scholarly journals Influence of Pore Residues on Permeation Properties in the Kv2.1 Potassium Channel. Evidence for a Selective Functional Interaction of K+ with the Outer Vestibule

2003 ◽  
Vol 121 (2) ◽  
pp. 111-124 ◽  
Author(s):  
Joseph F. Consiglio ◽  
Payam Andalib ◽  
Stephen J. Korn
Keyword(s):  
Potassium Channel ◽  
Binding Site ◽  
Outward Current ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channels ◽  
Local Energy Minimum ◽  
Current Magnitude ◽  
Outer Vestibule ◽  
Permeation Properties

The Kv2.1 potassium channel contains a lysine in the outer vestibule (position 356) that markedly reduces open channel sensitivity to changes in external [K+]. To investigate the mechanism underlying this effect, we examined the influence of this outer vestibule lysine on three measures of K+ and Na+ permeation. Permeability ratio measurements, measurements of the lowest [K+] required for interaction with the selectivity filter, and measurements of macroscopic K+ and Na+ conductance, were all consistent with the same conclusion: that the outer vestibule lysine in Kv2.1 interferes with the ability of K+ to enter or exit the extracellular side of the selectivity filter. In contrast to its influence on K+ permeation properties, Lys 356 appeared to be without effect on Na+ permeation. This suggests that Lys 356 limited K+ flux by interfering with a selective K+ binding site. Combined with permeation studies, results from additional mutagenesis near the external entrance to the selectivity filter indicated that this site was located external to, and independent from, the selectivity filter. Protonation of a naturally occurring histidine in the same outer vestibule location in the Kv1.5 potassium channel produced similar effects on K+ permeation properties. Together, these results indicate that a selective, functional K+ binding site (e.g., local energy minimum) exists in the outer vestibule of voltage-gated K+ channels. We suggest that this site is the location of K+ hydration/dehydration postulated to exist based on the structural studies of KcsA. Finally, neutralization of position 356 enhanced outward K+ current magnitude, but did not influence the ability of internal K+ to enter the pore. These data indicate that in Kv2.1, exit of K+ from the selectivity filter, rather than entry of internal K+ into the channel, limits outward current magnitude. We discuss the implications of these findings in relation to the structural basis of channel conductance in different K+ channels.

scholarly journals Inhibition of the Collapse of the Shaker K+ Conductance by Specific Scorpion Toxins

2004 ◽  
Vol 123 (3) ◽  
pp. 265-279 ◽  
Author(s):  
Froylan Gómez-Lagunas ◽  
Cesar V.F. Batista ◽  
Timoteo Olamendi-Portugal ◽  
Martha E. Ramírez-Domínguez ◽  
Lourival D. Possani
Keyword(s):  
Binding Site ◽  
Selectivity Filter ◽  
Channel Block ◽  
Scorpion Toxins ◽  
K Channels ◽  
Outer Vestibule ◽  
Plausible Model ◽  
K Current ◽  
Do So ◽  
External K

The Shaker B K+ conductance (GK) collapses when the channels are closed (deactivated) in Na+ solutions that lack K+ ions. Also, it is known that external TEA (TEAo) impedes the collapse of GK (Gómez-Lagunas, F. 1997. J. Physiol. 499:3–15; Gómez-Lagunas, F. 2001. J. Gen. Physiol. 118:639–648), and that channel block by TEAo and scorpion toxins are two mutually exclusive events (Goldstein, S.A.N., and C. Miller. 1993. Biophys. J. 65:1613–1619). Therefore, we tested the ability of scorpion toxins to inhibit the collapse of GK in 0 K+. We have found that these toxins are not uniform regarding the capacity to protect GK. Those toxins, whose binding to the channels is destabilized by external K+, are also effective inhibitors of the collapse of GK. In addition to K+, other externally added cations also destabilize toxin block, with an effectiveness that does not match the selectivity sequence of K+ channels. The inhibition of the drop of GK follows a saturation relationship with [toxin], which is fitted well by the Michaelis-Menten equation, with an apparent Kd bigger than that of block of the K+ current. However, another plausible model is also presented and compared with the Michaelis-Menten model. The observations suggest that those toxins that protect GK in 0 K+ do so by interacting either with the most external K+ binding site of the selectivity filter (suggesting that the K+ occupancy of only that site of the pore may be enough to preserve GK) or with sites capable of binding K+ located in the outer vestibule of the pore, above the selectivity filter.

scholarly journals Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels

2004 ◽  
Vol 124 (6) ◽  
pp. 729-739 ◽  
Author(s):  
Yu-Yang Zhang ◽  
Janice L. Robertson ◽  
Daniel A. Gray ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
Single Amino Acid ◽  
Structural Basis ◽  
K Channels ◽  
Chord Conductance ◽  
In Series ◽  
Carboxy Terminal

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at Vm = −100 mV from 34 to 21 pS. The slope conductance measured between −60 and −140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba2+, suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl+:K+, accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K+ transport.

scholarly journals Control of Single Channel Conductance in the Outer Vestibule of the Kv2.1 Potassium Channel

2006 ◽  
Vol 128 (2) ◽  
pp. 231-246 ◽  
Author(s):  
Josef G. Trapani ◽  
Payam Andalib ◽  
Joseph F. Consiglio ◽  
Stephen J. Korn
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Markov Modeling ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Current Magnitude ◽  
Hidden Markov Modeling ◽  
Outer Vestibule ◽  
Dependent Change ◽  
Conduction Pathway

Current magnitude in Kv2.1 potassium channels is modulated by external [K+]. In contrast to behavior expected from the change in electrochemical driving force, outward current through Kv2.1 channels becomes larger when extracellular [K+] is increased within the physiological range. The mechanism that underlies this unusual property involves the opening of Kv2.1 channels into one of two different outer vestibule conformations, which are defined by their sensitivity to TEA. Channels that open into a TEA-sensitive conformation generate larger macroscopic currents, whereas channels that open into a TEA-insensitive conformation generate smaller macroscopic currents. At higher [K+], more channels open into the TEA-sensitive conformation. In this manuscript, we examined the mechanism by which the conformational change produced a change in current magnitude. We started by testing the simplest hypothesis: that each pharmacologically defined channel conformation produces a different single channel conductance, one smaller and one larger, and that the [K+]-dependent change in current magnitude reflects the [K+]-dependent change in the percentage of channels that open into each of the two conformations. Using single channel and macroscopic recordings, as well as hidden Markov modeling, we were able to quantitatively account for [K+]-dependent regulation of macroscopic current with this model. Combined with previously published work, these results support a model whereby an outer vestibule lysine interferes with K+ flux through the channel, and that the [K+]-dependent change in orientation of this lysine alters single channel conductance by changing the level of this interference. Moreover, these results provide an experimental example of single channel conductance being modulated at the outer end of the conduction pathway by a mechanism that involves channel activation into open states with different outer vestibule conformations.

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

2013 ◽  
Vol 142 (5) ◽  
pp. 523-542 ◽  
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.

scholarly journals Functional Consequences of a Decreased Potassium Affinity in a Potassium Channel Pore

1999 ◽  
Vol 113 (2) ◽  
pp. 347-358 ◽  
Author(s):  
Eva M. Ogielska ◽  
Richard W. Aldrich
Keyword(s):  
Potassium Channel ◽  
Binding Site ◽  
Conformational Change ◽  
Electrostatic Interactions ◽  
Selectivity Filter ◽  
Inactivation Rate ◽  
Ion Binding ◽  
Present Evidence ◽  
Channel Pore ◽  
Functional Consequences

Ions bound near the external mouth of the potassium channel pore impede the C-type inactivation conformational change (Lopez-Barneo, J., T. Hoshi, S. Heinemann, and R. Aldrich. 1993. Receptors Channels. 1:61– 71; Baukrowitz, T., and G. Yellen. 1995. Neuron. 15:951–960). In this study, we present evidence that the occupancy of the C-type inactivation modulatory site by permeant ions is not solely dependent on its intrinsic affinity, but is also a function of the relative affinities of the neighboring sites in the potassium channel pore. The A463C mutation in the S6 region of Shaker decreases the affinity of an internal ion binding site in the pore (Ogielska, E.M., and R.W. Aldrich, 1998). However, we have found that this mutation also decreases the C-type inactivation rate of the channel. Our studies indicate that the C-type inactivation effects observed with substitutions at position A463 most likely result from changes in the pore occupancy of the channel, rather than a change in the C-type inactivation conformational change. We have found that a decrease in the potassium affinity of the internal ion binding site in the pore results in lowered (electrostatic) interactions among ions in the pore and as a result prolongs the time an ion remains bound at the external C-type inactivation site. We also present evidence that the C-type inactivation constriction is quite local and does not involve a general collapse of the selectivity filter. Our data indicate that in A463C potassium can bind within the selectivity filter without interfering with the process of C-type inactivation.

scholarly journals Influence of Permeant Ions on Voltage Sensor Function in the Kv2.1 Potassium Channel

2004 ◽  
Vol 123 (4) ◽  
pp. 387-400 ◽  
Author(s):  
Joseph F. Consiglio ◽  
Stephen J. Korn
Keyword(s):  
Potassium Channel ◽  
Ionic Current ◽  
Ionic Currents ◽  
Voltage Sensor ◽  
Selectivity Filter ◽  
Charge Movement ◽  
Channel Activation ◽  
Outer Vestibule ◽  
Specific Selectivity ◽  
Current Activation

We previously demonstrated that the outer vestibule of activated Kv2.1 potassium channels can be in one of two conformations, and that K+occupancy of a specific selectivity filter site determines which conformation the outer vestibule is in. These different outer vestibule conformations result in different sensitivities to internal and external TEA, different inactivation rates, and different macroscopic conductances. The [K+]-dependent switch in outer vestibule conformation is also associated with a change in rate of channel activation. In this paper, we examined the mechanism by which changes in [K+] modulate the rate of channel activation. Elevation of symmetrical [K+] or [Rb+] from 0 to 3 mM doubled the rate of on-gating charge movement (Qon), measured at 0 mV. Cs+produced an identical effect, but required 40-fold higher concentrations. All three permeant ions occupied the selectivity filter over the 0.03–3 mM range, so simple occupancy of the selectivity filter was not sufficient to produce the change in Qon. However, for each of these permeant ions, the speeding of Qonoccurred with the same concentration dependence as the switch between outer vestibule conformations. Neutralization of an amino acid (K356) in the outer vestibule, which abolishes the modulation of channel pharmacology and ionic currents by the K+-dependent reorientation of the outer vestibule, also abolished the K+-dependence of Qon. Together, the data indicate that the K+-dependent reorientation in the outer vestibule was responsible for the change in Qon. Moreover, similar [K+]-dependence and effects of mutagenesis indicate that the K+-dependent change in rate of Qoncan account for the modulation of ionic current activation rate. Simple kinetic analysis suggested that K+reduced an energy barrier for voltage sensor movement. These results provide strong evidence for a direct functional interaction, which is modulated by permeant ions acting at the selectivity filter, between the outer vestibule of the Kv2.1 potassium channel and the voltage sensor.

scholarly journals Structural basis for ion selectivity in TMEM175 K+ channels

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Janine D Brunner ◽  
Roman P Jakob ◽  
Tobias Schulze ◽  
Yvonne Neldner ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Ph Regulation ◽  
Ion Selectivity ◽  
Selectivity Filter ◽  
Structural Basis ◽  
K Channels ◽  
X Ray ◽  
Lysosomal Ph ◽  
Additional Layer ◽  
Channel Opening

The TMEM175 family constitutes recently discovered K+channels that are important for autophagosome turnover and lysosomal pH regulation and are associated with the early onset of Parkinson Disease. TMEM175 channels lack a P-loop selectivity filter, a hallmark of all known K+ channels, raising the question how selectivity is achieved. Here, we report the X-ray structure of a closed bacterial TMEM175 channel in complex with a nanobody fusion-protein disclosing bound K+ ions. Our analysis revealed that a highly conserved layer of threonine residues in the pore conveys a basal K+ selectivity. An additional layer comprising two serines in human TMEM175 increases selectivity further and renders this channel sensitive to 4-aminopyridine and Zn2+. Our findings suggest that large hydrophobic side chains occlude the pore, forming a physical gate, and that channel opening by iris-like motions simultaneously relocates the gate and exposes the otherwise concealed selectivity filter to the pore lumen.

scholarly journals Computational study of non-conductive selectivity filter conformations and C-type inactivation in a voltage-dependent potassium channel

2021 ◽  
Vol 153 (9) ◽  
Author(s):  
Jing Li ◽  
Rong Shen ◽  
Ahmed Rohaim ◽  
Ramon Mendoza Uriarte ◽  
Mikolai Fajer ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Computational Study ◽  
Homology Model ◽  
Selectivity Filter ◽  
K Channels ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Signature Sequence ◽  
Dynamics Simulations ◽  
Pore Domain

C-type inactivation is a time-dependent process of great physiological significance that is observed in a large class of K+ channels. Experimental and computational studies of the pH-activated KcsA channel show that the functional C-type inactivated state, for this channel, is associated with a structural constriction of the selectivity filter at the level of the central glycine residue in the signature sequence, TTV(G)YGD. The structural constriction is allosterically promoted by the wide opening of the intracellular activation gate. However, whether this is a universal mechanism for C-type inactivation has not been established with certainty because similar constricted structures have not been observed for other K+ channels. Seeking to ascertain the general plausibility of the constricted filter conformation, molecular dynamics simulations of a homology model of the pore domain of the voltage-gated potassium channel Shaker were performed. Simulations performed with an open intracellular gate spontaneously resulted in a stable constricted-like filter conformation, providing a plausible nonconductive state responsible for C-type inactivation in the Shaker channel. While there are broad similarities with the constricted structure of KcsA, the hypothetical constricted-like conformation of Shaker also displays some subtle differences. Interestingly, those are recapitulated by the Shaker-like E71V KcsA mutant, suggesting that the residue at this position along the pore helix plays a pivotal role in determining the C-type inactivation behavior. Free energy landscape calculations show that the conductive-to-constricted transition in Shaker is allosterically controlled by the degree of opening of the intracellular activation gate, as observed with the KcsA channel. The behavior of the classic inactivating W434F Shaker mutant is also characterized from a 10-μs MD simulation, revealing that the selectivity filter spontaneously adopts a nonconductive conformation that is constricted at the level of the second glycine in the signature sequence, TTVGY(G)D.

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