A novel conus peptide ligand for K+ channels

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.     

Energetic and structural interactions between delta-dendrotoxin and a voltage-gated potassium channel

Dendrotoxin proteins isolated from Mamba snake venom block potassium channels with a high degree of specificity and selectivity. Using site-directed mutagenesis we have identified residues that constitute the functional interaction surfaces of delta-dendrotoxin and its voltage-gated potassium channel receptor. delta-Dendrotoxin uses a triangular patch formed by seven side-chains (Lys3, Tyr4, Lys6, Leu7, Pro8, Arg10, Lys26) to block K(+) currents carried by a Shaker potassium channel variant. The inhibitory surface of the toxin interacts with channel residues at Shaker positions 423, 425, 427, 431, and 449 near the pore. Amino acid mutations that interact across the toxin-channel interface were identified by mutant cycle analysis. These results constrain the possible orientation of dendrotoxin with respect to the K(+) channel structure. We propose that dendrotoxin binds near the pore entryway but does not act as a physical plug.     

Solution structure of hanatoxin1, a gating modifier of voltage-dependent K(+) channels: common surface features of gating modifier toxins

The three-dimensional structure of hanatoxin1 (HaTx1) was determined by using NMR spectroscopy. HaTx1 is a 35 amino acid residue peptide toxin that inhibits the drk1 voltage-gated K(+) channel not by blocking the pore, but by altering the energetics of gating. Both the amino acid sequence of HaTx1 and its unique mechanism of action distinguish this toxin from the previously described K(+) channel inhibitors. Unlike most other K(+) channel-blocking toxins, HaTx1 adopts an "inhibitor cystine knot" motif and is composed of two beta-strands, strand I for residues 19-21 and strand II for residues 28-30, connected by four chain reversals. A comparison of the surface features of HaTx1 with those of other gating modifier toxins of voltage-gated Ca(2+) and Na(+) channels suggests that the combination of a hydrophobic patch and surrounding charged residues is principally responsible for the binding of gating modifier toxins to voltage-gated ion channels.     

Sequence-function analysis of the K+-selective family of ion channels using a comprehensive alignment and the KcsA channel structure

Sequence-function analysis of K(+)-selective channels was carried out in the context of the 3.2 A crystal structure of a K(+) channel (KcsA) from Streptomyces lividans (Doyle et al., 1998). The first step was the construction of an alignment of a comprehensive set of K(+)-selective channel sequences forming the putative permeation path. This pathway consists of two transmembrane segments plus an extracellular linker. Included in the alignment are channels from the eight major classes of K(+)-selective channels from a wide variety of species, displaying varied rectification, gating, and activation properties. Segments of the alignment were assigned to structural motifs based on the KcsA structure. The alignment's accuracy was verified by two observations on these motifs: 1), the most variability is shown in the turret region, which functionally is strongly implicated in susceptibility to toxin binding; and 2), the selectivity filter and pore helix are the most highly conserved regions. This alignment combined with the KcsA structure was used to assess whether clusters of contiguous residues linked by hydrophobic or electrostatic interactions in KcsA are conserved in the K(+)-selective channel family. Analysis of sequence conservation patterns in the alignment suggests that a cluster of conserved residues is critical for determining the degree of K(+) selectivity. The alignment also supports the near-universality of the "glycine hinge" mechanism at the center of the inner helix for opening K channels. This mechanism has been suggested by the recent crystallization of a K channel in the open state. Further, the alignment reveals a second highly conserved glycine near the extracellular end of the inner helix, which may be important in minimizing deformation of the extracellular vestibule as the channel opens. These and other sequence-function relationships found in this analysis suggest that much of the permeation path architecture in KcsA is present in most K(+)-selective channels. Because of this finding, the alignment provides a robust starting point for homology modeling of the permeation paths of other K(+)-selective channel classes and elucidation of sequence-function relationships therein. To assay these applications, a homology model of the Shaker A channel permeation path was constructed using the alignment and KcsA as the template, and its structure evaluated in light of established structural criteria.     

KcsA crystal structure as framework for a molecular model of the Na(+) channel pore

The crystal structure of the pore-forming part of the KcsA bacterial K(+)-selective channel suggests a possible motif for related voltage-gated channels. We examined the hypothesis that the spacial orientation of the KcsA M1 and M2 alpha-helices also predicts the backbone location of S5 and S6 helices of the voltage-gated Na(+) channel. That channel's P region structure is expected to be different because selectivity is determined by side-chain interactions rather than by main-chain carbonyls, and its outer vestibule accommodates relatively large toxin molecules, tetrodotoxin (TTX) and saxitoxin (STX), which interact with selectivity ring residues. The Na(+) channel P loop was well-modeled by the alpha-helix-turn-beta-strand motif, which preserves the relationships for toxin interaction with the Na(+) channel found experimentally. This outer vestibule was docked into the extracellular part of the inverted teepee structure formed by the S5 and S6 helices that were spacially located by coordinates of the KcsA M1 and M2 helix main chains [Doyle et al. (1998) Science 280, 69-74], but populated with side chains of the respective S5 and S6 structures. van der Waals contacts were optimized with minimal adjustment of the S5, S6, and P loop structures, forming a densely packed pore structure. Nonregular external S5-P and P-S6 segments were not modeled here, except the P-S6 segment of domain II. The resulting selectivity region structure is consistent with Na(+) channel permeation properties, offering suggestions for the molecular processes involved in selectivity. The ability to construct a Na(+) channel pore model consistent with most of the available biophysical and mutational information suggests that the KcsA structural framework may be conserved in voltage-gated channels.     

Interactions of the H5 pore region and hydroxylamine with N-type inactivation in the Shaker K+ channel.

Mutations at sites in the H5 region of the Shaker B K+ channel were used to analyze the influence of the pore on N-type inactivation. Single-channel and two-electrode voltage clamp analyses showed that mutations at residues T441 and T442, which are thought to lie at the internal mouth of the pore, produced opposite effects on inactivation: the inactivated state is stabilized by T441S and destabilized by T442S. In addition, an ammonium derivative, hydroxylamine (OH-(NH3)+), appears to bind in the pore region of T441S and further decreases the rate of recovery from N-type inactivation. This effect relies on the presence of the amino-terminal. The effect of hydroxylamine on the T441S mutation of this K+ channel shows several properties analogous to those of local anesthetics on the Na+ channel. These results can be interpreted to suggest that part of the H5 region contributes to the receptor for the inactivation particle and that a hydroxylamine ion trapped near that site can stabilize their interaction.     

Probing the outer mouth structure of the HERG channel with peptide toxin footprinting and molecular modeling

Previous studies have shown that the unusually long S5-P linker lining human ether a-go-go related gene's (hERG's) outer vestibule is critical for its channel function: point mutations at high-impact positions here can interfere with the inactivation process and, in many cases, also reduce the pore's K+ selectivity. Because no data are available on the equivalent region in the available K channel crystal structures to allow for homology modeling, we used alternative approaches to model its three-dimensional structure. The first part of this article describes mutant cycle analysis used to identify residues on hERG's outer vestibule that interact with specific residues on the interaction surface of BeKm-1, a peptide toxin with known NMR structure and a high binding affinity to hERG. The second part describes molecular modeling of hERG's pore domain. The transmembrane region was modeled after the crystal structure of KvAP pore domain. The S5-P linker was docked to the transmembrane region based on data from previous NMR and mutagenesis experiments, as well as a set of modeling criteria. The models were further restrained by contact points between hERG's outer vestibule and the bound BeKm-1 toxin molecule deduced from the mutant cycle analysis. Based on these analyses, we propose a working model for the open conformation of the outer vestibule of the hERG channel, in which the S5-P linkers interact with the pore loops to influence ion flux through the pore.     

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.     

Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel

Hanatoxin inhibits voltage-gated K(+) channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K(+) channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.     

N-type inactivation of the potassium channel KcsA by the Shaker B "ball" peptide: mapping the inactivating peptide-binding epitope

The effects of the inactivating peptide from the eukaryotic Shaker BK(+) channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr(8), which exhibit the strongest STD effects; the C4H in the imidazole ring of His(16); the methyl protons of Val(4), Leu(7), and Leu(10) and the side chain amine protons of one, if not both, the Lys(18) and Lys(19) residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val(4) and Leu(10), whereas His(16) seems similarly affected as before. Conversely, STD effects on Tyr(8) are strongly diminished, and those on Lys(18) and/or Lys(19) are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of (13)C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a beta-hairpin structure when bound to the KcsA channel. Indeed, docking such a beta-hairpin structure into an open pore model for K(+) channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.     

Hanging gondola structure of the T1 domain in a voltage-gated K(+) channel

The T1 domain is a approximately 100-residue sequence in the cytoplasmic N-terminal region of K(v)-type K(+) channels. The structure of the isolated domain is known, but it is uncertain whether the structure of this domain is maintained in the fully assembled, membrane-associated, homotetrameric channel protein. We use the structure of the isolated domain as a guide for designing disulfide bonds to cross-link Shaker K(+) channels through the T1 domain. Six pairs of residues with side chains closely apposed across the T1 subunit interface were selected for replacement by cysteine. Of these, three pairs formed cross-links upon air oxidation of cysteine-substituted Shaker channels expressed in Xenopus oocyte membranes. Two of these cross-linked channels were examined electrophysiologically and were found to have gating properties only slightly altered from wild-type. The results show that the structure of the isolated T1 domain exists in the mature ion channel. They also demand that this domain is attached to the membrane-embedded part of the protein as a cytoplasmic "hanging gondola", and that ions gain access to the pore through four "windows" formed by the linker connecting T1 to the channel's first transmembrane helix.     

Exploring structural features of the interaction between the scorpion toxinCnErg1 and ERG K+ channels

The gamma-KTx-type scorpion toxins specific for K+ channels were found to interact with ERG channels on the turret region, while alpha-KTx3.2 Agitoxin-2 binds to the pore region of the Shaker K+ channel, and alpha-KTx5.3 BmP05 binds to the intermediate region of the small-conductance calcium-activated K-channel (SK(Ca)). In order to explore the critical residues for gamma-KTx binding, we determined the NMR structure of native gamma-KTx1.1 (CnErg1), a 42 amino acid residues scorpion toxin isolated from the venom of the Mexican scorpion Centruro?des noxius Hoffmann, and we used computational evolutionary trace (ET) analysis to predict possible structural and functional features of interacting surfaces. The 1H-NMR three-dimensional solution structure of native ergtoxin (CnErg1) was solved using a total of 452 distance constraints, 13 3J(NH-Halpha) and 10 hydrogen bonds. The structure is characterized by 2 segments of alpha-helices and a triple-stranded antiparallel beta-sheet stabilized by 4 disulfide bridges. The ET and structural analysis provided indication of the presence of two important amino acid residue clusters, one hydrophobic and the other hydrophilic, that should be involved in the surface contact between the toxin and the channel. Some features of the proposed interacting surface are discussed.     

Molecular determinants of ion permeation and selectivity in inositol 1,4,5-trisphosphate receptor Ca2+ channels

We tested the hypothesis that key residues in a putative intraluminal loop contribute to determination of ion permeation through the intracellular Ca(2+) release channel (inositol 1,4,5-trisphosphate receptors (IP(3)Rs)) that is gated by the second messenger inositol 1,4,5-trisphosphate (IP(3)). To accomplish this, we mutated residues within the putative pore forming region of the channel and analyzed the functional properties of mutant channels using a (45)Ca(2+) flux assay and single channel electrophysiological analyses. Two IP(3)R mutations, V2548I and D2550E, retained the ability to release (45)Ca(2+) in response to IP(3). When analyzed at the single channel level; both recombinant channels had IP(3)-dependent open probabilities similar to those observed in wild-type channels. The mutation V2548I resulted in channels that exhibited a larger K(+) conductance (489 +/- 13 picosiemens (pS) for V2548I versus 364 +/- 5 pS for wild-type), but retained a Ca(2+) selectivity similar to wild-type channels (P(Ca(2+)):P(K(+)) approximately 4:1). Conversely, D2550E channels were nonselective for Ca(2+) over K(+) (P(Ca(2+)):P(K(+)) approximately 0.6:1), while the K(+) conductance was effectively unchanged (391 +/- 4 pS). These results suggest that amino acid residues Val(2548) and Asp(2550) contribute to the ion conduction pathway. We propose that the pore of IP(3)R channels has two distinct sites that control monovalent cation permeation (Val(2548)) and Ca(2+) selectivity (Asp(2550)).     

NMR studies of the fifth transmembrane segment of Na+,K+-ATPase reveals a non-helical ion-binding region

The structure of a synthetic peptide corresponding to the fifth membrane-spanning segment (M5) in Na(+),K(+)-ATPase in sodium dodecyl sulfate (SDS) micelles was determined using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The spectra reveal that this peptide is substantially less alpha-helical than the corresponding M5 peptide of Ca(2+)-ATPase. A well-defined alpha-helix is shown in the C-terminal half of the peptide. Apart from a short helical stretch at the N-terminus, the N-terminal half contains a non-helical region with two proline residues and sequence similarity to a non-structured transmembrane element of the Ca(2+)-ATPase. Furthermore, this region spans the residues implicated in Na(+) and K(+) transport, where they are likely to offer the flexibility needed to coordinate Na(+) as well as K(+) during active transport.     

A specific two-pore domain potassium channel blocker defines the structure of the TASK-1 open pore

Two-pore domain potassium (K(2P)) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K(2P) channels. We describe A1899 as a potent and highly selective blocker of the K(2P) channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K(2P) open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K(2P) channels.     

Molecular cloning and characterization of four scorpion K(+)-toxin-like peptides: a new subfamily of venom peptides (alpha-KTx14) and genomic analysis of a member.

Four full-length cDNAs encoding the precursors of four K(+)-toxin-like peptides (named BmKK(1), BmKK(2), BmKK(3) and BmmKK(4), respectively) were first isolated from a venom gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The deduced precursors of BmKK(1), BmKK(2) and BmKK(3) are all made of 54 amino acid residues including a signal peptide of 23 residues, and a mature toxin of 31 residues with three disulfide bridges. The precursor of BmKK(4) is composed of 55 amino acid residues including a signal peptide of 23 residues, a mature toxin of 30 residues cross-linked by three disulfide bridges, and an extra Gly-Lys tail which should be removed in the processing step. The four peptides displayed 24-97% sequence identity with each other, and less than 27% homology with any other scorpion toxins described. However, they shared a common disulfide bridge pattern, which was consistent with that of most short-chain K(+)-toxins, suggesting they represent a new class of scorpion toxins and their target receptors may be a subfamily of K(+) channels. We classified the BmKK toxin subfamily as alpha-KTx14 according to the classification rules. The genomic sequence of BmKK(2) was also cloned and sequenced. It consisted of two exons, disrupted by an intron of 79 bp inserted in the region encoding the C-terminal part of the signal peptide. This structure was very similar to that of other K(+)-toxins described previously.     

Structural basis of the Na+/H+ exchanger regulatory factor PDZ1 interaction with the carboxyl-terminal region of the cystic fibrosis transmembrane conductance regulator

The PDZ1 domain of the Na(+)/H(+) exchanger regulatory factor (NHERF) binds with nanomolar affinity to the carboxyl-terminal sequence QDTRL of the cystic fibrosis transmembrane conductance regulator (CFTR) and plays a central role in the cellular localization and physiological regulation of this chloride channel. The crystal structure of human NHERF PDZ1 bound to the carboxyl-terminal peptide QDTRL has been determined at 1.7-A resolution. The structure reveals the specificity and affinity determinants of the PDZ1-CFTR interaction and provides insights into carboxyl-terminal leucine recognition by class I PDZ domains. The peptide ligand inserts into the PDZ1 binding pocket forming an additional antiparallel beta-strand to the PDZ1 beta-sheet, and an extensive network of hydrogen bonds and hydrophobic interactions stabilize the complex. Remarkably, the guanido group of arginine at position -1 of the CFTR peptide forms two salt bridges and two hydrogen bonds with PDZ1 residues Glu(43) and Asn(22), respectively, providing the structural basis for the contribution of the penultimate amino acid of the peptide ligand to the affinity of the interaction.     

Structure of the amino terminus of a gap junction protein

Charged amino acid residues in the amino terminus of gap junction forming proteins (connexins) form part, if not all, of the transjunctional voltage sensor of gap junction channels and play a fundamental role in ion permeation. Results from studies of the voltage dependence of N-terminal mutants predict that residues 1-10 of Group I connexins lie within the channel pore and that the N-terminus forms the channel vestibule by the creation of a turn initiated by the conserved G12 residue. Here we report that intercellular channels containing mutations of G12 in Cx32 to residues that are likely to interfere with flexibility of this locus (G12S, G12Y, and G12V) do not express junctional currents, whereas a connexin containing a proline residue at G12 (Cx32G12P), which is expected to maintain a structure similar to that of the G12 locus, forms nearly wild-type channels. We have solved the structure of an N-terminal peptide of Cx26 (MDWGTLQSILGGVNK) using 1H 2D NMR. The peptide contains two structured domains connected by a flexible hinge (domain-hinge-domain motif) that would allow the placement of the amino terminus within the channel pore. Residues 1-10 adopt a helical conformation and line the channel entrance while residues 12-15 form an open turn. Overall, there is good agreement between the structural and dynamic features of the N-terminal peptide provided by NMR and the functional studies of the voltage dependence of channels formed by wild-type and N-terminal mutations.     

Synthesis, 1H NMR structure, and activity of a three-disulfide-bridged maurotoxin analog designed to restore the consensus motif of scorpion toxins

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus. The toxin displays an exceptionally wide range of pharmacological activity since it binds onto small conductance Ca(2+)-activated K(+) channels and also blocks Kv channels (Shaker, Kv1.2 and Kv1.3). MTX possesses 53-68% sequence identity with HsTx1 and Pi1, two other K(+) channel short chain scorpion toxins cross-linked by four disulfide bridges. These three toxins differ from other K(+)/Cl(-)/Na(+) channel scorpion toxins cross-linked by either three or four disulfide bridges by the presence of an extra half-cystine residue in the middle of a consensus sequence generally associated with the formation of an alpha/beta scaffold (an alpha-helix connected to an antiparallel beta-sheet by two disulfide bridges). Because MTX exhibits an uncommon disulfide bridge organization among known scorpion toxins (C1-C5, C2-C6, C3-C4, and C7-C8 instead of C1-C4, C2-C5, and C3-C6 for three-disulfide-bridged toxins or C1-C5, C2-C6, C3-C7, and C4-C8 for four-disulfide-bridged toxins), we designed and chemically synthesized an MTX analog with three instead of four disulfide bridges ([Abu(19),Abu(34)]MTX) and in which the entire consensus motif of scorpion toxins was restored by the substitution of the two half-cystines in positions 19 and 34 (corresponding to C4 and C8) by two isosteric alpha-aminobutyrate (Abu) derivatives. The three-dimensional structure of [Abu(19), Abu(34)]MTX in solution was solved by (1)H NMR. This analog adopts the alpha/beta scaffold with now conventional half-cystine pairings connecting C1-C5, C2-C6, and C3-C7 (with C4 and C8 replaced by Abu derivatives). This novel arrangement in half-cystine pairings that concerns the last disulfide bridge results mainly in a reorientation of the alpha-helix regarding the beta-sheet structure. In vivo, [Abu(19),Abu(34)]MTX remains lethal in mice as assessed by intracerebroventricular injection of the peptide (LD(50) value of 0. 25 microg/mouse). The structural variations are also accompanied by changes in the pharmacological selectivity of the peptide, suggesting that the organization pattern of disulfide bridges should affect the three-dimensional presentation of certain key residues critical to the blockage of K(+) channel subtypes.     

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.