Activation of the Ca2+-sensing receptors increases currents through inward rectifier K+ channels via activation of phosphatidylinositol 4-kinase.

Inward rectifier K+ channels are important for maintaining normal electrical function in many cell types. The proper function of these channels requires the presence of membrane phosphoinositide 4,5-bisphosphate (PIP2). Stimulation of the Ca2+-sensing receptor CaR, a pleiotropic G protein-coupled receptor, activates both Gq/11, which decreases PIP2, and phosphatidylinositol 4-kinase (PI-4-K), which, conversely, increases PIP2. How membrane PIP2 levels are regulated by CaR activation and whether these changes modulate inward rectifier K+ are unknown. In this study, we found that activation of CaR by the allosteric agonist, NPSR568, increased inward rectifier K+ current (I K1) in guinea pig ventricular myocytes and currents mediated by Kir2.1 channels exogenously expressed in HEK293T cells with a similar sensitivity. Moreover, using the fluorescent PIP2 reporter tubby-R332H-cYFP to monitor PIP2 levels, we found that CaR activation in HEK293T cells increased membrane PIP2 concentrations. Pharmacological studies showed that both phospholipase C (PLC) and PI-4-K are activated by CaR stimulation with the latter played a dominant role in regulating membrane PIP2 and, thus, Kir currents. These results provide the first direct evidence that CaR activation upregulates currents through inward rectifier K+ channels by accelerating PIP2 synthesis. The regulation of I K1 plays a critical role in the stability of the electrical properties of many excitable cells, including cardiac myocytes and neurons. Further, synthetic allosteric modulators that increase CaR activity have been used to treat hyperparathyroidism, and negative CaR modulators are of potential importance in the treatment of osteoporosis. Thus, our results provide further insight into the roles played by CaR in the cardiovascular system and are potentially valuable for heart disease treatment and drug safety.

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.

Extracellular K+ elevates outward currents through Kir2.1 channels by increasing single-channel conductance.

Outward currents through inward rectifier K+ channels (Kir) play a pivotal role in determining resting membrane potential and in controlling excitability in many cell types. Thus, the regulation of outward Kir current (IK1) is important for appropriate physiological functions. It is known that outward IK1 increases with increasing extracellular K+ concentration ([K+]o), but the underlying mechanism is not fully understood. A "K+-activation of K+-channel" hypothesis and a "blocking-particle" model have been proposed to explain the [K+]o-dependence of outward IK1. Yet, these mechanisms have not been examined at the single-channel level. In the present study, we explored the mechanisms that determine the amplitudes of outward IK1 at constant driving forces [membrane potential (Vm) minus reversal potential (EK)]. We found that increases in [K+]o elevated the single-channel current to the same extent as macroscopic IK1 but did not affect the channel open probability at a constant driving force. In addition, spermine-binding kinetics remained unchanged when [K+]o ranged from 1 to 150 mM at a constant driving force. We suggest the regulation of K+ permeation by [K+]o as a new mechanism for the [K+]o-dependence of outward IK1.

The extracellular K+ concentration dependence of outward currents through Kir2.1 channels is regulated by extracellular Na+ and Ca2+.

It has been known for more than three decades that outward Kir currents (I(K1)) increase with increasing extracellular K(+) concentration ([K(+)](o)). Although this increase in I(K1) can have significant impacts under pathophysiological cardiac conditions, where [K(+)](o) can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K(+)](o) dependence of Kir2.1-mediated outward I(K1) was due to [K(+)](o)-dependent inhibition of outward I(K1) by extracellular Na(+) and Ca(2+). This could be accounted for by Na(+)/Ca(2+) inhibition of I(K1) through screening of local negative surface charges. Consistent with this, extracellular Na(+) and Ca(2+) reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I(K1) in a similar [K(+)](o)-dependent manner as Na(+)/Ca(2+). Site-directed mutagenesis studies identified Asp(114) and Glu(153) as the source of surface charges. Reducing K(+) activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na(+) and Ca(2+), suggesting that in addition to exerting a surface electrostatic effect, Na(+) and Ca(2+) might inhibit outward I(K1) by inhibiting K(+) activation. This study identified interactions of K(+) with Na(+) and Ca(2+) that are important for the [K(+)](o) dependence of Kir2.1-mediated outward I(K1).

Oligomerization is crucial for the stability and function of heme oxygenase-1 in the endoplasmic reticulum.

Heme oxygenase-1 (HO-1), a stress-inducible enzyme anchored in the endoplasmic reticulum (ER) by a single transmembrane segment (TMS) located at the C terminus, interacts with NADPH cytochrome P450 reductase and biliverdin reductase to catalyze heme degradation to biliverdin and its metabolite, bilirubin. Previous studies suggested that HO-1 functions as a monomer. Using chemical cross-linking, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments, here we showed that HO-1 forms dimers/oligomers in the ER. However, oligomerization was not observed with a truncated HO-1 lacking the C-terminal TMS (amino acids 266-285), which exhibited cytosolic and nuclear localization, indicating that the TMS is essential for the self-assembly of HO-1 in the ER. To identify the interface involved in the TMS-TMS interaction, residue Trp-270, predicted by molecular modeling as a potential interfacial residue of TMS alpha-helices, was mutated, and the effects on protein subcellular localization and activity assessed. The results showed that the W270A mutant was present exclusively in the ER and formed oligomers with similar activity to those of the wild type HO-1. Interestingly, the W270N mutant was localized not only in the ER, but also in the cytosol and nucleus, suggesting it is susceptible to proteolytic cleavage. Moreover, the microsomal HO activity of the W270N mutant was significantly lower than that of the wild type. The W270N mutation appears to interfere with the oligomeric state, as revealed by a lower FRET efficiency. Collectively, these data suggest that oligomerization, driven by TMS-TMS interactions, is crucial for the stabilization and function of HO-1 in the ER.

K+ binding in the G-loop and water cavity facilitates Ba2+ 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.

Structural changes in the cytoplasmic pore of the Kir1.1 channel during pHi-gating probed by FRET.

Kir1.1 channels are important in maintaining K+ homeostasis in the kidney. Intracellular acidification reversibly closes the Kir1.1 channel and thus decreases K+ secretion. In this study, we used Foster resonance energy transfer (FRET) to determine whether the conformation of the cytoplasmic pore changes in response to intracellular pH (pHi)-gating in Kir1.1 channels fused with enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (ECFP-Kir1.1-EYFP). Because the fluorescence intensities of ECFP and EYFP were affected at pHi < 7.4 where pHi-gating occurs in the ECFP-Kir1.1-EYFP construct, we examined the FRET efficiencies of an ECFP-S219R-EYFP mutant, which is completed closed at pHi 7.4 and open at pHi 10.0. FRET efficiency was increased from 25% to 40% when the pHi was decreased from 10.0 to 7.4. These results suggest that the conformation of the cytoplasmic pore in the Kir1.1 channel changes in response to pHi gating such that the N- and C-termini move apart from each other at pHi 7.4, when the channel is open.

Characterization of a novel Nav1.5 channel mutation, A551T, associated with Brugada syndrome.

Brugada syndrome is a life-threatening, inherited arrhythmia disorder associated with autosomal dominant mutations in SCN5A, the gene encoding the human cardiac Na+ channel alpha subunit (Nav1.5). Here, we characterized the biophysical properties of a novel Brugada syndrome-associated Nav1.5 mutation, A551T, identified in a proband who was successfully resuscitated from an episode of ventricular fibrillation with sudden collapse. Whole-cell currents through wild-type (WT) Nav1.5 and mutant (A551T) channels were recorded and compared in the human embryonic kidney cell line HEK293T transfected with SCN5A cDNA and SCN1B cDNA, using the patch-clamp technique. Current density was decreased in the A551T mutant compared to the WT. In addition, the A551T mutation reduced Nav1.5 activity by promoting entry of the channel into fast inactivation from the closed state, thereby shifting the steady-state inactivation curve by -5 mV. Furthermore, when evaluated at -90 mV, the resting membrane potential, but not at the conventionally used -120 mV, both the percentage, and rate, of channel recovery from inactivation were reduced in the mutant. These results suggest that the DI-DII linker may be involved in the stability of inactivation gating process. This study supports the notion that a reduction in Nav1.5 channel function is involved in the pathogenesis of Brugada syndrome. The structural-functional study of the Nav1.5 channel advances our understanding of its pathophysiolgocial function.

Linkage analysis reveals allosteric coupling in Kir2.1 channels.

Potassium-selective inward rectifier (Kir) channels are a class of membrane proteins necessary for maintaining stable resting membrane potentials, controlling excitability, and shaping the final repolarization of action potentials in excitable cells. In addition to the strong inward rectification of the ionic current caused by intracellular blockers, Kir2.1 channels possess "weak" inward rectification observed in inside-out patches after prolonged washout of intracellular blockers. The mechanisms underlying strong inward rectification have been attributed to voltage-dependent block by intracellular Mg2+ and polyamines; however, the mechanism responsible for weak rectification remains elusive. Hypotheses include weak voltage-dependent block and intrinsic voltage-dependent gating. Here, we performed a conductance Hill analysis of currents recorded with a double-ramp protocol to evaluate different mechanisms proposed for weak inward rectification of Kir2.1 channels. Linkage analysis in the form of a Hill plot revealed that the ramp currents could be best explained by allosteric coupling between a mildly voltage-dependent pore gate (gating charge ∼0.18 eo) and a voltage sensor (gating charge ∼1.7 eo). The proposed voltage sensor stabilized the closing of the pore gate (coupling factor ∼31). We anticipate that the use of linkage analysis will broaden understanding of functional coupling in ion channels and proteins in general.

A common SCN5A polymorphism attenuates a severe cardiac phenotype caused by a nonsense SCN5A mutation in a Chinese family with an inherited cardiac conduction defect.

The SCN5A mutations have been associated with a variety of arrhythmic disorders, including type 3 long QT syndrome (LQT3), Brugada syndrome and inherited cardiac conduction defects. The relationship between genotype and phenotype in SCN5A mutations is complex. Some SCN5A mutations may cause death or severe manifestations in some people and may not cause any symptoms or arrhythmias in others. The causes of these unpredictable clinical manifestations remain incompletely understood. The molecular basis of a four-generation family with cardiac conduction abnormalities was studied and whether variants in the SCN5A gene could account for the cardiac phenotypic variability observed in this family was determined. A novel mutation (W1421X) of SCN5A was identified in a four-generation family with cardiac conduction abnormalities and several cases of sudden death. Most family members who carry this W1421X mutation have developed major clinical manifestations or electrocardiographic abnormalities, both of which became more prominent as the patients grew older. However, the 73-year-old grandfather, who carried both the W1421X and R1193Q mutations, had thus far remained healthy and presented with only subtle electrocardiographic abnormalities, whereas most of his offspring, who carried a single mutation (W1421X), had died early or had major disease manifestations. This observation suggests that the R1193Q mutation has a complementary role in alleviating the deleterious effects conferred by W1421X in the function of the SCN5A gene. This report provides a good model to explain the mechanism of penetrance of genetic disorders.

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 the glutamate gate of CLC-2 chloride channel by pore occupancy.

CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H(+). Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl(-), Br(-), SCN(-), and I(-)) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl(-)]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H(+) plays a minor role in dislodging the glutamate gate.

Mechanism for attenuated outward conductance induced by mutations in the cytoplasmic pore of Kir2.1 channels.

Outward currents through Kir2.1 channels regulate the electrical properties of excitable cells. These currents are subject to voltage-dependent attenuation by the binding of polyamines to high- and low-affinity sites, which leads to inward rectification, thereby controlling cell excitability. To examine the effects of positive charges at the low-affinity site in the cytoplasmic pore on inward rectification, we studied a mutant Kir channel (E224K/H226E) and measured single-channel currents and streaming potentials (Vstream), the latter provide the ratio of water to ions queued in a single-file permeation process in the selectivity filter. The water-ion coupling ratio was near one at a high K(+) concentration ([K(+)]) for the wild-type channel and increased substantially as [K(+)] decreased. On the other hand, fewer ions occupied the selectivity filter in the mutant at all [K(+)]. A model for the Kir channel involving a K(+) binding site in the wide pore was introduced. Model analyses revealed that the rate constants associated with the binding and release to and from the wide-pore K(+) binding site was modified in the mutant. These effects lead to the reduced contribution of a conventional two-ion permeation mode to total conductance, especially at positive potentials, thereby inward rectification.

Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes.

Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling.

Solution structure of a K(+)-channel blocker from the scorpion Tityus cambridgei.

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.

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.

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.

A ring of negative charges in the intracellular vestibule of Kir2.1 channel modulates K+ permeation.

The glutamate at site 224 of a Kir2.1 channel plays an important role in K+ permeation. The single-channel inward current flickers with reduced conductance in an E224G mutant. We show that open-channel fluctuations can also be observed in E224C, E224K, and E224Q mutants. Yet, open-channel fluctuations were not observed in either the wild-type or an E224D mutant. Introducing a negatively charged methanethiosulfonate reagent to the E224C mutant irreversibly increased channel conductance and eliminated open-channel fluctuations. These results suggest that although the negatively charged residue 224 is located at the internal vestibule, it is important for smooth inward K+ conduction. We identified a substate in the E224G mutant and showed that open-channel fluctuations are mainly attributed to rapid transitions between the substate and the main state. Also, we characterized the voltage- and ion-dependence of the substate kinetics. The open-channel fluctuations decreased in internal NH4+ or Tl+ as compared to internal K+. These results suggest that NH4+ and Tl+ gate the E224G mutant in a more stable state. Based on an ion-conduction model, we propose that the appearance of the substate in the E224G mutant is due to changes of ion gating in association with variations of ion-ion interaction in the permeation pathway.

Conformational changes in Kir2.1 channels during NH4+-induced inactivation.

We have shown previously that NH(4)(+) binding to the external pore of a Kir2.1 channel induces channel inactivation possibly through conformational changes. In this study, we performed further biophysical analyses of the NH(4)(+)-induced inactivation modeled by a refined kinetic scheme. Also, we investigated the conformational change hypothesis by examining whether the chemical modification of single-cysteine substitution of amino acids located at the internal pore alters the kinetics of the NH(4)(+)-induced inactivation. In addition, we examined whether the mutation of amino acids located at various parts of a Kir2.1 channel influences the NH(4)(+)-induced inactivation. Kir2.1 channels were expressed in Xenopus oocytes and studied using patch-clamp techniques. The gating of the NH(4)(+)-induced inactivation was affected by mutation of several amino acids located at various regions of the Kir2.1 channel. These results suggest that amino acids from different parts of a Kir2.1 channel are involved in the channel closure. Furthermore, internal chemical modification of several cysteine mutants resulted in the block of inward currents and changes in the on and off rate for the NH(4)(+)-induced inactivation, suggesting that the internal pore mouth is involved in the closure of a Kir2.1 channel. Taken together these results provide new evidence for conformational changes affecting the NH(4)(+)-induced inactivation in the Kir2.1 channel.

The effects of spermine on the accessibility of residues in the M2 segment of Kir2.1 channels expressed in Xenopus oocytes.

We examined the effects of spermine binding to aspartate at site 172 on the accessibility of internal trimethylammonioethylmethane thiosulphonate (MTSET) to substituted cysteines within the pore of a Kir2.1 channel. Spermine prevented MTSET modification in Q164C and G168C mutants, indicating that sites 164 and 168 are located externally to the spermine binding site. The rates of MTSET modification were significantly reduced by spermine in I176C mutants, indicating that site 176 is located internally to D172 and that the bound spermine hinders the reaction of MTSET with cysteine at site 176. Spermidine, putrescine and Mg2+ also decreased MTSET modification at site 176. The order of effect is putrescine > spermidine approximately = spermine approximately = Mg2+. To account for the electrostatic and physical repulsion between MTSET and polyamines, possible locations of polyamines in the pore are discussed. In D172C mutants, the spermine that bound to sites 224 and 299 completely inhibited channels at +40 mV, yet MTSET remained accessible to site 172. In addition, in the D172C mutant, spermine did not affect the exit rate of Ba2+ bound to the threonine at the site 141. These results indicate that spermine bound at the cytoplasmic pore induces channel closure at positions 141-172. The effects of spermine on the accessibility of amino acids in the pore may shed light on the structural and functional relationships of the Kir2.1 channels during inward rectification.