous cell physiological processes ranging from gene transcription to secretion to learning and memory. The InsP3R is a calcium-selective cation channel whose gating is regulated not only by InsP3, but by other ligands as well, in particular cytoplasmic Ca2+. Over the last decade, detailed quantitative studies of InsP3R channel function and its regulation by ligands and interacting proteins have provided new insights into a remarkable richness of channel regulation and of the structural aspects that underlie signal transduction and permeation. Here, we focus on these developments and review and synthesize the literature regarding the structure and single-channel properties of the InsP3R.

scholarly journals Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits

2018 ◽  
Vol 150 (10) ◽  
pp. 1373-1385 ◽  
Author(s):  
Michelle Yen ◽  
Richard S. Lewis
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Functional Coupling ◽  
Open Probability ◽  
Channel Activation ◽  
Channel Current ◽  
Crac Channels ◽  
Crac Channel ◽  
Pore Properties

The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca2+ selectivity. Although STIM1 dimers are known to bind directly to the cytosolic C termini of the six Orai1 subunits (SUs) that form the channel hexamer, the dependence of channel activation and selectivity on the number of occupied binding sites is not well understood. Here we address these questions using dimeric and hexameric Orai1 concatemers in which L273D mutations were introduced to inhibit STIM1 binding to specific Orai1 SUs. By measuring FRET between fluorescently labeled STIM1 and Orai1, we find that homomeric L273D mutant channels fail to bind STIM1 appreciably; however, the L273D SU does bind STIM1 and contribute to channel activation when located adjacent to a WT SU. These results suggest that STIM1 dimers can interact with pairs of neighboring Orai1 SUs. Surprisingly, a single L273D mutation within the Orai1 hexamer reduces channel open probability by ∼90%, triples the size of the single-channel current, weakens the Ca2+ binding affinity of the selectivity filter, and lowers the selectivity for Na+ over Cs+ in the absence of divalent cations. These findings reveal a surprisingly strong functional coupling between STIM1 binding and CRAC channel gating and pore properties. We conclude that under physiological conditions, all six Orai1 SUs of the native CRAC channel bind STIM1 to effectively open the pore and generate the signature properties of extremely low conductance and high ion selectivity.

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