Site-directed glycosylation tagging of functional Kir2.1 reveals that the putative pore-forming segment is extracellular

Inwardly rectifying K+ channels or Kirs are a large gene family and have been predicted to have two transmembrane segments, M1 and M2, intracellular N and C termini, and two extracellular loops, E1 and E2, separated by an intramembranous pore-forming segment, H5. H5 contains a stretch of eight residues that are similar in voltage-dependent K+ channels, Kvs, and this stretch is called the signature sequence of K+ channels. Because mutations in this sequence altered selectivity in Kvs, it has been designated as the selectivity filter. Previously, we used N-glycosylation substitution mutants to map the extracellular topology of a weak inwardly rectifying K+ channel, Kir1.1 or ROMK1, and found that the entire H5 segment was extracellular. We now report utilization of introduced N-glycosylation sites, NX(S/T), at positions Ser(128) in E1, and Gln(140), Ileu(143), and Phe(147) in the H5 sequence of a strong inwardly rectifying K+ channel, Kir2.1. Furthermore, we show that biotinylated channel proteins with N-linked oligosaccharides attached at positions 140 and 143 in the signature sequence are located at the cell surface. Mutant channels were functional as detected by whole-cell and single-channel recordings. Unlike Kir1.1, position Lys(117) was not occupied. We conclude that, for yet another K+ channel, the invariant G(Y/F)G sequence is extracellular rather than intramembranous.     

Selectivity Changes during Activation of Mutant Shaker Potassium Channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH₂-terminal–truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179–182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in ∼40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of ∼1.6 e₀. The fully open T442S chimeric channel has the conductance sequence Rb⁺ > NH₄ ⁺ > K⁺. The opposite conductance sequence, K⁺ > NH₄ ⁺ > Rb⁺, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb⁺.     

The role of the cytoplasmic pore in inward rectification of Kir2.1 channels

Steeply voltage-dependent block by intracellular polyamines underlies the strong inward rectification properties of Kir2.1 and other Kir channels. Mutagenesis studies have identified several negatively charged pore-lining residues (D172, E224, and E299, in Kir2.1) in the inner cavity and cytoplasmic domain as determinants of the properties of spermine block. Recent crystallographic determination of the structure of the cytoplasmic domains of Kir2.1 identified additional negatively charged residues (D255 and D259) that influence inward rectification. In this study, we have characterized the kinetic and steady-state properties of spermine block in WT Kir2.1 and in mutations of the D255 residue (D255E, A, K, R). Despite minimal effects on steady-state blockade by spermine, D255 mutations have profound effects on the blocking kinetics, with D255A marginally, and D255R dramatically, slowing the rate of block. In addition, these mutations result in the appearance of a sustained current (in the presence of spermine) at depolarized voltages. These features are reproduced with a kinetic model consisting of a single open state, two sequentially linked blocked states, and a slow spermine permeation step, with residue D255 influencing the spermine affinity and rate of entry into the shallow blocked state. The data highlight a "long-pore" effect in Kir channels, and emphasize the importance of considering blocker permeation when assessing the effects of mutations on apparent blocker affinity.     

Extracellular links in Kir subunits control the unitary conductance of SUR/Kir6.0 ion channels.

Potassium (K+) channels are highly selective for K+ ions but their unitary conductances are quite divergent. Although Kir6.1 and Kir6.2 are highly homologous and both form functional K+ channels with sulfonylurea receptors, their unitary conductances measured with 150 mM extracellular K+ are approximately 35 and 80 pS, respectively. We found that a chain of three amino acid residues N123-V124-R125 of Kir6.1 and S113-I114-H115 of Kir6.2 in the M1-H5 extracellular link and single residues M148 of Kir6.1 and V138 of Kir6.2 in the H5-M2 link accounted for the difference. By using a 3D structure model of Kir6.2, we were able to recognize two independent plausible mechanisms involved in the determination of single channel conductance of the Kir6.0 subunits: (i) steric effects at Kir6.2V138 or Kir6.1M148 in the H5-M2 link influence directly the diffusion of K+ ions; and (ii) structural constraints between Kir6.2S113 or Kir6. 1N123 in the M1-H5 link and Kir6.2R136 or Kir6.1R146 near the H5 region control the conformation of the permeation pathway. These mechanisms represent a novel and possibly general aspect of the control of ion channel permeability.     

K channel subconductance levels result from heteromeric pore conformations

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (K(v)2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the "open" state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open-close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.     

A role for the middle C terminus of G-protein-activated inward rectifier potassium channels in regulating gating

We have used sulfhydryl-modifying reagents to investigate the regulation of G-protein-activated inward rectifier potassium (GIRK) channels via their cytoplasmic domains. Modification of either the conserved N-terminal cysteines (GIRK1C53 and GIRK2C65) or the middle C-terminal cysteines (GIRK1C310 and GIRK2C321) independently inhibited GIRK1/GIRK2 heteromeric channels. With the exception of GIRK2C65, these cysteines were relatively inaccessible to large modifying reagents. The accessibility was further reduced by a mutation at the end of the second transmembrane domain that stabilized the open state of the channel. Thus it is unlikely that these cysteines line the permeation pathway of the open pore. Cysteines introduced 3 and 6 amino acids upstream of GIRK2C321 (G318C and E315C) were considerably more accessible. The effect of modification was dependent on the charge of the reagent. Modification of E315C in GIRK2 and E304C in GIRK1 by sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) increased the current by approximately 17-fold, whereas modification by 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA(+)), abolished the current. There was no effect on single-channel conductance. Thus a switch in charge at this middle C-terminal position was sufficient to gate the channel open and closed. This glutamate is conserved in all members of the Kir family. The E303K mutation in Kir2.1 inhibits channel function and causes Andersen's syndrome in humans (Plaster, N. M., Tawil, R., Tristani-Firouzi, M., Canun, S., Bendahhou, S., Tsunoda, A., Donaldson, M. R., Iannaccone, S. T., Brunt, E., Barohn, R., Clark, J., Deymeer, F., George, A. L., Jr., Fish, F. A., Hahn, A., Nitu, A., Ozdemir, C., Serdaroglu, P., Subramony, S. H., Wolfe, G., Fu, Y. H., and Ptacek, L. J. (2001) Cell 105, 511-519 and Preisig-Muller, R., Schlichthorl, G., Goerge, T., Heinen, S., Bruggemann, A., Rajan, S., Derst, C., Veh, R. W., and Daut, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 7774-7779). Our results suggest that this residue regulates channel gating through an electrostatic mechanism.     

Determination of the functional unit of the cystic fibrosis transmembrane conductance regulator chloride channel. One polypeptide forms one pore

The magnitudes and distributions of subconductance states were studied in chloride channels formed by the wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and in CFTRs bearing amino acid substitutions in transmembrane segment 6. Within an open burst, it was possible to distinguish three distinct conductance states referred to as the full conductance, subconductance 1, and subconductance 2 states. Amino acid substitutions in transmembrane segment 6 altered the duration and probability of occurrence of these subconductance states but did not greatly alter their relative amplitudes. Results from real time measurements indicated that covalent modification of single R334C-CFTR channels by [2-(trimethylammonium)ethyl]methanethiosulfonate resulted in the simultaneous modification of all three conductance levels in what appeared to be a single step, without changing the proportion of time spent in each state. This behavior suggests that at least a portion of the conduction path is common to all three conducting states. The time course for the modification of R334C-CFTR, measured in outside-out macropatches using a rapid perfusion system, was also consistent with a single modification step as if each pore contained only a single copy of the cysteine at position 334. These results are consistent with a model for the CFTR conduction pathway in which a single anion-conducting pore is formed by a single CFTR polypeptide.     

Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification

Inwardly rectifying K+ channels bind intracellular magnesium and polyamines to generate inward rectification. We have examined the architecture of the inner pore of Kir2.1 channels by covalently attaching a constrained number (from one to four) of positively charged moieties of different sizes to the channel. Our results indicate that the inner pore is formed solely by the second transmembrane segment and is unprecedentedly wide. At a position critical for inward rectification (D172), the pore is sufficiently wide to bind three Mg2+ ions or polyamine molecules simultaneously. Single-channel recordings directly demonstrate that partially modified channels exhibit distinct subconductance levels. Such a wide inner pore may greatly facilitate ion permeation and high-affinity binding of multiple pore blockers to generate strong inward rectification.     

Topogenesis of two transmembrane type K+ channels,Kir 2.1 and KcsA

Potassium channels, which control the passage of K+ across cell membranes, have two transmembrane segments, M1 and M2, separated by a hydrophobic P region containing a highly conserved signature sequence. Here we analyzed the membrane topogenesis characteristics of the M1, M2, and P regions in two animal and bacterial two-transmembrane segment-type K+ channels, Kir 2.1 and KcsA, using an in vitro translation and translocation system. In contrast to the equivalent transmembrane segment, S5, in the voltage-dependent K+ channel, KAT1, the M1 segment in KcsA, was found to have a strong type II signal-anchor function, which favors the Ncyt/Cexo topology. The N-terminal cytoplasmic region was required for efficient, correctly orientated integration of M1 in Kir 2.1. Analysis of N-terminal modification by in vitro metabolic labeling showed that the N terminus in Kir 2.1 was acetylated. The hydrophobic P region showed no topogenic function, allowing it to form a loop, but not a transmembrane structure in the membrane; this region was transiently exposed in the endoplasmic reticulum lumen during the membrane integration process. M2 was found to possess a stop-transfer function and a type I signal-anchor function, enabling it to span the membrane. The C-terminal cytoplasmic region in KcsA was found to affect the efficiency with which the M2 achieved their final structure. Comparative topogenesis studies of Kir 2.1 and KcsA allowed quantification of the relative contributions of each segment and the cytoplasmic regions to the membrane topology of these two proteins. The membrane topogenesis of the pore-forming structure is discussed using results for Kir 2.1, KcsA, and KAT1.     

Kir5.1 underlies long-lived subconductance levels in heteromeric Kir4.1/Kir5.1 channels from Xenopus tropicalis

The inwardly-rectifying potassium channel subunit Kir5.1 selectively co-assembles with members of the Kir4.0 subfamily to form novel pH-sensitive heteromeric channels with unique single channel properties. In this study, we have cloned orthologs of Kir4.1 and Kir5.1 from the genome of the amphibian, Xenopus tropicalis (Xt). Heteromeric XtKir4.1/XtKir5.1 channels exhibit similar macroscopic current properties to rat Kir4.1/Kir5.1 with a faster time-dependent rate of activation. However, single channel analysis of heteromeric XtKir4.1/XtKir5.1 channels reveals that they have markedly different long-lived, multi-level subconductance states. Furthermore, we demonstrate that the XtKir5.1 subunit is responsible for these prominent subconductance levels. These results are consistent with a model in which the slow transitions between sublevel states represent the movement of individual subunits. These novel channels now provide an excellent model system to determine the structural basis of subconductance levels and contribution of heteromeric pore architecture to this process.     

Stable cation coordination at a single outer pore residue defines permeation properties in Kir channels

In epithelial Kir7.1 channels a non-conserved methionine in the outer pore region adjacent to the G-Y-G selectivity filter (position +2) was found to determine unique properties for permeant and blocking ions characteristic of a K(+) channel in a single-occupancy state. The monovalent cation permeability sequence of Kir7.1 channels expressed in Xenopus oocytes was Tl(+)>K(+)>Rb(+)NH(4)(+)>Cs(+)>Na(+)>Li(+), but the macroscopic conductance for Rb(+) was approximately 8-fold larger than for the smaller K(+) ions, and decreased approximately 40-fold with the conserved arginine at the +2 position (Kir7.1M125R). Moreover, in Kir7.1 Rb(+) restored the typical permeation properties of other multi-ion channels indicating that a stable coordination of permeant ions at the +2 position defines the initial step in the conduction pathway of Kir channels.     

Identification of gamma-aminobutyric acid receptor-interacting factor 1 (TRAK2) as a trafficking factor for the K+ channel Kir2.1

To identify proteins that regulate potassium channel activity and expression, we performed functional screening of mammalian cDNA libraries in yeast that express the mammalian K(+) channel Kir2.1. Growth of Kir2.1-expressing yeast in media with low K(+) concentration is a function of K(+) uptake via Kir2.1 channels. Therefore, the host strain was transformed with a human cDNA library, and cDNA clones that rescued growth at low K(+) concentration were selected. One of these clones was identical to the protein of unknown function isolated previously as gamma-aminobutyric acid receptor-interacting factor 1 (GRIF-1) (Beck, M., Brickley, K., Wilkinson, H., Sharma, S., Smith, M., Chazot, P., Pollard, S., and Stephenson, F. (2002) J. Biol. Chem. 277, 30079-30090). GRIF-1 specifically enhanced Kir2.1-dependent growth in yeast and Kir2.1-mediated (86)Rb(+) efflux in HEK293 cells. Quantitative microscopy and flow cytometry analysis of immunolabeled surface Kir2.1 channel showed that GRIF-1 significantly increased the number of Kir2.1 channels in the plasma membrane of COS and HEK293 cells. Physical interaction of Kir2.1 channel and GRIF-1 was demonstrated by co-immunoprecipitation from HEK293 lysates and yeast two-hybrid assay. In vivo association of Kir2.1 and GRIF-1 was demonstrated by co-immunoprecipitation from brain lysate. Yeast two-hybrid assays showed that an N-terminal region of GRIF-1 interacts with a C-terminal region of Kir2.1. These results indicate that GRIF-1 binds to Kir2.1 and facilitates trafficking of this channel to the cell surface.     

Gain-of-function mutations reveal expanded intermediate states and a sequential action of two gates in MscL

The tension-driven gating transition in the large mechanosensitive channel MscL proceeds through detectable states of intermediate conductance. Gain-of-function (GOF) mutants with polar or charged substitutions in the main hydrophobic gate display altered patterns of subconducting states, providing valuable information about gating intermediates. Here we present thermodynamic analysis of several GOF mutants to clarify the nature and position of low-conducting conformations in the transition pathway. Unlike wild-type (WT) MscL, which predominantly occupies the closed and fully open states with very brief substates, the mild V23T GOF mutant frequently visits a multitude of short-lived subconducting states. Severe mutants V23D and G22N open in sequence: closed (C) --> low-conducting substate (S) --> open (O), with the first subtransition occurring at lower tensions. Analyses of equilibrium state occupancies as functions of membrane tension show that the C-->S subtransition in WT MscL is associated with only a minor conductance increment, but the largest in-plane expansion and free energy change. The GOF substitutions strongly affect the first subtransition by reducing area ((Delta)A) and energy ((Delta)E) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger (Delta)E associated with the second (S-->O) subtransition, but a (Delta)A similar to WT. The area changes indicate that closed conformations of GOF mutants are physically preexpanded. The tension dependencies of rate constants for channel closure (k(off)) predict different positions of rate-limiting barriers on the energy-area profiles for WT and GOF MscL. The data support the two-gate mechanism in which the first subtransition (C-->S) can be viewed as opening of the central (M1) gate, resulting in an expanded water-filled "leaky" conformation. Strong facilitation of this step by polar GOF substitutions suggests that separation of M1 helices associated with hydration of the pore in WT MscL is the major energetic barrier for opening. Mutants with a stabilized S1 gate demonstrate impeded transitions from low-conducting substates to the fully open state, whereas extensions of S1-M1 linkers result in a much higher probability of reverse O-->S transitions. These data strongly suggest that the bulk of conductance gain in the second subtransition (S-->O) occurs through the opening of the NH2-terminal (S1) gate and the linkers are coupling elements between the M1 and S1 gates.     

Mutations stabilizing an open conformation within the external region of the permeation pathway of the potassium channel KcsA

Four subunits of the bacterial Streptomyces lividans protein KcsA form a K+ channel which can be functionally reconstituted in vitro. Here we show that substitution of the tyrosine residue 82 by cysteine, valine or threonine, but not by glycine, led to functional channel types. Like the wild-type (WT) and an L81C channel, the mutant channels exhibit an internal pH-sensitive side and are cation selective. Based on the relative positions of the blocker tetraethylammonium within the electric field, the external entryways of the channels are concluded to have similar dimensions. For inward currents, the WT and the mutant channels vary in the occupancy of their subconductance states and concomitantly in their mean currents. Rectification properties are scarcely (L81C), little (Y82C) or considerably (Y82T and Y82V) altered. The data suggest that the amino acid type in position 82 stabilizes to varying degrees an open conformation within the external region of the permeation pathway.     

Ser165 in the second transmembrane region of the Kir2.1 channel determines its susceptibility to blockade by intracellular Mg2+

The strong inward rectification of Kir2.1 currents is reportedly due to blockade of the outward current by cytoplasmic magnesium (Mg(2+)(i)) and polyamines, and is known to be determined in part by three negatively charged amino acid residues: Asp172, Glu224, and Glu299 (D172, E224, E299). Our aim was to identify additional sites contributing to the inward rectification of Kir2.1 currents. To accomplish this, we introduced into wild-type Kir2.1 and its D172N and D172N & E224G & E299S mutants various point mutations selected on the basis of a comparison of the sequences of Kir2.1 and the weak rectifier sWIRK. By analyzing macroscopic currents recorded from Xenopus oocytes using two-electrode voltage clamp, we determined that S165L mutation decreases inward rectification, especially with the triple mutant. The susceptibility to blockade by intracellular blockers was examined using HEK293 transfectants and the inside-out patch clamp configuration. The sensitivity to spermine was significantly diminished in the D172N and triple mutant, but not the S165L mutant. Both the S165L and D172N mutants were less susceptible to blockade by Mg(2+)(i) than the wild-type channel, and the susceptibility was still lower in the D172N & S165L double mutant. These results suggest that S165 is situated deeper into the pore from inside than D172, where it is accessible to Mg(2+)(i) but not to spermine. The single channel conductance of the D172N mutant was similar to that of the wild-type Kir2.1, whereas the conductance of the S165L mutant was significantly lower. Permeation by extracellular Rb+ (Rb(+)(o)) was dramatically increased by S165L mutation, but was increased only slightly by D172N mutation. By contrast, the Rb+/K+ permeability ratio was increased equally by D172N and S165L mutation. We therefore propose that S165 forms the narrowest part of the Kir2.1 pore, where both extracellular and intracellular blockers plug the permeation pathway.     

Ion channels formed by a highly charged peptide.

A peptide (MA-beta) corresponding to a segment of the nicotinic acetylcholine receptor (AChR) that has amphipathic alpha-helical periodicity forms ion channels in artificial phospholipid bilayers. The MA-beta ion channels are very stable, comprise two discrete conductance states, and undergo rapid, flickering-type closings. The discrete-conductance ion channels formed by MA-beta contrast with the continuous-conductance ion channels formed by a peptide (M2-delta) identical in sequence with M2 [Oiki, S., Danho, W., Madison, V., & Montal, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8703-8707], a putative transmembrane segment of the AChR. Neither MA-beta nor M2-delta sufficiently mimics the electrophysiological properties of the native AChR. We suggest that peptide ion channels can be classified into at least three general groups: discrete-conductance channels, such as MA-beta; continuous-conductance channels, such as M2-delta; and membrane disruptors, such as those formed by short, amphipathic alpha-helical peptides.     

Subconductance States of a Mutant NMDA Receptor Channel Kinetics,Calcium, and Voltage Dependence

The kinetic properties of main and subconductance states of a mutant mouse N-methyl-d-aspartate (NMDA) receptor channel were examined. Recombinant receptors made of ζ-ε₂ (NR1-NR2B) subunits having asparagine-to-glutamine mutations in the M2 segment (ζN598Q /ε₂N589Q) were expressed in Xenopus oocytes. Single channel currents recorded from outside-out patches were analyzed using hidden Markov model techniques. In Ca²⁺-free solutions, an open receptor channel occupies a main conductance (93 pS) and a subconductance (62 pS) with about equal probability. There are both brief and long-lived subconductance states, but only a single main level state. At −80 mV, the lifetime of the main and the longer-lived sub level are both ∼3.3 ms. The gating of the pore and the transition between conductance levels are essentially independent processes. Surprisingly, hyperpolarization speeds both the sub-to-main and main-to-sub transition rate constants (∼120 mV/e-fold change), but does not alter the equilibrium occupancies. Extracellular Ca²⁺ does not influence the transition rate constants. We conclude that the subconductance levels arise from fluctuations in the energetics of ion permeation through a single pore, and that the voltage dependence of these fluctuations reflects the modulation by the membrane potential of the barrier between the main and subconductance conformations of the pore.     

Cysteine residues in the nucleotide binding domains regulate the conductance state of CFTR channels

Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATPgammaS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance.     

Cytoplasmic acidosis induces multiple conductance states in ATP-sensitive potassium channels of cardiac myocytes

We studied the effect of cytoplasmic acidosis on the ionic conducting states of ATP-sensitive potassium channels in heart ventricular cells of guinea pigs and rabbits by using a patch-clamp technique with inside-out patch configuration. Under normal conditions (pH 7.4), the channel alternated between a closed state and a main open state in the absence of nucleotides on the cytoplasmic side. As internal pH was reduced below 6.5, the single channel current manifested distinct subconductance levels. The probability of the appearance of these subconductance levels was pH dependent with a greater probability of subconductance states at lower pH. A variance-mean amplitude analysis technique revealed two subconductance levels approximately equally spaced between the main open level and the closed level (63 and 33%). A current-voltage plot of the two subconductance levels and the main level showed that they had similar reversal potentials and rectification properties. An intrinsic flickering gating property characteristic of these ATP-sensitive channels was found unchanged in the 63% subconductance state, suggesting that this subconductance state and the main conductance state share similar ion pore properties (including ion selection and block) and similar gating mechanisms. The appearance of the subconductance states decreased as ionic strength was increased, and the subconductance states were also slightly voltage dependent, suggesting an electrostatic interaction between the protons and the negative surface charge in the vicinity of the binding sites, which may be close to the inner entrance of the ion pore. Proteolytic modification of the channel on the cytoplasmic side with trypsin did not abolish the subconductance levels. External acidosis did not induce subconductance levels. These results suggest that protons bound to the negatively charged group at the inner entrance of the channel ion pore may induce conformational changes, leading to partially reduced conductance states.     

Role of conserved glycines in pH gating of Kir1.1 (ROMK)

Gating of inward rectifier Kir1.1 potassium channels by internal pH is believed to occur when large hydrophobic leucines, on each of the four subunits, obstruct the permeation path at the cytoplasmic end of the inner transmembrane helices (TM2). In this study, we examined whether closure of the channel at this point involves bending of the inner helix at one or both of two highly conserved glycine residues (corresponding to G134 and G143 in KirBac1.1) that have been proposed as putative "gating hinges" for potassium channels. Replacement of these conserved inner helical glycines by less flexible alanines did not abolish gating but shifted the apparent pKa from 6.6 +/- 0.01 (wild-type) to 7.1 +/- 0.01 for G157A-Kir1.1b, and to 7.3 +/- 0.01 for G148A-Kir1.1b. When both glycines were mutated the effect was additive, shifting the pKa by 1.2 pH units to 7.8 +/- 0.04 for the double mutant: G157A+G148A. At this pKa, the double mutant would remain completely closed under physiological conditions. In contrast, when the glycine at G148 was replaced by a proline, the pKa was shifted in the opposite direction from 6.6 +/- 0.01 (wild-type) to 5.7 +/- 0.01 for G148P. Although conserved glycines at G148 and G157 made it significantly easier to open the channel, they were not an absolute requirement for pH gating in Kir1.1. In addition, none of the glycine mutants produced more than small changes in either the cell-attached or excised single-channel kinetics which, in this channel, argues against changes in the selectivity filter. The putative pH sensor at K61-Kir1.1b, (equivalent to K80-Kir1.1a) was also examined. Mutation of this lysine to an untitratable methionine did not abolish pH gating, but shifted the pKa into an acid range from 6.6 +/- 0.01 to 5.4 +/- 0.04, similar to pH gating in Kir2.1. Hence K61-Kir1.1b cannot function as the exclusive pH sensor for the channel, although it may act as one of multiple pH sensors, or as a link between a cytoplasmic sensor and the channel gate. K61-Kir1.1b also interacted differently with the two glycine mutations. Gating of the double mutant: K61M+G148A was indistinguishable from K61M alone, whereas gating of K61M+G157A was midway between the alkaline pKa of G157A and the acid pKa of K61M. Finally, closure of ROMK, G148A, G157A, and K61M all required the same L160-Kir1.1b residue at the cytoplasmic end of the inner transmembrane helix. Hence in wild-type and mutant channels, closure occurs by steric occlusion of the permeation path by four leucine side chains (L160-Kir1.1b) at the helix bundle crossing. This is facilitated by the conserved glycines on TM2, but pH gating in Kir1.1 does not absolutely require glycine hinges in this region.