scholarly journals Probing the Cavity of the Slow Inactivated Conformation of Shaker Potassium Channels

2007 ◽  
Vol 129 (5) ◽  
pp. 403-418 ◽  
Author(s):  
Gyorgy Panyi ◽  
Carol Deutsch
Keyword(s):  
Potassium Channels ◽  
Binding Site ◽  
Conformational Change ◽  
Marked Decrease ◽  
Selectivity Filter ◽  
Slow Inactivation ◽  
Voltage Gated ◽  
Activation Gate ◽  
Shaker Potassium Channels

Slow inactivation involves a local rearrangement of the outer mouth of voltage-gated potassium channels, but nothing is known regarding rearrangements in the cavity between the activation gate and the selectivity filter. We now report that the cavity undergoes a conformational change in the slow-inactivated state. This change is manifest as altered accessibility of residues facing the aqueous cavity and as a marked decrease in the affinity of tetraethylammonium for its internal binding site. These findings have implications for global alterations of the channel during slow inactivation and putative coupling between activation and slow-inactivation gates.

scholarly journals Cross Talk between Activation and Slow Inactivation Gates of Shaker Potassium Channels

2006 ◽  
Vol 128 (5) ◽  
pp. 547-559 ◽  
Author(s):  
Gyorgy Panyi ◽  
Carol Deutsch
Keyword(s):  
Potassium Channels ◽  
Cross Talk ◽  
Cysteine Residue ◽  
Slow Inactivation ◽  
Fundamental Factor ◽  
Functional Assays ◽  
Activation Gate ◽  
The Status ◽  
Shaker Potassium Channels ◽  
Rate Of Activation

This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs+ ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.

scholarly journals A Novel Interaction in the Selectivity Filter of Voltage-Gated Potassium Channels Crucial for Slow Inactivation

2013 ◽  
Vol 104 (2) ◽  
pp. 122a-123a
Author(s):  
Stephan A. Pless ◽  
Ana P. Niciforovic ◽  
Christopher A. Ahern
Keyword(s):  
Potassium Channels ◽  
Selectivity Filter ◽  
Slow Inactivation ◽  
Voltage Gated

scholarly journals Disruption of the Key Ca2+ Binding Site in the Selectivity Filter of Neuronal Voltage-Gated Calcium Channels Inhibits Channel Trafficking

Cell Reports ◽  
2019 ◽  
Vol 29 (1) ◽  
pp. 22-33.e5 ◽  
Author(s):  
James O. Meyer ◽  
Shehrazade Dahimene ◽  
Karen M. Page ◽  
Laurent Ferron ◽  
Ivan Kadurin ◽  
...  
Keyword(s):  
Calcium Channels ◽  
Binding Site ◽  
Selectivity Filter ◽  
Channel Trafficking ◽  
Voltage Gated ◽  
Voltage Gated Calcium Channels

scholarly journals Evidence for a Deep Pore Activation Gate in Small Conductance Ca2+-activated K+ Channels

2007 ◽  
Vol 130 (6) ◽  
pp. 601-610 ◽  
Author(s):  
Andrew Bruening-Wright ◽  
Wei-Sheng Lee ◽  
John P. Adelman ◽  
James Maylie
Keyword(s):  
Cysteine Residue ◽  
Selectivity Filter ◽  
K Channel ◽  
Homologous Region ◽  
Sk Channels ◽  
Voltage Gated ◽  
Kv Channels ◽  
Substituted Cysteine Accessibility ◽  
Activation Gate ◽  
Small Conductance

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (Kv) channels, but are distinct in that they are gated solely by calcium (Ca2+), not voltage. For Kv channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd2+) and barium (Ba2+), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd2+ applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd2+ and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba2+ applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba2+ is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba2+ pre-block slows MTSEA modification of A384C in open but not in closed (Ba2+-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.

scholarly journals Tail End of the S6 Segment

2002 ◽  
Vol 120 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Shinghua Ding ◽  
Richard Horn
Keyword(s):  
Potassium Channels ◽  
Energy Barrier ◽  
Single Channel ◽  
Noise Analysis ◽  
Selectivity Filter ◽  
Open Probability ◽  
Cytoplasmic Extension ◽  
Activation Gate ◽  
Single Channel Currents ◽  
Channel Currents

The permeation pathway in voltage-gated potassium channels has narrow constrictions at both the extracellular and intracellular ends. These constrictions might limit the flux of cations from one side of the membrane to the other. The extracellular constriction is the selectivity filter, whereas the intracellular bundle crossing is proposed to act as the activation gate that opens in response to a depolarization. This four-helix bundle crossing is composed of S6 transmembrane segments, one contributed by each subunit. Here, we explore the cytoplasmic extension of the S6 transmembrane segment of Shaker potassium channels, just downstream from the bundle crossing. We substituted cysteine for each residue from N482 to T489 and determined the amplitudes of single channel currents and maximum open probability (Po,max) at depolarized voltages using nonstationary noise analysis. One mutant, F484C, significantly reduces Po,max, whereas Y483C, F484C, and most notably Y485C, reduce single channel conductance (γ). Mutations of residue Y485 have no effect on the Rb+/K+ selectivity, suggesting a local effect on γ rather than an allosteric effect on the selectivity filter. Y485 mutations also reduce pore block by tetrabutylammonium, apparently by increasing the energy barrier for blocker movement through the open activation gate. Replacing Rb+ ions for K+ ions reduces the amplitude of single channel currents and makes γ insensitive to mutations of Y485. These results suggest that Rb+ ions increase an extracellular energy barrier, presumably at the selectivity filter, thus making it rate limiting for flux of permeant ions. These results indicate that S6T residues have an influence on the conformation of the open activation gate, reflected in both the stability of the open state and the energy barriers it presents to ions.

scholarly journals An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels

2009 ◽  
Vol 134 (6) ◽  
pp. 461-469 ◽  
Author(s):  
Christopher A. Ahern ◽  
Amy L. Eastwood ◽  
Dennis A. Dougherty ◽  
Richard Horn
Keyword(s):  
Aromatic Ring ◽  
Conformational Changes ◽  
Time Course ◽  
Selectivity Filter ◽  
Nonsense Suppression ◽  
Quadrupole Moments ◽  
Slow Inactivation ◽  
Single Mutant ◽  
Shaker Potassium Channels

Slow inactivation of Kv1 channels involves conformational changes near the selectivity filter. We examine such changes in Shaker channels lacking fast inactivation by considering the consequences of mutating two residues, T449 just external to the selectivity filter and V438 in the pore helix near the bottom of the selectivity filter. Single mutant T449F channels with the native V438 inactivate very slowly, and the canonical foot-in-the-door effect of extracellular tetraethylammonium (TEA) is not only absent, but the time course of slow inactivation is accelerated by TEA. The V438A mutation dramatically speeds inactivation in T449F channels, and TEA slows inactivation exactly as predicted by the foot-in-the-door model. We propose that TEA has this effect on V438A/T449F channels because the V438A mutation produces allosteric consequences within the selectivity filter and may reorient the aromatic ring at position 449. We investigated the possibility that the blocker promotes the collapse of the outer vestibule (spring-in-the-door) in single mutant T449F channels by an electrostatic attraction between a cationic TEA and the quadrupole moments of the four aromatic rings. To test this idea, we used in vivo nonsense suppression to serially fluorinate the introduced aromatic ring at the 449 position, a manipulation that withdraws electrons from the aromatic face with little effect on the shape, net charge, or hydrophobicity of the aromatic ring. Progressive fluorination causes monotonically enhanced rates of inactivation. In further agreement with our working hypothesis, increasing fluorination of the aromatic gradually transforms the TEA effect from spring-in-the-door to foot-in-the-door. We further substantiate our electrostatic hypothesis by quantum mechanical calculations.

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