Structural Model of the Outer Vestibule and Selectivity Filter of the Shaker Voltage-gated K + Channel

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.

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Structural basis for C-type inactivation in a Shaker family voltage gated K+ channel

10.1101/2021.10.01.462615 ◽

2021 ◽

Author(s):

Ravikumar Reddi ◽

Kimberly Matulef ◽

Erika A. Riederer ◽

Matthew R. Whorton ◽

Francis I. Valiyaveetil

Keyword(s):

Crystal Structure ◽

Binding Sites ◽

Structural Changes ◽

Selectivity Filter ◽

Structural Basis ◽

K Channel ◽

Ion Binding ◽

Channel Pore ◽

Voltage Gated ◽

Ion Binding Sites

AbstractC-type inactivation is a process by which ion flux through a voltage-gated K+ (Kv) channel is regulated at the selectivity filter. While prior studies have indicated that C-type inactivation involves structural changes at the selectivity filter, the nature of the changes have not been resolved. Here we report the crystal structure of the Kv1.2 channel in a C-type inactivated state. The structure shows that C-type inactivation involves changes in the selectivity filter that disrupt the outer two ion binding sites in the filter. The changes at the selectivity filter propagate to the extracellular mouth and the turret regions of the channel pore. The structural changes observed are consistent with the functional hallmarks of C-type inactivation. This study highlights the intricate interplay between K+ occupancy at the ion binding sites and the interactions of the selectivity filter in determining the balance between the conductive and the inactivated conformations of the filter.

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Structural basis of ion permeation gating in Slo2.1 K+ channels

The Journal of General Physiology ◽

10.1085/jgp.201311064 ◽

2013 ◽

Vol 142 (5) ◽

pp. 523-542 ◽

Cited By ~ 16

Author(s):

Priyanka Garg ◽

Alison Gardner ◽

Vivek Garg ◽

Michael C. Sanguinetti

Keyword(s):

Selectivity Filter ◽

Structural Basis ◽

K Channel ◽

Xenopus Laevis Oocytes ◽

Ion Permeation ◽

Channel Activation ◽

K Channels ◽

Voltage Gated ◽

Channel Function ◽

Activation Gate

The activation gate of ion channels controls the transmembrane flux of permeant ions. In voltage-gated K+ channels, the aperture formed by the S6 bundle crossing can widen to open or narrow to close the ion permeation pathway, whereas the selectivity filter gates ion flux in cyclic-nucleotide gated (CNG) and Slo1 channels. Here we explore the structural basis of the activation gate for Slo2.1, a weakly voltage-dependent K+ channel that is activated by intracellular Na+ and Cl−. Slo2.1 channels were heterologously expressed in Xenopus laevis oocytes and activated by elevated [NaCl]i or extracellular application of niflumic acid. In contrast to other voltage-gated channels, Slo2.1 was blocked by verapamil in an activation-independent manner, implying that the S6 bundle crossing does not gate the access of verapamil to its central cavity binding site. The structural basis of Slo2.1 activation was probed by Ala scanning mutagenesis of the S6 segment and by mutation of selected residues in the pore helix and S5 segment. Mutation to Ala of three S6 residues caused reduced trafficking of channels to the cell surface and partial (K256A, I263A, Q273A) or complete loss (E275A) of channel function. P271A Slo2.1 channels trafficked normally, but were nonfunctional. Further mutagenesis and intragenic rescue by second site mutations suggest that Pro271 and Glu275 maintain the inner pore in an open configuration by preventing formation of a tight S6 bundle crossing. Mutation of several residues in S6 and S5 predicted by homology modeling to contact residues in the pore helix induced a gain of channel function. Substitution of the pore helix residue Phe240 with polar residues induced constitutive channel activation. Together these findings suggest that (1) the selectivity filter and not the bundle crossing gates ion permeation and (2) dynamic coupling between the pore helix and the S5 and S6 segments mediates Slo2.1 channel activation.

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Potassium-dependent Changes in the Conformation of the Kv2.1 Potassium Channel Pore

The Journal of General Physiology ◽

10.1085/jgp.113.6.819 ◽

1999 ◽

Vol 113 (6) ◽

pp. 819-836 ◽

Cited By ~ 71

Author(s):

David Immke ◽

Michael Wood ◽

Laszlo Kiss ◽

Stephen J. Korn

Keyword(s):

Outer Edge ◽

Selectivity Filter ◽

Inactivation Rate ◽

K Channel ◽

Outer Vestibule ◽

Na Currents ◽

Conduction Pathway ◽

Conformational Rearrangements ◽

Low K ◽

K Currents

The voltage-gated K+ channel, Kv2.1, conducts Na+ in the absence of K+. External tetraethylammonium (TEAo) blocks K+ currents through Kv2.1 with an IC50 of 5 mM, but is completely without effect in the absence of K+. TEAo block can be titrated back upon addition of low [K+]. This suggested that the Kv2.1 pore undergoes a cation-dependent conformational rearrangement in the external vestibule. Individual mutation of lysine (Lys) 356 and 382 in the outer vestibule, to a glycine and a valine, respectively, increased TEAo potency for block of K+ currents by a half log unit. Mutation of Lys 356, which is located at the outer edge of the external vestibule, significantly restored TEAo block in the absence of K+ (IC50 = 21 mM). In contrast, mutation of Lys 382, which is located in the outer vestibule near the TEA binding site, resulted in very weak (extrapolated IC50 = ∼265 mM) TEAo block in the absence of K+. These data suggest that the cation-dependent alteration in pore conformation that resulted in loss of TEA potency extended to the outer edge of the external vestibule, and primarily involved a repositioning of Lys 356 or a nearby amino acid in the conduction pathway. Block by internal TEA also completely disappeared in the absence of K+, and could be titrated back with low [K+]. Both internal and external TEA potencies were increased by the same low [K+] (30–100 μM) that blocked Na+ currents through the channel. In addition, experiments that combined block by internal and external TEA indicated that the site of K+ action was between the internal and external TEA binding sites. These data indicate that a K+-dependent conformational change also occurs internal to the selectivity filter, and that both internal and external conformational rearrangements resulted from differences in K+ occupancy of the selectivity filter. Kv2.1 inactivation rate was K+ dependent and correlated with TEAo potency; as [K+] was raised, TEAo became more potent and inactivation became faster. Both TEAo potency and inactivation rate saturated at the same [K+]. These results suggest that the rate of slow inactivation in Kv2.1 was influenced by the conformational rearrangements, either internal to the selectivity filter or near the outer edge of the external vestibule, that were associated with differences in TEA potency.

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Atomic Constraints between the Voltage Sensor and the Pore Domain in a Voltage-gated K+ Channel of Known Structure

The Journal of General Physiology ◽

10.1085/jgp.200809962 ◽

2008 ◽

Vol 131 (6) ◽

pp. 549-561 ◽

Cited By ~ 20

Author(s):

Anthony Lewis ◽

Vishwanath Jogini ◽

Lydia Blachowicz ◽

Muriel Lainé ◽

Benoît Roux

Keyword(s):

Structural Model ◽

Voltage Sensor ◽

K Channel ◽

Structural Reorganization ◽

Voltage Gated ◽

Transmembrane Segments ◽

Close Physical Proximity ◽

Activated State ◽

Dynamics Simulations ◽

Pore Domain

In voltage-gated K+ channels (Kv), membrane depolarization promotes a structural reorganization of each of the four voltage sensor domains surrounding the conducting pore, inducing its opening. Although the crystal structure of Kv1.2 provided the first atomic resolution view of a eukaryotic Kv channel, several components of the voltage sensors remain poorly resolved. In particular, the position and orientation of the charged arginine side chains in the S4 transmembrane segments remain controversial. Here we investigate the proximity of S4 and the pore domain in functional Kv1.2 channels in a native membrane environment using electrophysiological analysis of intersubunit histidine metallic bridges formed between the first arginine of S4 (R294) and residues A351 or D352 of the pore domain. We show that histidine pairs are able to bind Zn2+ or Cd2+ with high affinity, demonstrating their close physical proximity. The results of molecular dynamics simulations, consistent with electrophysiological data, indicate that the position of the S4 helix in the functional open-activated state could be shifted by ∼7–8 Å and rotated counterclockwise by 37° along its main axis relative to its position observed in the Kv1.2 x-ray structure. A structural model is provided for this conformation. The results further highlight the dynamic and flexible nature of the voltage sensor.

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K+ channel gating: C-type inactivation is enhanced by calcium or lanthanum outside

The Journal of General Physiology ◽

10.1085/jgp.201411223 ◽

2014 ◽

Vol 144 (3) ◽

pp. 221-230 ◽

Cited By ~ 12

Author(s):

Clay M. Armstrong ◽

Toshinori Hoshi

Keyword(s):

Channel Gating ◽

Selectivity Filter ◽

K Channel ◽

Slow Process ◽

K Channels ◽

Inward Current ◽

Voltage Gated ◽

Positive Shift ◽

High Affinity ◽

Opening And Closing

Many voltage-gated K+ channels exhibit C-type inactivation. This typically slow process has been hypothesized to result from dilation of the outer-most ring of the carbonyls in the selectivity filter, destroying this ring’s ability to bind K+ with high affinity. We report here strong enhancement of C-type inactivation upon extracellular addition of 10–40 mM Ca2+ or 5–50 µM La3+. These multivalent cations mildly increase the rate of C-type inactivation during depolarization and markedly promote inactivation and/or suppress recovery when membrane voltage (Vm) is at resting levels (−80 to −100 mV). At −80 mV with 40 mM Ca2+ and 0 mM K+ externally, ShBΔN channels with the mutation T449A inactivate almost completely within 2 min or less with no pulsing. This behavior is observed only in those mutants that show C-type inactivation on depolarization and is distinct from the effects of Ca2+ and La3+ on activation (opening and closing of the Vm-controlled gate), i.e., slower activation of K+ channels and a positive shift of the mid-voltage of activation. The Ca2+/La3+ effects on C-type inactivation are antagonized by extracellular K+ in the low millimolar range. This, together with the known ability of Ca2+ and La3+ to block inward current through K+ channels at negative voltage, strongly suggests that Ca2+/La3+ acts at the outer mouth of the selectivity filter. We propose that at −80 mV, Ca2+ or La3+ ions compete effectively with K+ at the channel’s outer mouth and prevent K+ from stabilizing the filter’s outer carbonyl ring.

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