scholarly journals Electrostatics in the Cytoplasmic Pore Produce Intrinsic Inward Rectification in Kir2.1 Channels

2005 ◽  
Vol 126 (6) ◽  
pp. 551-562 ◽  
Author(s):  
Shih-Hao Yeh ◽  
Hsueh-Kai Chang ◽  
Ru-Chi Shieh
Keyword(s):  
Cell Types ◽  
Inward Rectifier ◽  
Inward Rectification ◽  
Membrane Excitability ◽  
Channel Inhibition ◽  
Charged Residues ◽  
Voltage Dependent ◽  
Inwardly Rectifying ◽  
Kir2.1 Channel ◽  
Positively Charged

Inward rectifier K+ channels are important in regulating membrane excitability in many cell types. The physiological functions of these channels are related to their unique inward rectification, which has been attributed to voltage-dependent block. Here, we show that inward rectification can also be induced by neutral and positively charged residues at site 224 in the internal vestibule of tetrameric Kir2.1 channels. The order of extent of inward rectification is E224K mutant > E224G mutant > wild type in the absence of internal blockers. Mutating the glycines at the equivalent sites to lysines also rendered weak inward rectifier Kir1.1 channels more inwardly rectifying. Also, conjugating positively charged methanethiosulfonate to the cysteines at site 224 induced strong inward rectification, whereas negatively charged methanethiosulfonate alleviated inward rectification in the E224C mutant. These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel. In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel. In this and our previous studies we showed that the M2 bundle crossing and selectivity filter were not involved in the inward rectification induced by spermine interacting with E224 and E299. We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.

scholarly journals Locale and chemistry of spermine binding in the archetypal inward rectifier Kir2.1

2010 ◽  
Vol 135 (5) ◽  
pp. 495-508 ◽  
Author(s):  
Harley T. Kurata ◽  
Emily A. Zhu ◽  
Colin G. Nichols
Keyword(s):  
Binding Site ◽  
Voltage Dependence ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
Before And After ◽  
Voltage Dependent ◽  
Inwardly Rectifying ◽  
Kinetics Of

Polyamine block of inwardly rectifying potassium (Kir) channels underlies their steep voltage dependence observed in vivo. We have examined the potency, voltage dependence, and kinetics of spermine block in dimeric Kir2.1 constructs containing one nonreactive subunit and one cysteine-substituted subunit before and after modification by methanethiosulfonate (MTS) reagents. At position 169C (between the D172 “rectification controller” and the selectivity filter), modification by either 2-aminoethyl MTS (MTSEA) or 2-(trimethylammonium)ethyl MTS (MTSET) reduced the potency and voltage dependence of spermine block, consistent with this position overlapping the spermine binding site. At position 176C (between D172 and the M2 helix bundle crossing), modification by MTSEA also weakened spermine block. In contrast, MTSET modification of 176C dramatically slowed the kinetics of spermine unblock, with almost no effect on potency or voltage dependence. The data are consistent with MTSET modification of 176C introducing a localized barrier in the inner cavity, resulting in slower spermine entry into and exit from a “deep” binding site (likely between the D172 rectification controller and the selectivity filter), but leaving the spermine binding site mostly unaffected. These findings constrain the location of deep spermine binding that underlies steeply voltage-dependent block, and further suggest important chemical details of high affinity binding of spermine in Kir2.1 channels—the archetypal model of strong inward rectification.

Serotonin and forskolin increase an inwardly rectifying potassium conductance in cultured identified Aplysia neurons

1987 ◽  
Vol 58 (5) ◽  
pp. 909-921 ◽  
Author(s):  
D. P. Lotshaw ◽  
I. B. Levitan
Keyword(s):  
Voltage Clamp ◽  
Potassium Conductance ◽  
Phosphodiesterase Inhibitor ◽  
Identified Neurons ◽  
Inward Rectification ◽  
Current Response ◽  
Aplysia Neurons ◽  
Voltage Dependent ◽  
K Concentration ◽  
Inwardly Rectifying

1. The effect of serotonin (5-HT) and forskolin on an inwardly rectifying K+ conductance (IKR) was studied using voltage-clamp techniques in several identified Aplysia neurons isolated and maintained in primary cell culture. 2. Inward rectification was observed in the current-voltage relationship of the identified neurons R15, R2, B1, and B2 and was predominately due to IKR, as demonstrated by the dependence of inward rectification on the extracellular K+ concentration, instantaneous kinetics of the membrane current response to hyperpolarizing voltage clamp pulses, and voltage-dependent Ba2+ block of the inwardly rectifying current. 3. 5-HT increased IKR conductance between 100 and 400% in the identified neuron R15 in culture and increased IKR conductance approximately 50% in the identified neurons B1, B2, and R2 in culture. The adenylate cyclase activator, forskolin, plus a phosphodiesterase inhibitor, Ro 20-1724, also increased IKR conductance in these neurons. 4. 5-HT and forskolin modulated other ion conductances as well in all of these cultured neurons.

scholarly journals Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine

Cell ◽  
1995 ◽  
Vol 80 (1) ◽  
pp. 149-154 ◽  
Author(s):  
B Fakler ◽  
U Brändle ◽  
E Glowatzki ◽  
S Weidemann ◽  
H.-P Zenner ◽  
...  
Keyword(s):  
Inward Rectifier ◽  
Inward Rectification ◽  
K Channels ◽  
Voltage Dependent

Characterization of a transient outward K+ current with inward rectification in canine ventricular myocytes

1998 ◽  
Vol 274 (3) ◽  
pp. C577-C585 ◽  
Author(s):  
Gui-Rong Li ◽  
Haiying Sun ◽  
Stanley Nattel
Keyword(s):  
Action Potential ◽  
Ventricular Myocytes ◽  
Reversal Potential ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Inward Rectification ◽  
Transient Outward Current ◽  
Membrane Excitability ◽  
Voltage Dependent ◽  
Patch Technique

The threshold potential for the classical depolarization-activated transient outward K+ current and Cl− current is positive to −30 mV. With the whole cell patch technique, a transient outward current was elicited in the presence of 5 mM 4-aminopyridine (4-AP) and 5 μM ryanodine at voltages positive to the K+ equilibrium potential in canine ventricular myocytes. The current was abolished by 200 μM Ba2+ or omission of external K+([Formula: see text]) and showed biexponential inactivation. The current-voltage relation for the peak of the transient outward component showed moderate inward rectification. The transient outward current demonstrated voltage-dependent inactivation (half-inactivation voltage: −43.5 ± 3.2 mV) and rapid, monoexponential recovery from inactivation (time constant: 13.2 ± 2.5 ms). The reversal potential responded to the changes in[Formula: see text] concentration. Action potential clamp revealed two phases of Ba2+-sensitive current during the action potential, including a large early transient component after the upstroke and a later outward component during phase 3 repolarization. The present study demonstrates that depolarization may elicit a Ba2+- and[Formula: see text]-sensitive, 4-AP-insensitive, transient outward current with inward rectification in canine ventricular myocytes. The properties of this K+ current suggest that it may carry a significant early outward current upon depolarization that may play a role in determining membrane excitability and action potential morphology.

Inward rectification in Limulus ventral photoreceptors

1992 ◽  
Vol 8 (1) ◽  
pp. 19-25 ◽  
Author(s):  
Cynthia L. Phillips ◽  
Juan Bacigalupo ◽  
Peter M. O'Day
Keyword(s):  
Late Phase ◽  
Inward Rectifier ◽  
Inward Rectification ◽  
Voltage Range ◽  
Inward Current ◽  
Intracellular Ca ◽  
Dynamic Voltage ◽  
Inward Currents ◽  
Two Phases ◽  
Inwardly Rectifying

AbstractWe examined inward rectification in Limulus ventral photoreceptors using the two-microelectrode voltage clamp. Hyperpolarization in the dark induced an inward current whose magnitude was distinctly dependent on extracellular K+ concentration, [K+0]. The [K+0] dependence resembled the characteristic [K+0] dependence of other inward rectifiers. The inward current was not dependent on extracellular Ca2+ or Na+, and it was unaffected by intracellular injection of Cl−. The hyperpolarization induced currents had two phases, an early nearly instantaneous phase and a slowly developing late phase. The currents were sensitive to extracellular barium and cesium. In voltage-pulse experiments, the magnitudes of the inwardly rectifying currents were variable from cell to cell, with some cells exhibiting negligible inward currents. Large hyperpolarizations (to membrane potentials more negative than about – 140 mV) caused unstable inward current recordings, irreversible desensitization, and irreversible elevation of intracellular Ca2+ concentration. The inward rectifier provides negative feedback by tending to depolarize the cell (with inward current) in response to hyperpolarization. We suggest that the inward rectifier reduces the amount of hyperpolarization that would otherwise be generated by electrogenic processes. This feature would restrict the dynamic voltage range of the photoreceptors at very hyperpolarized potentials.

Inwardly rectifying K+ current in osteoclasts

1989 ◽  
Vol 256 (6) ◽  
pp. C1277-C1282 ◽  
Author(s):  
S. M. Sims ◽  
S. J. Dixon
Keyword(s):  
Membrane Potential ◽  
Cell Types ◽  
Membrane Properties ◽  
Inward Rectification ◽  
Patch Clamp Recording ◽  
Current Voltage ◽  
K Current ◽  
Inwardly Rectifying ◽  
Current Voltage Relationship ◽  
Voltage Relationship

Membrane properties of freshly isolated rat osteoclasts were studied using the whole cell patch-clamp recording technique. The membrane potential could switch between two stable levels, approximately -70 and -15 mV. Voltage-clamp studies indicated that osteoclasts exhibited marked inward rectification, with hyperpolarizing voltage commands from -70 mV activating large inward currents. No voltage-dependent currents were observed in response to depolarization. An increase in external K+ concentration shifted the current-voltage relationship positive in a manner predicted for K+ current. Furthermore, barium and cesium reversibly suppressed the inward current. Thus the dominant current evident in osteoclasts was inwardly rectifying K+ current, resembling that found in a number of cell types, including cardiac and skeletal muscle and oocytes. The current-voltage relationship of osteoclasts was "N-shaped" and could intersect the zero-current level at three potentials, accounting for two stable membrane potentials. Switching of membrane potential between these two levels may regulate a number of the cellular processes involved in bone resorption.

Variants with increased negative electrostatic potential in the Cx50 gap junction pore increased unitary channel conductance and magnesium modulation

2018 ◽  
Vol 475 (21) ◽  
pp. 3315-3330 ◽  
Author(s):  
Mary Grace Tejada ◽  
Swathy Sudhakar ◽  
Nicholas K. Kim ◽  
Hiroshi Aoyama ◽  
Brian H. Shilton ◽  
...  
Keyword(s):  
Gap Junction ◽  
Structural Models ◽  
Channel Conductance ◽  
Intracellular Magnesium ◽  
Amino Terminal ◽  
Molecular Determinants ◽  
Charged Residues ◽  
Voltage Dependent ◽  
Pore Lining ◽  
Positively Charged

Gap junction (GJ) channels are oligomers of connexins forming channels linking neighboring cells. GJs formed by different connexins show distinct unitary channel conductance (γj), transjunctional voltage-dependent gating (Vj-gating) properties, and modulation by intracellular magnesium ([Mg2+]i). The underlying molecular determinants are not fully clear. Previous experimental evidence indicates that residues in the amino terminal (NT) and initial segment of the first extracellular (E1) domain influence the γj, Vj-gating, and/or [Mg2+]i modulation in several GJs. Increasing negatively charged residues in Cx50 (connexin50) E1 (G46D or G46E) increased γj, while increasing positively charged residue (G46K) reduced the γj. Sequence alignment of Cx50 and Cx37 in the NT and E1 domains revealed that in Cx50 G8 and V53, positions are negatively charged residues in Cx37 (E8 and E53, respectively). To evaluate these residues together, we generated a triple variant in Cx50, G8E, G46E, and V53E simultaneously to study its γj, Vj-gating properties, and modulation by [Mg2+]i. Our data indicate that the triple variant and individual variants G8E, G46E, and V53E significantly increased Cx50 GJ γj without a significant change in the Vj gating. In addition, elevated [Mg2+]i reduced γj in Cx50 and all the variant GJs. These results and our homology structural models suggest that these NT/E1 residues are likely to be pore-lining and the variants increased the negative electrostatic potentials along the GJ pore to facilitate the γj of this cation-preferring GJ channel. Our results indicate that electrostatic properties of the Cx50 GJ pore are important for the γj and the [Mg2+]i modulation.

scholarly journals Mg2+-dependent Gating and Strong Inward Rectification of the Cation Channel TRPV6

2003 ◽  
Vol 121 (3) ◽  
pp. 245-260 ◽  
Author(s):  
Thomas Voets ◽  
Annelies Janssens ◽  
Jean Prenen ◽  
Guy Droogmans ◽  
Bernd Nilius
Keyword(s):  
Divalent Cations ◽  
Inward Rectification ◽  
Electrical Field ◽  
Gating Mechanism ◽  
Voltage Dependent ◽  
A Site ◽  
Inwardly Rectifying ◽  
Inside Out ◽  
Aspartate Residue

TRPV6 (CaT1/ECaC2), a highly Ca2+-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg2+. Mg2+ blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg2+ is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg2+, outward conductance is still ∼10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg2+-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg2+ sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg2+. The effects of intracellular Mg2+ on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K+ channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.

scholarly journals Pore Block versus Intrinsic Gating in the Mechanism of Inward Rectification in Strongly Rectifying Irk1 Channels

2000 ◽  
Vol 116 (4) ◽  
pp. 561-568 ◽  
Author(s):  
Donglin Guo ◽  
Zhe Lu
Keyword(s):  
Channel Gating ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Ion Conduction ◽  
Pore Blocking ◽  
Voltage Dependent ◽  
Excised Patches ◽  
Ph Buffer ◽  
Inwardly Rectifying

The IRK1 channel is inhibited by intracellular cations such as Mg2+ and polyamines in a voltage-dependent manner, which renders its I-V curve strongly inwardly rectifying. However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg2+ and polyamines, the macroscopic I-V curve of the channels displays modest rectification. This observation forms the basis of a hypothesis, alternative to the pore-blocking hypothesis, that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations. We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity. Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.

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