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.     

Multi-ion versus single-ion conduction mechanisms can yield current rectification in biological ion channels

There is clear evidence that the net magnitude of negative charge at the intracellular end of inwardly rectifying potassium channels helps to generate an asymmetry in the magnitude of the current that will pass in each direction. However, a complete understanding of the physical mechanism that links these charges to current rectification has yet to be obtained. Using Brownian dynamics, we compare the conduction mechanism and binding sites in rectifying and non-rectifying channel models. We find that in our models, rectification is a consequence of asymmetry in the hydrophobicity and charge of the pore lining. As a consequence, inward conduction can occur by a multi-ion conduction mechanism. However, outward conduction is restricted, since there are fewer ions at the intracellular entrance and outwardly moving ions must cross the pore on their own. We pose the question as to whether the same mechanism could be at play in inwardly rectifying potassium channels.     

Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 A resolution

Inward rectifier K(+) channels govern the resting membrane voltage in many cells. Regulation of these ion channels via G protein-coupled receptor signaling underlies the control of heart rate and the actions of neurotransmitters in the central nervous system. We have determined the protein structure formed by the intracellular N- and C termini of the G protein-gated inward rectifier K(+) channel GIRK1 at 1.8 A resolution. A cytoplasmic pore, conserved among inward rectifier K(+) channels, extends the ion pathway to 60 A, nearly twice the length of a canonical transmembrane K(+) channel. The cytoplasmic pore is lined by acidic and hydrophobic amino acids, creating a favorable environment for polyamines, which block the pore. These results explain in structural and chemical terms the basis of inward rectification, and they also have implications for G protein regulation of GIRK channels.     

External [K+] and the block of the K+ inward rectifier by external Cs+ in frog skeletal muscle.

Frog skeletal muscle has a K+ channel called the inward rectifier, which passes inward current more readily than outward current. Gay and Stanfield (1977) described a voltage-dependent block of inward K+ currents through the inward rectifier by external Cs+ in frog muscle. Here, frog single muscle fibers were voltage clamped using the vaseline-gap voltage-clamp technique to study the effect of external [K+] on the voltage-dependent block of inward K+ currents through the inward rectifier by external Cs+. The block of inward K+ currents through the channel by external Cs+ was found to depend on external [K+], such that increasing the external concentration of the permeant ion K+ potentiated the block produced by the impermeant external Cs+. These findings are not consistent with a one-ion channel model for the inward rectifier. The Eyring rate theory formalism for channels, viewed as single-file multi-ion pores (Hille and Schwarz, 1978), was used to develop a two-site multi-ion model for the inward rectifier. This model successfully reproduced the experimentally observed potentiation of the Cs+ block of the channel by external K+, thus lending further support to the view of the inward rectifier as a multi-ion channel.     

The mechanism of inward rectification of potassium channels: "long-pore plugging" by cytoplasmic polyamines

The mechanism of inward rectification was examined in cell-attached and inside-out membrane patches from Xenopus oocytes expressing the cloned strong inward rectifier HRK1. Little or no outward current was measured in cell-attached patches. Inward currents reach their maximal value in two steps: an instantaneous phase followed by a time-dependent "activation" phase, requiring at least two exponentials to fit the time- dependent phase. After an activating pulse, the quasi-steady state current-voltage (I-V) relationship could be fit with a single Boltzmann equation (apparent gating charge, Z = 2.0 +/- 0.1, n = 3). Strong rectification and time-dependent activation were initially maintained after patch excision into high [K+] (K-INT) solution containing 1 mM EDTA, but disappeared gradually, until only a partial, slow inactivation of outward current remained. Biochemical characterization (Lopatin, A. N., E. N. Makhina, and C. G. Nichols, 1994. Nature. 372:366-396.) suggests that the active factors are naturally occurring polyamines (putrescine, spermidine, and spermine). Each polyamine causes reversible, steeply voltage-dependent rectification of HRK1 channels. Both the blocking affinity and the voltage sensitivity increased as the charge on the polyamine increased. The sum two Boltzmann functions is required to fit the spermine and spermidine steady state block. Putrescine unblock, like Mg2+ unblock, is almost instantaneous, whereas the spermine and spermidine unblocks are time dependent. Spermine and spermidine unblocks (current activation) can each be fit with single exponential functions. Time constants of unblock change e-fold every 15.0 +/- 0.7 mV (n = 3) and 33.3 +/- 6.4 mV (n = 5) for spermine and spermidine, respectively, matching the voltage sensitivity of the two time constants required to fit the activation phase in cell-attached patches. It is concluded that inward rectification in intact cells can be entirely accounted for by channel block. Putrescine and Mg2+ ions can account for instantaneous rectification; spermine and spermidine provide a slower rectification corresponding to so-called intrinsic gating of inward rectifier K channels. The structure of spermine and spermidine leads us to suggest a specific model in which the pore of the inward rectifier channel is plugged by polyamines that enter deeply into the pore and bind at sites within the membrane field. We propose a model that takes into account the linear structure of the natural polyamines and electrostatic repulsion between two molecules inside the pore. Experimentally observed instantaneous and steady state rectification of HRK1 channels as well as the time-dependent behavior of HRK1 currents are then well fit with the same set of parameters for all tested voltages and concentrations of spermine and spermidine.     

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.     

The pore helix dipole has a minor role in inward rectifier channel function

Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.     

Novel gating mechanism of polyamine block in the strong inward rectifier K channel Kir2.1

Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.     

Block of tetrodotoxin-resistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence

Tetrodotoxin-resistant (TTX-R) Na(+) channels are much less susceptible to external TTX but more susceptible to external Cd(2+) block than tetrodotoxin-sensitive (TTX-S) Na(+) channels. Both TTX and Cd(2+) seem to block the channel near the "DEKA" ring, which is probably part of a multi-ion single-file region adjacent to the external pore mouth and is involved in the selectivity filter of the channel. In this study we demonstrate that other multivalent transitional metal ions such as La(3+), Zn(2+), Ni(2+), Co(2+), and Mn(2+) also block the TTX-R channels in dorsal root ganglion neurons. Just like Cd(2+), the blocking effect has little intrinsic voltage dependence, but is profoundly influenced by Na(+) flow. The apparent dissociation constants of the blocking ions are always significantly smaller in inward Na(+) currents than those in outward Na(+) current, signaling exit of the blocker along with the Na(+) flow and a high internal energy barrier for "permeation" of these multivalent blocking ions through the pore. Most interestingly, the activation and especially the inactivation kinetics are slowed by the blocking ions. Moreover, the gating changes induced by the same concentration of a blocking ion are evidently different in different directions of Na(+) current flow, but can always be correlated with the extent of pore block. Further quantitative analyses indicate that the apparent slowing of channel activation is chiefly ascribable to Na(+) flow-dependent unblocking of the bound La(3+) from the open Na(+) channel, whereas channel inactivation cannot happen with any discernible speed in the La(3+)-blocked channel. Thus, the selectivity filter of Na(+) channel is probably contiguous to a single-file multi-ion region at the external pore mouth, a region itself being nonselective in terms of significant binding of different multivalent cations. This region is "open" to the external solution even if the channel is "closed" ("deactivated"), but undergoes imperative conformational changes during the gating (especially the inactivation) process of the channel.     

Inward rectification of the IRK1 K+ channel reconstituted in lipid bilayers.

Inwardly rectifying potassium (K+) channels (IRK1) were incorporated into lipid bilayers to test the relative contributions of various mechanisms to inward rectification. IRK1 channels were expressed in Xenopus laevis oocytes and oocyte membrane vesicles containing the channels were fused with lipid bilayers. The major properties of the IRK1 channel were similar whether measured in the oocyte membrane or lipid bilayer; the single channel conductance was 21 pS in 140 mM symmetrical [K+] and varied as a square root of external [K+]. Importantly, IRK1 channels display voltage-dependent inward rectification in the absence of divalent ions or charged regulators such as spermine, indicating that they possess an intrinsic rectification mechanism. Although rectification was significantly increased by either Mg2+ or spermine added to the cytoplasmic face of the channel, their effects could not be explained by simple block of the open pore. The Hille and Schwartz (1978) model, originally proposed to explain inward rectification by singly charged blocking particles, cannot be used to explain rectification by multiply charged blocking particles. As an alternative, we propose that in addition to a slow gating mechanism producing long lasting open and closed states, there is a distinct, intrinsic fast gating process amplified by cytoplasmic Mg2+ and/or polyamine binding to the channel.     

Contributions of a negatively charged residue in the hydrophobic domain of the IRK1 inwardly rectifying K+ channel to K(+)-selective permeation.

Inwardly rectifying K+ channels are highly selective for K+ ions and show strong interaction with ions in the pore. Both features are important for the physiological functions of these channels and pose intriguing mechanistic questions of ion permeation. The aspartate residue in the second putative transmembrane segment of the IRK1 inwardly rectifying K+ channel, previously implicated in inward rectification gating due to cytoplasmic Mg2+ and polyamine block, is found in this study to be crucial for the channel's ability to distinguish between K+ and Rb+ ions. Mutation of this residue also perturbs the interaction between the channel pore and the Sr2+ blocking ion. Our studies suggest that this aspartate residue contributes to a selectivity filter near the cytoplasmic end of the pore.     

Ionic permeation and conduction properties of neuronal KCNQ2/KCNQ3 potassium channels

Heteromeric KCNQ2/3 potassium channels are thought to underlie the M-current, a subthreshold potassium current involved in the regulation of neuronal excitability. KCNQ channel subunits are structurally unique, but it is unknown whether these structural differences result in unique conduction properties. Heterologously expressed KCNQ2/3 channels showed a permeation sequence of while showing a conduction sequence of A differential contribution of component subunits to the properties of heteromeric KCNQ2/3 channels was demonstrated by studying homomeric KCNQ2 and KCNQ3 channels, which displayed contrasting ionic selectivities. KCNQ2/3 channels did not exhibit an anomalous mole-fraction effect in mixtures of K(+) and Rb(+). However, extreme voltage-dependence of block by external Cs(+) was indicative of multi-ion pore behavior. Block of KCNQ2/3 channels by external Ba(2+) ions was voltage-independent, demonstrating unusual ionic occupation of the outer pore. Selectivity properties and block of KCNQ2 were altered by mutation of outer pore residues in a manner consistent with the presence of multiple ion-binding sites. KCNQ2/3 channel deactivation kinetics were slowed exclusively by Rb(+), whereas activation of KCNQ2/3 channels was altered by a variety of external permeant ions. These data indicate that KCNQ2/3 channels are multi-ion pores which exhibit distinctive mechanisms of ion conduction and gating.     

Internal Na+ and Mg2+ blockade of DRK1 (Kv2.1) potassium channels expressed in Xenopus oocytes. Inward rectification of a delayed rectifier

Delayed rectifier potassium channels were expressed in the membrane of Xenopus oocytes by injection of rat brain DRK1 (Kv2.1) cRNA, and currents were measured in cell-attached and inside-out patch configurations. In intact cells the current-voltage relationship displayed inward going rectification at potentials > +100 mV. Rectification was abolished by excision of membrane patches into solutions containing no Mg2+ or Na+ ions, but was restored by introducing Mg2+ or Na+ ions into the bath solution. At +50 mV, half- maximum blocking concentrations for Mg2+ and Na+ were 4.8 +/- 2.5 mM (n = 6) and 26 +/- 4 mM (n = 3) respectively. Increasing extracellular potassium concentration reduced the degree of rectification of intact cells. It is concluded that inward going rectification resulting from voltage-dependent block by internal cations can be observed with normally outwardly rectifying DRK1 channels.     

Base of pore loop is important for rectification, activation, permeation, and block of Kir3.1/Kir3.4

The Kir3.1/Kir3.4 channel is an inward rectifier, agonist-activated K(+) channel. The location of the binding site within the channel pore that coordinates polyamines (and is thus responsible for inward rectification) and the location of the gate that opens the channel in response to agonist activation is unclear. In this study, we show, not surprisingly, that mutation of residues at the base of the selectivity filter in the pore loop and second transmembrane domain weakens Cs(+) block and decreases selectivity (as measured by Rb(+) and spermine permeation). However, unexpectedly, the mutations also weaken inward rectification and abolish agonist activation of the channel. In the wild-type channel and 34 mutant channels, there are significant (p < 0.05) correlations among the K(D) for Cs(+) block, Rb(+) and spermine permeation, inward rectification, and agonist activation. The significance of these findings is discussed. One possible conclusion is that the selectivity filter is responsible for inward rectification and agonist activation as well as permeation and block.     

Pore block versus intrinsic gating in the mechanism of inward rectification in strongly rectifying IRK1 channels

The IRK1 channel is inhibited by intracellular cations such as Mg(2+) and polyamines in a voltage-dependent manner, which renders its I-V curve strongly inwardly rectifying. However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg(2+) and polyamines, the macroscopic I-V curve of the channels displays modest rectification. This observation forms the basis of a hypothesis, alternative to the pore-blocking hypothesis, that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations. We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity. Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.     

Ion binding in the open HCN pacemaker channel pore: fast mechanisms to shape "slow" channels

I(H) pacemaker channels carry a mixed monovalent cation current that, under physiological ion gradients, reverses at approximately -34 mV, reflecting a 4:1 selectivity for K over Na. However, I(H) channels display anomalous behavior with respect to permeant ions such that (a) open channels do not exhibit the outward rectification anticipated assuming independence; (b) gating and selectivity are sensitive to the identity and concentrations of externally presented permeant ions; (c) the channels' ability to carry an inward Na current requires the presence of external K even though K is a minor charge carrier at negative voltages. Here we show that open HCN channels (the hyperpolarization-activated, cyclic nucleotide sensitive pore forming subunits of I(H)) undergo a fast, voltage-dependent block by intracellular Mg in a manner that suggests the ion binds close to, or within, the selectivity filter. Eliminating internal divalent ion block reveals that (a) the K dependence of conduction is mediated via K occupancy of site(s) within the pore and that asymmetrical occupancy and/or coupling of these sites to flux further shapes ion flow, and (b) the kinetics of equilibration between K-vacant and K-occupied states of the pore (10-20 micros or faster) is close to the ion transit time when the pore is occupied by K alone ( approximately 0.5-3 micros), a finding that indicates that either ion:ion repulsion involving Na is adequate to support flux (albeit at a rate below our detection threshold) and/or the pore undergoes rapid, permeant ion-sensitive equilibration between nonconducting and conducting configurations. Biophysically, further exploration of the Mg site and of interactions of Na and K within the pore will tell us much about the architecture and operation of this unusual pore. Physiologically, these results suggest ways in which "slow" pacemaker channels may contribute dynamically to the shaping of fast processes such as Na-K or Ca action potentials.     

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.     

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.     

Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.     

An ion''s view of the potassium channel. The structure of the permeation pathway as sensed by a variety of blocking ions

We have studied the block of potassium channels in voltage-clamped squid giant axons by nine organic and alkali cations, in order to learn how the channel selects among entering ions. When added to the internal solution, all of the ions blocked the channels, with inside-positive voltages enhancing the block. Cesium blocked the channels from the outside as well, with inside-negative voltages favoring block. We compared the depths to which different ions entered the channel by estimating the "apparent electrical distance" to the blocking site. Simulations with a three-barrier, double-occupancy model showed that the "apparent electrical distance," expressed as a fraction of the total transmembrane voltage, appears to be less than the actual value if the blocking ion can pass completely through the channel. These calculations strengthen our conclusion that sodium and cesium block at sites further into the channel than those occupied by lithium and the organic blockers. Our results, considered together with earlier work, demonstrate that the depth to which an ion can readily penetrate into the potassium channel depends both on its size and on the specific chemical groups on its molecular surface. The addition of hydroxyl groups to alkyl chains on a quaternary ammonium ion can both decrease the strength of binding and allow deeper penetration into the channel. For alkali cations, the degree of hydration is probably crucial in determining how far an ion penetrates. Lithium, the most strongly hydrated, appeared not to penetrate as far as sodium and cesium. Our data suggest that there are, minimally, four ion binding sites in the permeation pathway of the potassium channel, with simultaneous occupancy of at least two.