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.     

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 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.     

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.     

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.     

Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg(2+). Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.     

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.     

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.     

Interaction of Ba2+ with the pores of the cloned inward rectifier K+ channels Kir2.1 expressed in Xenopus oocytes.

Interactions of Ba2+ with K+ and molecules contributing to inward rectification were studied in the cloned inward rectifier K+ channels, Kir2.1. Extracellular Ba2+ blocked Kir2.1 channels with first-order kinetics in a Vm-dependent manner. At Vm more negative than -120 mV, the Kd-Vm relationship became less steep and the dissociation rate constants were larger, suggesting Ba2+ dissociation into the extracellular space. Both depolarization and increasing [K+]i accelerated the recovery from extracellular Ba2+ blockade. Intracellular K+ appears to relieve Ba2+ blockade by competitively slowing the Ba2+ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100 microM) reduced, whereas 1 mM [Mg2+]i only slightly reduced, the ability of intracellular K+ to repulse Ba2+ from the channel pore. Intracellular Ba2+ also blocked outward IKir2.1 in a voltage-dependent fashion. At Vm >/= +40 mV, where intrinsic inactivation is prominent, intracellular Ba2+ accelerated the inactivation rate of the outward IKir2.1 in a Vm-independent manner, suggesting interaction of Ba2+ with the intrinsic gate of Kir2.1 channels.     

Inward-rectifier potassium channels in basolateral membranes of frog skin epithelium

Inward-rectifier K channel: using macroscopic voltage clamp and single- channel patch clamp techniques we have identified the K+ channel responsible for potassium recycling across basolateral membranes (BLM) of principal cells in intact epithelia isolated from frog skin. The spontaneously active K+ channel is an inward rectifier (Kir) and is the major component of macroscopic conductance of intact cells. The current- voltage relationship of BLM in intact cells of isolated epithelia, mounted in miniature Ussing chambers (bathed on apical and basolateral sides in normal amphibian Ringer solution), showed pronounced inward rectification which was K(+)-dependent and inhibited by Ba2+, H+, and quinidine. A 15-pS Kir channel was the only type of K(+)-selective channel found in BLM in cell-attached membrane patches bathed in physiological solutions. Although the channel behaves as an inward rectifier, it conducts outward current (K+ exit from the cell) with a very high open probability (Po = 0.74-1.0) at membrane potentials less negative than the Nernst potential for K+. The Kir channel was transformed to a pure inward rectifier (no outward current) in cell- attached membranes when the patch pipette contained 120 mM KCl Ringer solution (normal NaCl Ringer in bath). Inward rectification is caused by Mg2+ block of outward current and the single-channel current-voltage relation was linear when Mg2+ was removed from the cytosolic side. Whole-cell current-voltage relations of isolated principal cells were also inwardly rectified. Power density spectra of ensemble current noise could be fit by a single Lorentzian function, which displayed a K dependence indicative of spontaneously fluctuating Kir channels. Conclusions: under physiological ionic gradients, a 15-pS inward- rectifier K+ channel generates the resting BLM conductance in principal cells and recycles potassium in parallel with the Na+/K+ ATPase pump.     

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.     

Mg2+-dependent gating and strong inward rectification of the cation channel TRPV6

TRPV6 (CaT1/ECaC2), a highly Ca(2+)-selective member of the TRP superfamily of cation channels, becomes permeable to monovalent cations in the absence of extracellular divalent cations. The monovalent currents display characteristic voltage-dependent gating and almost absolute inward rectification. Here, we show that these two features are dependent on the voltage-dependent block/unblock of the channel by intracellular Mg(2+). Mg(2+) blocks the channel by binding to a site within the transmembrane electrical field where it interacts with permeant cations. The block is relieved at positive potentials, indicating that under these conditions Mg(2+) is able to permeate the selectivity filter of the channel. Although sizeable outward monovalent currents were recorded in the absence of intracellular Mg(2+), outward conductance is still approximately 10 times lower than inward conductance under symmetric, divalent-free ionic conditions. This Mg(2+)-independent rectification was preserved in inside-out patches and not altered by high intracellular concentrations of spermine, indicating that TRPV6 displays intrinsic rectification. Neutralization of a single aspartate residue within the putative pore loop abolished the Mg(2+) sensitivity of the channel, yielding voltage-independent, moderately inwardly rectifying monovalent currents in the presence of intracellular Mg(2+). The effects of intracellular Mg(2+) on TRPV6 are partially reminiscent of the gating mechanism of inwardly rectifying K(+) channels and may represent a novel regulatory mechanism for TRPV6 function in vivo.     

C-terminus determinants for Mg2+ and polyamine block of the inward rectifier K+ channel IRK1.

Critical loci for ion conduction in inward rectifier K+ channels are only now being discovered. The C-terminal region of IRK1 plays a crucial role in Mg2+i blockade and single-channel K+ conductance. A negatively charged aspartate in the putative second transmembrane domain (position 172) is essential for time-dependent block by the cytoplasmic polyamines spermine and spermidine. We have now localized the C-terminus effect in IRK1 to a single, negatively charged residue (E224). Mutation of E224 to G, Q and S drastically reduced rectification. Furthermore, the IRK1 E224G mutation decreased block by Mg2+i and spermidine and, like the E224Q mutation, caused a dramatic reduction in the apparent single-channel K+ conductance. The double mutation IRK1 D172N+ E224G was markedly insensitive to spermidine block, displaying an affinity similar to ROMK1. The results are compatible with a model in which the negatively charged residue at position 224, E224, is a major determinant of pore properties in IRK1. By means of a specific interaction with the negatively charged residue at position 172, D172, E224 contributes to the formation of the binding pocket for Mg2+ and polyamines, a characteristic of strong inward rectifiers.     

Long polyamines act as cofactors in PIP2 activation of inward rectifier potassium (Kir2.1) channels

Phosphatidylinosital-4,5-bisphosphate (PIP2) acts as an essential factor regulating the activity of all Kir channels. In most Kir members, the dependence on PIP2 is modulated by other factors, such as protein kinases (in Kir1), G(betagamma) (in Kir3), and the sulfonylurea receptor (in Kir6). So far, however, no regulator has been identified in Kir2 channels. Here we show that polyamines, which cause inward rectification by selectively blocking outward current, also regulate the interaction of PIP2 with Kir2.1 channels to maintain channel availability. Using spermine and diamines as polyamine analogs, we demonstrate that both spontaneous and PIP2 antibody-induced rundown of Kir2.1 channels in excised inside-out patches was markedly slowed by long polyamines; in contrast, polyamines with shorter chain length were ineffective. In K188Q mutant channels, which have a low PIP2 affinity, application PIP2 (10 microM) was unable to activate channel activity in the absence of polyamines, but markedly activated channels in the presence of long diamines. Using neomycin as a measure of PIP2 affinity, we found that long polyamines were capable of strengthening either the wild type or K188Q channels' interaction with PIP2. The negatively charged D172 residue inside the transmembrane pore region was critical for the shift of channel-PIP2 binding affinity by long polyamines. Sustained pore block by polyamines was neither sufficient nor necessary for this effect. We conclude that long polyamines serve a dual role as both blockers and coactivators (with PIP2) of Kir2.1 channels.     

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.     

Revisiting inward rectification: K ions permeate through Kir2.1 channels during high-affinity block by spermidine

Outward currents through Kir2.1 channels play crucial roles in controlling the electrical properties of excitable cells, and such currents are subjected to voltage-dependent block by intracellular Mg(2+) and polyamines that bind to both high- and low-affinity sites on the channels. Under physiological conditions, high-affinity block is saturated and yet outward Kir2.1 currents can still occur, implying that high-affinity polyamine block cannot completely eliminate outward Kir2.1 currents. However, the underlying molecular mechanism remains unknown. Here, we show that high-affinity spermidine block, rather than completely occluding the single-channel pore, induces a subconducting state in which conductance is 20% that of the fully open channel. In a D172N mutant lacking the high-affinity polyamine-binding site, spermidine does not induce such a substate. However, the kinetics for the transitions between the substate and zero-current state in wild-type channels is the same as that of low-affinity block in the D172N mutant, supporting the notion that these are identical molecular events. Thus, the residual outward current after high-affinity spermidine block is susceptible to low-affinity block, which determines the final amplitude of the outward current. This study provides a detailed insight into the mechanism underlying the emergence of outward Kir2.1 currents regulated by inward rectification attributed to high- and low-affinity polyamine blocks.     

Polyamine permeation and rectification of Kir4.1 channels

Inward rectifier K(+) (Kir) channels are expressed in multiple neuronal and glial cells. Recent studies have equated certain properties of exogenously expressed Kir4.1 channels with those of native K(+) currents in brain cells, as well as demonstrating the expression of Kir4.1 subunits in these tissues. There are nagging problems however with assigning native currents to Kir4.1 channels. One major concern is that in many native tissues, the putatively correlated currents show much weaker rectification than typically reported for cloned Kir4.1 channels. We have now examined the polyamine-dependence of Kir4.1 channels expressed at high density in Cosm6 cells, using inside-out membrane patches. The experiments reveal a complex and variable rectification that can help explain the variability reported for candidate Kir4.1 currents in native cells. Most importantly, rectification seems to be incomplete, even at high polyamine concentrations. In excised membrane patches, with high levels of expression, and high concentrations of spermine, there is approximately 15% residual conductance that is insensitive to spermine. From a biophysical perspective, this is a striking finding, and indicates either that a bound spermine fails to completely block permeation or that significant spermine permeation (i.e. 'punchthrough') is occurring. To examine this further, we have examined block by philanthotoxin (PhTx, essentially spermine with a bulky tail). PhTx block, while less potent, is more complete than spermine block. This leads us to propose that spermine 'punchthrough' may be significant in Kir4 channels, and that this may be a major contributor to the weak rectification observed under physiological conditions.     

Crystal structure of a Kir3.1-prokaryotic Kir channel chimera

The Kir3.1 K(+) channel participates in heart rate control and neuronal excitability through G-protein and lipid signaling pathways. Expression in Escherichia coli has been achieved by replacing three fourths of the transmembrane pore with the pore of a prokaryotic Kir channel, leaving the cytoplasmic pore and membrane interfacial regions of Kir3.1 origin. Two structures were determined at 2.2 A. The selectivity filter is identical to the Streptomyces lividans K(+) channel within error of measurement (r.m.s.d.<0.2 A), suggesting that K(+) selectivity requires extreme conservation of three-dimensional structure. Multiple K(+) ions reside within the pore and help to explain voltage-dependent Mg(2+) and polyamine blockade and strong rectification. Two constrictions, at the inner helix bundle and at the apex of the cytoplasmic pore, may function as gates: in one structure the apex is open and in the other, it is closed. Gating of the apex is mediated by rigid-body movements of the cytoplasmic pore subunits. Phosphatidylinositol 4,5-biphosphate-interacting residues suggest a possible mechanism by which the signaling lipid regulates the cytoplasmic pore.     

Interactions of cations with the cytoplasmic pores of inward rectifier K(+) channels in the closed state

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore.     

Exaggerated Mg2+ inhibition of Kir2.1 as a consequence of reduced PIP2 sensitivity in Andersen syndrome

Andersen syndrome is an autosomal dominant disorder characterized by cardiac arrhythmias, periodic paralysis and dysmorphic features. Many Andersen syndrome cases have been associated with loss-of-function mutations in the inward rectifier K(+) channel Kir2.1 encoded by KCNJ2. Using engineered concatenated tetrameric channels we determined the mechanism for dominant loss-of-function associated with a trafficking-competent missense mutation, Kir2.1-T74A. This mutation alters a conserved threonine residue in an N-terminal domain analogous to the slide helix identified in the structure of a bacterial inward rectifier. Incorporation of a single mutant subunit in channel tetramers was sufficient to cause a selective impairment of whole-cell outward current, but no difference in the level of inward current compared with wild-type (WT) tetramers. The presence of two mutant subunits resulted in greatly reduced outward and impaired inward currents. Experiments using excised inside-out membrane patches revealed that tetramers with one mutant subunit exhibited increased Mg(2+) inhibition. Additional experiments demonstrated that concatenated tetramers containing one T74A subunit had reduced PIP(2) sensitivity, and that outward current carried by mutant tetramers could be restored by addition of PIP(2) in the absence of Mg(2+). Our results are consistent with the involvement of the Kir2.1 N-terminus in PIP(2) modulation of channel activity and support the existence of an inverse relationship between PIP(2) sensitivity and Mg(2+) inhibition of Kir2.1 channels. Our data also indicate that a single mutant subunit is sufficient to explain dominant-negative behavior of Kir2.1-T74A in Andersen syndrome.