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 Permeation Properties of Inward-Rectifier Potassium Channels and Their Molecular Determinants

2000 ◽  
Vol 115 (4) ◽  
pp. 391-404 ◽  
Author(s):  
Han Choe ◽  
Henry Sackin ◽  
Lawrence G. Palmer
Keyword(s):  
Single Channel ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Site Directed Mutagenesis ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Single Channels ◽  
Extracellular Loop ◽  
K Channels ◽  
Inward Rectifier Potassium Channels

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K+ channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K+ conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH2 terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl+ > K+ > Rb+ > NH4+. The ion selectivity sequence for ROMK2 based on conductance ratios was NH4+ (1.6) > K+ (1) > Tl+ (0.5) > Rb+ (0.4). For IRK1, the sequence was K+ (1) > Tl+ (0.8) > NH4+ (0.6) >> Rb+ (0.1). The difference in the NH4+/ K+ conductance (1.6) and permeability (0.09) ratios can be explained if NH4+ binds with lower affinity than K+ to sites within the pore. The relatively low conductances of NH4+ and Rb+ through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl+ conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.

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 Mutations reveal voltage gating of CNGA1 channels in saturating cGMP

2009 ◽  
Vol 134 (2) ◽  
pp. 151-164 ◽  
Author(s):  
Juan Ramón Martínez-François ◽  
Yanping Xu ◽  
Zhe Lu
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Open Probability ◽  
K Channels ◽  
Voltage Gated ◽  
Open Conformation ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Low Probability ◽  
Inherent Voltage

Activity of cyclic nucleotide–gated (CNG) cation channels underlies signal transduction in vertebrate visual receptors. These highly specialized receptor channels open when they bind cyclic GMP (cGMP). Here, we find that certain mutations restricted to the region around the ion selectivity filter render the channels essentially fully voltage gated, in such a manner that the channels remain mostly closed at physiological voltages, even in the presence of saturating concentrations of cGMP. This voltage-dependent gating resembles the selectivity filter-based mechanism seen in KcsA K+ channels, not the S4-based mechanism of voltage-gated K+ channels. Mutations that render CNG channels gated by voltage loosen the attachment of the selectivity filter to its surrounding structure, thereby shifting the channel's gating equilibrium toward closed conformations. Significant pore opening in mutant channels occurs only when positive voltages drive the pore from a low-probability open conformation toward a second open conformation to increase the channels' open probability. Thus, the structure surrounding the selectivity filter has evolved to (nearly completely) suppress the expression of inherent voltage-dependent gating of CNGA1, ensuring that the binding of cGMP by itself suffices to open the channels at physiological voltages.

scholarly journals Mechanism of the Voltage Sensitivity of IRK1 Inward-rectifier K+ Channel Block by the Polyamine Spermine

2005 ◽  
Vol 125 (4) ◽  
pp. 413-426 ◽  
Author(s):  
Hyeon-Gyu Shin ◽  
Zhe Lu
Keyword(s):  
Steady State ◽  
Voltage Dependence ◽  
Ion Selectivity ◽  
Inward Rectifier ◽  
Selectivity Filter ◽  
K Channel ◽  
Voltage Sensitivity ◽  
Channel Block ◽  
Head Groups ◽  
Voltage Dependent

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence ∼5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QAC10) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider (∼6 Å) than the ion selectivity filter (∼3 Å). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QAC10 should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.

scholarly journals Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

2015 ◽  
Vol 112 (50) ◽  
pp. 15366-15371 ◽  
Author(s):  
Joshua B. Brettmann ◽  
Darya Urusova ◽  
Marco Tonelli ◽  
Jonathan R. Silva ◽  
Katherine A. Henzler-Wildman
Keyword(s):  
Ion Selectivity ◽  
Selectivity Filter ◽  
Structural Basis ◽  
Functional Coupling ◽  
Dynamic Coupling ◽  
Allosteric Coupling ◽  
Allosteric Communication ◽  
Dependent Inactivation ◽  
Ion Binding Sites ◽  
Essential Connection

Flux-dependent inactivation that arises from functional coupling between the inner gate and the selectivity filter is widespread in ion channels. The structural basis of this coupling has only been well characterized in KcsA. Here we present NMR data demonstrating structural and dynamic coupling between the selectivity filter and intracellular constriction point in the bacterial nonselective cation channel, NaK. This transmembrane allosteric communication must be structurally different from KcsA because the NaK selectivity filter does not collapse under low-cation conditions. Comparison of NMR spectra of the nonselective NaK and potassium-selective NaK2K indicates that the number of ion binding sites in the selectivity filter shifts the equilibrium distribution of structural states throughout the channel. This finding was unexpected given the nearly identical crystal structure of NaK and NaK2K outside the immediate vicinity of the selectivity filter. Our results highlight the tight structural and dynamic coupling between the selectivity filter and the channel scaffold, which has significant implications for channel function. NaK offers a distinct model to study the physiologically essential connection between ion conduction and channel gating.

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 A Carboxy-terminal Inter-Helix Linker As the Site of Phosphatidylinositol 4,5-Bisphosphate Action on Kv7 (M-type) K+ Channels

2008 ◽  
Vol 132 (3) ◽  
pp. 361-381 ◽  
Author(s):  
Ciria C. Hernandez ◽  
Oleg Zaika ◽  
Mark S. Shapiro
Keyword(s):  
Single Channel ◽  
Lipid Binding ◽  
Structural Motif ◽  
K Channels ◽  
Docking Simulations ◽  
Binding Domains ◽  
Action Point ◽  
Binding Surface ◽  
Carboxy Terminal ◽  
Structural Mechanisms

The regulation of M-type (KCNQ [Kv7]) K+ channels by phosphatidylinositol 4,5-bisphosphate (PIP2) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of PIP2 on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential PIP2 sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of PIP2 action. Point mutants within this linker in Kv7.2 and Kv7.3 identified a conserved cluster of basic residues that interact with the lipid using electrostatic and hydrogen bonds. Homology modeling of this putative PIP2-binding linker in Kv7.2 and Kv7.3 using the solved structure of Kir2.1 and Kir3.1 channels as templates predicts a structure of Kv7.2 and 7.3 very similar to the Kir channels, and to the seven-β-sheet barrel motif common to other PIP2-binding domains. Phosphoinositide-docking simulations predict affinities and interaction energies in accord with the experimental data, and furthermore indicate that the precise identity of residues in the interacting pocket alter channel–PIP2 interactions not only by altering electrostatic energies, but also by allosterically shifting the structure of the lipid-binding surface. The results are likely to shed light on the general structural mechanisms of phosphoinositide regulation of ion channels.

scholarly journals Conductance selectivity of Na+ across the K+ channel via Na+ trapped in a tortuous trajectory

2021 ◽  
Vol 118 (12) ◽  
pp. e2017168118
Author(s):  
Kenichiro Mita ◽  
Takashi Sumikama ◽  
Masayuki Iwamoto ◽  
Yuka Matsuki ◽  
Kenji Shigemi ◽  
...  
Keyword(s):  
Potassium Channel ◽  
Single Channel ◽  
Ion Selectivity ◽  
Carbonyl Oxygen ◽  
Selectivity Filter ◽  
K Channel ◽  
Selectivity Ratio ◽  
Channel Current ◽  
Dynamics Simulations ◽  
Kcsa Potassium Channel

Ion selectivity of the potassium channel is crucial for regulating electrical activity in living cells; however, the mechanism underlying the potassium channel selectivity that favors large K+ over small Na+ remains unclear. Generally, Na+ is not completely excluded from permeation through potassium channels. Herein, the distinct nature of Na+ conduction through the prototypical KcsA potassium channel was examined. Single-channel current recordings revealed that, at a high Na+ concentration (200 mM), the channel was blocked by Na+, and this blocking was relieved at high membrane potentials, suggesting the passage of Na+ across the channel. At a 2,000 mM Na+ concentration, single-channel Na+ conductance was measured as one-eightieth of the K+ conductance, indicating that the selectivity filter allows substantial conduit of Na+. Molecular dynamics simulations revealed unprecedented atomic trajectories of Na+ permeation. In the selectivity filter having a series of carbonyl oxygen rings, a smaller Na+ was distributed off-center in eight carbonyl oxygen-coordinated sites as well as on-center in four carbonyl oxygen-coordinated sites. This amphipathic nature of Na+ coordination yielded a continuous but tortuous path along the filter. Trapping of Na+ in many deep free energy wells in the filter caused slow elution. Conversely, K+ is conducted via a straight path, and as the number of occupied K+ ions increased to three, the concerted conduction was accelerated dramatically, generating the conductance selectivity ratio of up to 80. The selectivity filter allows accommodation of different ion species, but the ion coordination and interactions between ions render contrast conduction rates, constituting the potassium channel conductance selectivity.

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

2020 ◽  
Author(s):  
Janine D Brunner ◽  
Roman P Jakob ◽  
Tobias Schulze ◽  
Yvonne Neldner ◽  
Anna Moroni ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Author Response ◽  
Structural Basis ◽  
K Channels
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