Long-pore Electrostatics in Inward-rectifier Potassium Channels

Inward-rectifier potassium (Kir) channels differ from the canonical K⁺ channel structure in that they possess a long extended pore (~85 Å) for ion conduction that reaches deeply into the cytoplasm. This unique structural feature is presumably involved in regulating functional properties specific to Kir channels, such as conductance, rectification block, and ligand-dependent gating. To elucidate the underpinnings of these functional roles, we examine the electrostatics of an ion along this extended pore. Homology models are constructed based on the open-state model of KirBac1.1 for four mammalian Kir channels: Kir1.1/ROMK, Kir2.1/IRK, Kir3.1/GIRK, and Kir6.2/KATP. By solving the Poisson-Boltzmann equation, the electrostatic free energy of a K⁺ ion is determined along each pore, revealing that mammalian Kir channels provide a favorable environment for cations and suggesting the existence of high-density regions in the cytoplasmic domain and cavity. The contribution from the reaction field (the self-energy arising from the dielectric polarization induced by the ion's charge in the complex geometry of the pore) is unfavorable inside the long pore. However, this is well compensated by the electrostatic interaction with the static field arising from the protein charges and shielded by the dielectric surrounding. Decomposition of the static field provides a list of residues that display remarkable correspondence with existing mutagenesis data identifying amino acids that affect conduction and rectification. Many of these residues demonstrate interactions with the ion over long distances, up to 40 Å, suggesting that mutations potentially affect ion or blocker energetics over the entire pore. These results provide a foundation for understanding ion interactions in Kir channels and extend to the study of ion permeation, block, and gating in long, cation-specific pores.     

Characterization of Single Inward Rectifier Potassium Channels from Embryonic Xenopus laevis Myocytes

Single inward rectifier K⁺ channels were studied in Xenopus laevis embryonic myocytes. We have characterized in detail the channel which is most frequently observed (Kir) although we routinely observe three other smaller current levels with the properties of inward rectifier K⁺ channels (Kir_(0.3), Kir_(0.5) and Kir_(0.7)). For Kir, slope conductances of inward currents were 10.3, 20.3, and 27.9 pS, in 60, 120 and 200 mM [K⁺] _o respectively. Extracellular Ba²⁺ blocked the normally high channel activity in a concentration-dependent manner (K _A = 7.8 μm, −90 mV). In whole-cell recordings of inward rectifier K⁺ current, marked voltage dependence of Ba²⁺ block over the physiological range of potentials was observed. We also examined current rectification. Following step depolarizations to voltages positive to E _K , outward currents through Kir channels were not observed even when the cytoplasmic face of excised patches were exposed to Mg²⁺-free solution at pH 9.1. This was probably also true for Kir_(0.3), Kir_(0.5) and Kir_(0.7) channels. We then examined the possibility of modulation of Kir channel activity and found neither ATP nor GTP-γS had any effect on Kir channel activity when added to the solution perfusing the cytoplasmic face of a patch. Kinetic analysis revealed Kir channels with a single open state (mean dwell time 72 msec) and two closed states (time constants 1.4, 79 msec). These results suggest that the native Kir channels of Xenopus myocytes have similar properties to the cloned strong inward rectifier K⁺ channels, in terms of conductance, kinetics and barium block but does show some differences in the effects of modulators of channel activity. Furthermore, skeletal muscle may contain either different inward rectifier channels or a single-channel type which can exist in stable subconductance states. Received: 16 September 1996/Revised: 14 March 1997     

Block of the Kir2.1 Channel Pore by Alkylamine Analogues of Endogenous Polyamines

Inward rectification induced by mono- and diaminoalkane application to inside-out membrane patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines, increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a limiting minimal value of ∼1.5 (n = 2 methylene groups) to ∼4 (n = 10 methylene groups). These observations lead us to hypothesize that monoamines and diamines block inward rectifier K⁺ channels by entering deeply into a long, narrow pore, displacing K⁺ ions to the outside of the membrane, with this displacement of K⁺ ions contributing to “extra” charge movement. All monoamines are proposed to lie with the “head” amine at a fixed position in the pore, determined by electrostatic interaction, so that zδ is independent of monoamine alkyl chain length. The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases, thus displacing more K⁺ ions to the outside, resulting in charge movement (zδ) increasing with the increase in alkyl chain length.     

Kir4.1/Kir5.1 channels possess strong intrinsic inward rectification determined by a voltage-dependent K+-flux gating mechanism

Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg²⁺. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K⁺-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K⁺-flux is convergent with the gating of PIP₂ because, at a saturating concentration, PIP₂ greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K⁺-flux gating.     

Evidence for the Direct Interaction of Spermine with the Inwardly Rectifying Potassium Channel

The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K⁺ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg²⁺ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg²⁺ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein βγ-subunit (Gβγ). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 μm, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gβγ-induced conformational rearrangement.The inwardly rectifying K⁺ channel (Kir)³ plays a pivotal role in controlling resting membrane potential, K⁺ homeostasis, heart rate, and hormone secretion (1). The inward rectification property of Kir is reportedly due to the voltage-dependent blockade of the outward current by intracellular polyamines and Mg²⁺ (2–6). The importance of the electrostatic interactions of polyamines and Mg²⁺ with the acidic residues in Kirs has been indicated by electrophysiological studies in combination with site-directed mutagenesis, which have been accelerated by the recent progress in the structural analyses of Kirs (7–10). The crystal structures revealed that they form a symmetric tetramer, in which the transmembrane pore, containing the K⁺-selective filter and the cytoplasmic pore consisting of the N- and C-terminal regions form a long pore for the K⁺ pathway.In Kir2.1/IRK1, the strongest inward rectifier in the Kir family, negatively charged acidic residues that influence the inward rectification have been identified, including Asp-172 (11) in the transmembrane pore and Glu-224, Asp-255, Asp-259, and Glu-299 in the cytoplasmic pore (9, 12–19). These acidic residues are assumed to be responsible for the electrostatic interaction with polyamines, in which the nitrogen atoms are positively charged at neutral pH (20).In other members of the Kir family, some of the electronegative residues are replaced by neutral ones, such as Gly and Ser. Although the total number of acidic residues correlates with the strength of inward rectification, the correlation cannot be completely explained for the strong rectifiers (9). In Kir3.1/GIRK1, which exhibits relatively strong inward rectification among the Kir family proteins, two of the four important acidic residues in Kir2.1 (Glu-224 and Asp-255) are replaced by Ser (Ser-225 and Ser-256 in Kir3.1, respectively). This suggests that the binding site(s) and stoichiometry of polyamine binding differ among the Kir proteins.Although the binding of polyamines to the cytoplasmic pore of Kirs is considered to be required for the blockade of the transmembrane pore to enable the inward rectification (16, 19, 21), the functional role of the polyamine binding to the cytoplasmic pore is still unclear. In particular, Kir3/GIRK is activated by the binding of G-protein βγ-subunits (Gβγ) to the cytoplasmic pore through the conformational rearrangement of the channel (22). Thus, the elucidation of the binding mode of Kir and polyamines based on the detection of their direct interaction as well as the effects of the binding on the protein conformation provides insights into the functional roles of the cytoplasmic pore in the gating and the inward rectifying property of the channel.Here, we report the thermodynamic and structural analyses of the interaction between spermine and the intracellular regions of Kir3.1/GIRK1. Our isothermal titration calorimetry (ITC) results indicated that two spermine molecules bind to a tetramer of Kir3.1/GIRK1, with a dissociation constant (K_d) value of 26 μm. NMR analyses together with ITC results revealed the spermine binding mode, in which one spermine molecule bridges two Asp-260 side chains of two adjacent subunits, with the alteration of the relative orientations of the four subunits. The spermine binding mode revealed here suggests the functional roles of the spermine binding to the cytoplasmic pore: in the resting state of the channel, spermine stabilizes a certain channel conformation, and upon activation, spermine is dissociated from the cytoplasmic pore through the Gβγ-induced conformational rearrangement, leading to rapid translocation to the transmembrane pore to block the outward K⁺ current.     

Nonindependent K+ Movement through the Pore in IRK1 Potassium Channels

We measured unidirectional K⁺ in- and efflux through an inward rectifier K channel (IRK1) expressed in Xenopus oocytes. The ratio of these unidirectional fluxes differed significantly from expectations based on independent ion movement. In an extracellular solution with a K⁺ concentration of 25 mM, the data were described by a Ussing flux-ratio exponent, n′, of ∼2.2 and was constant over a voltage range from −50 to −25 mV. This result indicates that the pore of IRK1 channels may be simultaneously occupied by at least three ions. The IRK1 n′ value of 2.2 is significantly smaller than the value of 3.5 obtained for Shaker K channels under identical conditions. To determine if other permeation properties that reflect multi-ion behavior differed between these two channel types, we measured the conductance (at 0 mV) of single IRK1 channels as a function of symmetrical K⁺ concentration. The conductance could be fit by a saturating hyperbola with a half-saturation K⁺ activity of 40 mM, substantially less than the reported value of 300 mM for Shaker K channels. We investigated the ability of simple permeation models based on absolute reaction rate theory to simulate IRK1 current–voltage, conductance, and flux-ratio data. Certain classes of four-barrier, three-site permeation models are inconsistent with the data, but models with high lateral barriers and a deep central well were able to account for the flux-ratio and single channel data. We conclude that while the pore in IRK1 and Shaker channels share important similarities, including K⁺ selectivity and multi-ion occupancy, they differ in other properties, including the sensitivity of pore conductance to K⁺ concentration, and may differ in the number of K⁺ ions that can simultaneously occupy the pore: IRK1 channels may contain three ions, but the pore in Shaker channels can accommodate four or more ions.     

Conformational Changes Underlying Pore Dilation in the Cytoplasmic Domain of Mammalian Inward Rectifier K+ Channels

The cytoplasmic domain of inward rectifier K⁺ (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent βD and βH strands, respectively. We mutated both residues, expressed them with the m₂-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K⁺ currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the βH and βI strands of the adjacent subunit. Because the βH strand forms a β sheet with the βI and βD strands, the immobilization of the pore-forming β sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.     

Voltage-dependent inhibition of outward Kir2.1 currents by extracellular spermine

Outward currents through inward rectifier Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells. Extracellular monovalent and divalent cations have been shown to reduce outward K⁺ conductance. In the present study, we examined whether spermine, with four positive charges, also inhibits outward Kir2.1 currents. We found that extracellular spermine inhibits steady-state outward Kir2.1 currents, an effect that increases as the voltage becomes more depolarizing, similar to that observed for intracellular spermine. However, several lines of evidence suggest that extracellular spermine does not inhibit outward currents by entering the cytoplasmic pore. Site-directed mutagenesis studies support that extracellular spermine directly interacts with the extracellular domain. In addition, we found that the voltage-dependent decay of outward Kir2.1 currents was necessary for inhibition by extracellular spermine. Further, a region at or near the selectivity filter and the cytoplasmic pore are involved in the voltage-dependent decay and thus in the inhibition of outward currents by extracellular spermine. Taken together, the data suggest that extracellular spermine bound to the mouth of the extracellular pore may induce an allosteric effect on voltage-dependent decay of outward currents, a process in which a region in the vicinity of the selectivity filter and cytoplasmic pore are involved. This study reveals that the extracellular pore domain, the selectivity filter and the cytoplasmic pore are in communication and this coupling is involved in modulating K⁺ conduction in the Kir2.1 channel.     

Modulation of Kir4.2 rectification properties and pHi-sensitive run-down by association with Kir5.1

Inwardly rectifying K⁺ channels (Kir) comprise seven subfamilies that can be subdivided further on the basis of cytosolic pH (pHi) sensitivity, rectification strength and kinetics, and resistance to run-down. Although distinct residues within each channel subunit define these properties, heteromeric association with other Kir subunits can modulate them. We identified such an effect in the wild-type forms of Kir4.2 and Kir5.1 and used this to further understand how the functional properties of Kir channels relate to their structures. Kir4.2 and a Kir4.2-Kir5.1 fusion protein were expressed in HEK293 cells. Inward currents from Kir4.2 were stable over 10 min and pHi-insensitive (pH 6 to 8). Conversely, currents from Kir4.2-Kir5.1 exhibited a pHi-sensitive run-down at slightly acidic pHi. At pHi 7.2, currents in response to voltage steps positive to E_K were essentially time independent for Kir4.2 indicating rapid block by Mg²⁺. Coexpression with Kir5.1 significantly increased the blocking time constant, and increased steady-state outward current characteristic of weak rectifiers. Recovery from blockade at negative potentials was voltage dependent and 2 to 10 times slower in the homomeric channel. These results show that Kir5.1 converts Kir4.2 from a strong to a weak rectifier, rendering it sensitive to pHi, and suggesting that Kir5.1 plays a role in fine-tuning Kir4.2 activity.     

Interactions of external K+ and internal blockers in a weak inward-rectifier K+ channel

We investigated the effects of changing extracellular K⁺ concentrations on block of the weak inward-rectifier K⁺ channel Kir1.1b (ROMK2) by the three intracellular cations Mg²⁺, Na⁺, and TEA⁺. Single-channel currents were monitored in inside-out patches made from Xenopus laevis oocytes expressing the channels. With 110 mM K⁺ in the inside (cytoplasmic) solution and 11 mM K⁺ in the outside (extracellular) solution, these three cations blocked K⁺ currents with a range of apparent affinities (K_i (0) = 1.6 mM for Mg²⁺, 160 mM for Na⁺, and 1.8 mM for TEA⁺) but with similar voltage dependence (zδ = 0.58 for Mg²⁺, 0.71 for Na⁺, and 0.61 for TEA⁺) despite having different valences. When external K⁺ was increased to 110 mM, the apparent affinity of all three blockers was decreased approximately threefold with no significant change in the voltage dependence of block. The possibility that the transmembrane cavity is the site of block was explored by making mutations at the N152 residue, a position previously shown to affect rectification in Kir channels. N152D increased the affinity for block by Mg²⁺ but not for Na⁺ or TEA⁺. In contrast, the N152Y mutation increased the affinity for block by TEA⁺ but not for Na⁺ or Mg²⁺. Replacing the C terminus of the channel with that of the strong inward-rectifier Kir2.1 increased the affinity of block by Mg²⁺ but had a small effect on that by Na⁺. TEA⁺ block was enhanced and had a larger voltage dependence. We used an eight-state kinetic model to simulate these results. The effects of voltage and external K⁺ could be explained by a model in which the blockers occupy a site, presumably in the transmembrane cavity, at a position that is largely unaffected by changes in the electric field. The effects of voltage and extracellular K⁺ are explained by shifts in the occupancy of sites within the selectivity filter by K⁺ ions.     

Electrostatics in the cytoplasmic pore produce intrinsic inward rectification in kir2.1 channels

Inward rectifier K+ channels are important in regulating membrane excitability in many cell types. The physiological functions of these channels are related to their unique inward rectification, which has been attributed to voltage-dependent block. Here, we show that inward rectification can also be induced by neutral and positively charged residues at site 224 in the internal vestibule of tetrameric Kir2.1 channels. The order of extent of inward rectification is E224K mutant > E224G mutant > wild type in the absence of internal blockers. Mutating the glycines at the equivalent sites to lysines also rendered weak inward rectifier Kir1.1 channels more inwardly rectifying. Also, conjugating positively charged methanethiosulfonate to the cysteines at site 224 induced strong inward rectification, whereas negatively charged methanethiosulfonate alleviated inward rectification in the E224C mutant. These results suggest that charges at site 224 may control inward rectification in the Kir2.1 channel. In a D172N mutant, spermine interacting with E224 and E299 induced channel inhibition during depolarization but did not occlude the pore, further suggesting that a mechanism other than channel block is involved in the inward rectification of the Kir2.1 channel. In this and our previous studies we showed that the M2 bundle crossing and selectivity filter were not involved in the inward rectification induced by spermine interacting with E224 and E299. We propose that neutral and positively charged residues at site 224 increase a local energy barrier, which reduces K+ efflux more than K+ influx, thereby producing inward rectification.     

Gating Properties of Inward-Rectifier Potassium Channels: Effects of Permeant Ions

Two inward-rectifier K⁺ channels, ROMK2 (Kir1.1b) and IRK1 (Kir2.1), were expressed in Xenopus oocytes and their gating properties were studied in cell-attached membrane patches. The gating properties depended strongly on the ion being conducted (K⁺, NH₄ ⁺, Rb⁺, or Tl⁺), suggesting tight coupling between permeation and gating. Mean open times were strongly dependent on the nature of the conducted ion. For ROMK2 the order from the longest to the shortest times was K⁺ > Rb⁺ > Tl⁺ > NH₄ ⁺. For IRK1 the sequence was K⁺ > NH₄ ⁺ > Tl⁺. In both cases the open times decreased monotonically as the membrane voltage was hyperpolarized. Both the absolute values and the voltage dependence of closed times were dependent on the conducted species. ROMK2 showed a single closed state whose mean lifetimes were biphasic functions of voltage. The maxima were at various voltages for different ions. IRK1 had at least two closed states whose lifetimes decreased monotonically with K⁺, increased monotonically with Tl⁺, and were relatively constant with NH₄ ⁺ as the conducted ion. We explain the ion-dependence of gating by assuming that the ions bind to a site within the permeation pathway, resulting in a stable, ion-dependent, closed state of the channel. The patterns of voltage-dependence of closed-state lifetimes, which are specific for different ions, can be explained by variations in the rate at which the bound ions leave the pore toward the inside or the outside of the cell. Received: 18 April 2001/Revised: 28 June 2001     

Lack of Negatively Charged Residues at the External Mouth of Kir2.2 Channels Enable the Voltage-Dependent Block by External Mg2+

Kir channels display voltage-dependent block by cytosolic cations such as Mg²⁺ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K⁺ concentration. In contrast, extracellular Mg²⁺ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg²⁺ and the relationship between the Mg²⁺ blockade and activity potentiation by permeant K⁺ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg²⁺ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg²⁺ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg²⁺ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg²⁺ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K⁺ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg²⁺.     

Charges in the Cytoplasmic Pore Control Intrinsic Inward Rectification and Single-Channel Properties in Kir1.1 and Kir2.1 Channels

An E224G mutation of the Kir2.1 channel generates intrinsic inward rectification and single-channel fluctuations in the absence of intracellular blockers. In this study, we showed that positively charged residues H226, R228 and R260, near site 224, regulated the intrinsic inward rectification and single-channel properties of the E224G mutant. By carrying out systematic mutations, we found that the charge effect on the intrinsic inward rectification and single-channel conductance is consistent with a long-range electrostatic mechanism. A Kir1.1 channel where the site equivalent to E224 in the Kir2.1 channel is a glycine residue does not show inward rectification or single-channel fluctuations. The G223K and N259R mutations of the Kir1.1 channel induced intrinsic inward rectification and reduced the single-channel conductance but did not generate large open-channel fluctuations. Substituting the cytoplasmic pore of the E224G mutant into the Kir1.1 channel induced open-channel fluctuations and intrinsic inward rectification. The single-channel conductance of the E224G mutant showed inward rectification. Also, a voltage-dependent gating mechanism decreased open probability during depolarization and contributed to the intrinsic inward rectification in the E224G mutant. In addition to an electrostatic effect, a close interaction of K⁺ with channel pore may be required for generating open-channel fluctuations in the E224G mutant.     

Time-dependent Outward Currents through the Inward Rectifier Potassium Channel IRK1 : The Role of Weak Blocking Molecules

Outward currents through the inward rectifier K⁺ channel contribute to repolarization of the cardiac action potential. The properties of the IRK1 channel expressed in murine fibroblast (L) cells closely resemble those of the native cardiac inward rectifier. In this study, we added Mg²⁺ (0.44–1.1 mM) or putrescine (∼0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K⁺. Without internal Mg²⁺, small outward currents flowed only at potentials between −80 (the reversal potential) and ∼−40 mV during voltage steps applied from −110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg²⁺, small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between −40 and 0 mV were concurrent with the contribution of Mg²⁺ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to −50 mV after short depolarizing steps (>0 mV), a transient increase appeared in outward currents at −50 mV. Since the peak amplitude depended on the fraction of Mg²⁺-blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg²⁺ block, followed by a re-block of channels by spermine. Shift in the holding potential (−110 to −80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg²⁺-blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg²⁺. When both spermine (1 μM, an estimated free spermine level during whole-cell recordings) and putrescine (300 μM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg²⁺ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.     

K binding in the G-loop and water cavity facilitates Ba movement in the Kir2.1 channel

K⁺ are selectively coordinated in the selectivity filter and concerted K⁺ and water movements in this region ensure high conduction rates in K⁺ channels. In channels with long pores many K⁺ binding sites are located intracellular to the selectivity filter (inner vestibule), but their contribution to permeation has not been well studied. We investigated this phenomenon by slowing the ion permeation process via blocking inwardly rectifying Kir2.1 channels with Ba²⁺ in the selectivity filter and observing the effect of K⁺ in the inner vestibule on Ba²⁺ exit. The dose-response effect of the intracellular K⁺ concentration ([K⁺]_i) on Ba²⁺ exit was recorded with and without intracellular polyamines, which compete with K⁺ for binding sites. Ba²⁺ exit was facilitated by the cooperative binding of at least three K⁺. Site-directed mutagenesis studies suggest that K⁺ interacting with Ba²⁺ bound in the selectivity filter were located in the region between selectivity filter and cytoplasmic pore, i.e. the water cavity and G-loop. One of the K⁺ binding sites was located at residue D172 and another was possibly at M301. This study provides functional evidence for the three K⁺ binding sites in the inner vestibule previously identified by crystal structure study.     

[K+] dependence of open-channel conductance in cloned inward rectifier potassium channels (IRK1, Kir2.1).

Potassium conduction through unblocked inwardly rectifying (IRK1, Kir2.1) potassium channels was measured in inside-out-patches from Xenopus oocytes, after removal of polyamine-induced strong inward rectification. Unblocked IRK1 channel current-voltage (I-V) relations show very mild inward rectification in symmetrical solutions, are linearized in nonsymmetrical solutions that bring the K+ reversal potential to extreme negative values, and follow Goldman-Hodgkin-Katz constant field equation at extreme positive E alpha. When intracellular K+ concentration (KIN) was varied, at constant extracellular K+ concentration (KOUT) the conductance at the reversal potential (GREV) followed closely the predictions of the Goldman-Hodgkin-Katz constant field equation at low concentrations and saturated sharply at concentrations of > 150 mM. Similarly, when KOUT was varied, at constant KIN, GREV saturated at concentrations of > 150 mM. A square-root dependence of conductance on KOUT is a well-known property of inward rectifier potassium channels and is a property of the open channel. A nonsymmetrical two-site three-barrier model can qualitatively explain both the I-V relations and the [K+] dependence of conductance of open IRK1 (Kir2.1) channels.     

A Conserved Arginine Residue in the Pore Region of an Inward Rectifier K Channel (IRK1) as an External Barrier for Cationic Blockers

The number, sign, and distribution of charged residues in the pore-forming H5 domain for inward-rectifying K channels (IRK1) are different from the otherwise homologous H5 domains of other voltage-gated K channels. We have mutated Arg¹⁴⁸, which is perfectly conserved in all inward rectifiers, to His in the H5 of IRK1 (Kir2.1). Channel activity was lost by the mutation, but coexpression of the mutant (R148H) along with the wild-type (WT) mRNA revealed populations of channels with reduced single-channel conductances. Long-lasting and flickery sublevels were detected exclusively for the coexpressed channels. These findings indicated that the mutant subunit formed hetero-oligomers with the WT subunit. The permeability ratio was altered by the mutation, while the selectivity sequence (K⁺ > Rb⁺ > NH₄ ⁺ >> Na⁺) was preserved. The coexpression made the IRK1 channel more sensitive to extracellular block by Mg²⁺ and Ca²⁺, and turned this blockade from a voltage-independent to a -dependent process. The sensitivity of the mutant channels to Mg²⁺ was enhanced at higher pH and by an increased ratio of mutant:WT mRNA, suggesting that the charge on the Arg site controlled the sensitivity. The blocking rate of open channel blockers, such as Cs⁺ and Ba²⁺, was facilitated by coexpression without significant change in the steady state block. Evaluation of the electrical distance to the binding site for Mg²⁺ or Ca²⁺ and that to the barrier peak for block by Cs⁺ or Ba²⁺ suggest that Arg¹⁴⁸ is located between the external blocking site for Mg²⁺ or Ca²⁺ and the deeper blocking site for Cs⁺ or Ba²⁺ in the IRK1 channel. It is concluded that Arg¹⁴⁸ serves as a barrier to cationic blockers, keeping Mg²⁺ and Ca²⁺ out from the electric field of the membrane.     

Functional roles of charged amino acid residues on the wall of the cytoplasmic pore of Kir2.1

It is known that rectification of currents through the inward rectifier K(+) channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg(2+) and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K(+) permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg(2+) or spermine and the voltage dependence of the blockade. It also reduced K(+) permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg(2+), spermine, and K(+) so that the transmembrane pore is sufficiently filled with K(+) ions, which enables strong voltage-dependent blockade with adequate outward K(+) conductance.