Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex III.

Recent evidence has implicated succinate-driven reverse electron transport (RET) through complex I as a major source of damaging reactive oxygen species (ROS) underlying reperfusion injury after prolonged cardiac ischemia. However, this explanation may be incomplete, because RET on reperfusion is self-limiting and therefore transient. RET can only generate ROS when mitochondria are well polarized, and it ceases when permeability transition pores (PTP) open during reperfusion. Because prolonged ischemia/reperfusion also damages electron transport complexes, we investigated whether such damage could lead to ROS production after PTP opening has occurred. Using isolated cardiac mitochondria, we demonstrate a novel mechanism by which antimycin-inhibited complex III generates significant amounts of ROS in the presence of Mg2+ and NAD+ and the absence of exogenous substrates upon inner membrane pore formation by alamethicin or Ca2+-induced PTP opening. We show that H2O2 production under these conditions is related to Mg2+-dependent NADH generation by malic enzyme. H2O2 production is blocked by stigmatellin, indicating its origin from complex III, and by piericidin, demonstrating the importance of NADH-related ubiquinone reduction for ROS production under these conditions. For maximal ROS production, the rate of NADH generation has to be equal or below that of NADH oxidation, as further increases in [NADH] elevate ubiquinol-related complex III reduction beyond the optimal range for ROS generation. These results suggest that if complex III is damaged during ischemia, PTP opening may result in succinate/malate-fueled ROS production from complex III due to activation of malic enzyme by increases in matrix [Mg2+], [NAD+], and [ADP].

Reactive oxygen species production induced by pore opening in cardiac mitochondria: The role of complex II.

Succinate-driven reverse electron transport (RET) through complex I is hypothesized to be a major source of reactive oxygen species (ROS) that induces permeability transition pore (PTP) opening and damages the heart during ischemia/reperfusion. Because RET can only generate ROS when mitochondria are fully polarized, this mechanism is self-limiting once PTP opens during reperfusion. In the accompanying article (Korge, P., Calmettes, G., John, S. A., and Weiss, J. N. (2017) J. Biol. Chem. 292, 9882-9895), we showed that ROS production after PTP opening can be sustained when complex III is damaged (simulated by antimycin). Here we show that complex II can also contribute to sustained ROS production in isolated rabbit cardiac mitochondria following inner membrane pore formation induced by either alamethicin or calcium-induced PTP opening. Two conditions are required to maximize malonate-sensitive ROS production by complex II in isolated mitochondria: (a) complex II inhibition by atpenin A5 or complex III inhibition by stigmatellin that results in succinate-dependent reduction of the dicarboxylate-binding site of complex II (site IIf); (b) pore opening in the inner membrane resulting in rapid efflux of succinate/fumarate and other dicarboxylates capable of competitively binding to site IIf The decrease in matrix [dicarboxylate] allows O2 access to reduced site IIf, thereby making electron donation to O2 possible, explaining the rapid increase in ROS production provided that site IIf is reduced. Because ischemia is known to inhibit complexes II and III and increase matrix succinate/fumarate levels, we hypothesize that by allowing dicarboxylate efflux from the matrix, PTP opening during reperfusion may activate sustained ROS production by this mechanism after RET-driven ROS production has ceased.

Hexokinase-mitochondrial interactions regulate glucose metabolism differentially in adult and neonatal cardiac myocytes.

In mammalian tumor cell lines, localization of hexokinase (HK) isoforms to the cytoplasm or mitochondria has been shown to control their anabolic (glycogen synthesis) and catabolic (glycolysis) activities. In this study, we examined whether HK isoform differences could explain the markedly different metabolic profiles between normal adult and neonatal cardiac tissue. We used a set of novel genetically encoded optical imaging tools to track, in real-time in isolated adult (ARVM) and neonatal (NRVM) rat ventricular myocytes, the subcellular distributions of HKI and HKII, and the functional consequences on glucose utilization. We show that HKII, the predominant isoform in ARVM, dynamically translocates from mitochondria and cytoplasm in response to removal of extracellular glucose or addition of iodoacetate (IAA). In contrast, HKI, the predominant isoform in NRVM, is only bound to mitochondria and is not displaced by the above interventions. In ARVM, overexpression of HKI, but not HKII, increased glycolytic activity. In neonatal rat ventricular myocytes (NVRM), knockdown of HKI, but not HKII, decreased glycolytic activity. In conclusion, differential interactions of HKI and HKII with mitochondria underlie the different metabolic profiles of ARVM and NRVM, accounting for the markedly increased glycolytic activity of NRVM.

The cardiac Na+-Ca2+ exchanger has two cytoplasmic ion permeation pathways.

The Na(+)-Ca(2+) exchanger (NCX) is a ubiquitously expressed plasma membrane protein. It plays a fundamental role in Ca(2+) homeostasis by moving Ca(2+) out of the cell using the electrochemical gradient of Na(+) as the driving force. Recent structural studies of a homologous archaebacterial exchanger, NCX_Mj, revealed its outward configuration with two potential ion permeation pathways exposed to the extracellular environment. Based on the symmetry of NCX_Mj structure, an atomic model of an inward-facing conformation was generated showing similar pathways but directed to the cytoplasm. The presence of these water-filled cavities has yet to be confirmed experimentally, and it is unknown if the mammalian exchanger adopts the same structure. In this study, we mutated multiple residues within transmembrane segments 2 and 7 of NCX1.1 (cardiac isoform) to cysteines, allowing us to investigate their sensitivity to membrane-impermeable sulfhydryl reagents as exchanger current block. By trapping NCX1.1 in the inward-facing configuration, we have mapped two differently sized cytoplasmic aqueous cavities, the access of which is modified during exchange. This data reveals movements of the protein associated with ion transport. Electrophysiological characterization shows that the conserved residues within transmembrane segments 2 and 7, coordinating Na(+) and Ca(2+) ions in NCX_Mj, play a fundamental role in NCX1.1. Our results suggest a similar architecture between the mammalian and archaebacterial exchangers.

Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers.

Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.

Conformational changes of a Ca2+-binding domain of the Na+/Ca2+ exchanger monitored by FRET in transgenic zebrafish heart.

The Na(+)/Ca(2+) exchanger is the major Ca(2+) extrusion mechanism in cardiac myocytes. The activity of the cardiac Na(+)/Ca(2+) exchanger is dynamically regulated by intracellular Ca(2+). Previous studies indicate that Ca(2+) binding to a high-affinity Ca(2+)-binding domain (CBD1) in the large intracellular loop is involved in regulation. We generated transgenic zebrafish with cardiac-specific expression of CBD1 linked to yellow and cyan fluorescent protein. Ca(2+) binding to CBD1 induces conformational changes, as detected by fluorescence resonance energy transfer. With this transgenic fish model, we were able to monitor conformational changes of the Ca(2+) regulatory domain of Na(+)/Ca(2+) exchanger in intact hearts. Treatment with the positive inotropic agents ouabain and isoproterenol increased both Ca(2+) transients and Ca(2+)-induced changes in fluorescence resonance energy transfer. The results indicate that Ca(2+) regulation of the Na(+)/Ca(2+) exchanger domain CBD1 changes with inotropic state. The transgenic fish models will be useful to further characterize the regulatory properties of the Na(+)/Ca(2+) exchanger in vivo.

Phosphatidylinositol-4,5-bisphosphate (PIP2) regulation of strong inward rectifier Kir2.1 channels: multilevel positive cooperativity.

Inwardly rectifying potassium (Kir) channels are gated by the interaction of their cytoplasmic regions with membrane-bound phosphatidylinositol-4,5-bisphosphate (PIP(2)). In the present study, we examined how PIP(2) interaction regulates channel availability and channel openings to various subconductance levels (sublevels) as well as the fully open state in the strong inward rectifier Kir2.1 channel. Various Kir2.1 channel constructs were expressed in Xenopus oocytes and single channel or macroscopic currents were recorded from inside-out patches. The wild-type (WT) channel rarely visited the subconductance levels under control conditions. However, upon reducing Kir2.1 channel interaction with PIP(2) by a variety of interventions, including PIP(2) antibodies, screening PIP(2) with neomycin, or mutating PIP(2) binding sites (e.g. K188Q), visitation to the sublevels was markedly increased before channels were converted to an unavailable mode in which they did not open. No channel activity was detected in channels with the double mutation K188A/R189A, a mutant which exhibits extremely weak interaction with PIP(2). By linking subunits together in tandem dimers or tetramers containing mixtures of WT and K188A/R189A subunits, we demonstrate that one functional PIP(2)-interacting WT subunit is sufficient to convert channels from the unavailable to the available mode with a high open probability dominated by the fully open state, with similar kinetics as tetrameric WT channels. Occasional openings to sublevels become progressively less frequent as the number of WT subunits increases. Quantitative analysis reveals that the interaction of PIP(2) with WT subunits exerts strong positive cooperativity in both converting the channels from the unavailable to the available mode, and in promoting the fully open state over sublevels. We conclude that the interaction of PIP(2) with only one Kir2.1 subunit is sufficient for the channel to become available and to open to its full conductance state. Interaction with additional subunits exerts positive cooperativity at multiple levels to further enhance channel availability and promote the fully open state.

Dynamic modulation of intracellular glucose imaged in single cells using a FRET-based glucose nanosensor.

To study intracellular glucose homeostasis, the glucose nanosensor FLIPglu-600 microM, which undergoes changes in fluorescence resonance energy transfer (FRET) upon interaction with glucose, was expressed in four mammalian cell lines: COS-7, CHO, HEK293, and C2C12. Upon addition of extracellular glucose, the intracellular FRET ratio decreased rapidly as intracellular glucose increased. The kinetics were fast (tau=5 to 15 s) in COS and C2C12 cells and slow (tau=20 to 40 s) in HEK and CHO cells. Upon removal of extracellular glucose, the FRET ratio returned to its initial value at similar rates (tau=15 to 40 s) in all cell types. In all cell types, the glucose uptake FRET signal was blocked by the glucose transporter (GLUTx) inhibitor cytochalasin B and was not affected by the Na/glucose transporter inhibitor phlorizin. Glucose clearance was inhibited by the glycolytic inhibitor iodoacetate. Using beta-escin to permeabilize the cell, we found that the glucose gradient across the membrane was strongly dependent on the rates of glucose uptake versus glucose clearance. With 10 mM extracellular glucose and a high rate of glucose clearance, intracellular glucose level fell below 100 muM when glucose uptake rate was low, whereas it exceeded 0.5 mM when glucose uptake was high. Cells cultured in high glucose maintained lower basal intracellular glucose levels than cells cultured in low glucose, attributed to "reciprocal regulation" of glycolysis and gluconeogenesis. Basal glucose level also increased with elevated temperatures. Experiments performed with C2C12 cells demonstrated a shift from fast glucose uptake to slow glucose uptake in the absence of insulin during differentiation.

A novel role for connexin hemichannel in oxidative stress and smoking-induced cell injury.

Oxidative stress is linked to many pathological conditions, including ischemia, atherosclerosis and neurodegenerative disorders. The molecular mechanisms of oxidative stress induced pathophysiology and cell death are currently poorly understood. Our present work demonstrates that oxidative stress induced by reactive oxygen species and cigarette smoke extract depolarize the cell membrane and open connexin hemichannels. Under oxidative stress, connexin expression and connexin silencing resulted in increased and reduced cell deaths, respectively. Morphological and live/dead assays indicate that cell death is likely through apoptosis. Our studies provide new insights into the mechanistic role of hemichannels in oxidative stress induced cell injury.

Activation of inwardly rectifying potassium (Kir) channels by phosphatidylinosital-4,5-bisphosphate (PIP2): interaction with other regulatory ligands.

All members of the inwardly rectifying potassium channels (Kir1-7) are regulated by the membrane phospholipid, phosphatidylinosital-4,5-bisphosphate (PIP(2)). Some are also modulated by other regulatory factors or ligands such as ATP and G-proteins, which give them their common names, such as the ATP sensitive potassium (K(ATP)) channel and the G-protein gated potassium channel. Other more non-specific regulators include polyamines, kinases, pH and Na(+) ions. Recent studies have demonstrated that PIP(2) acts cooperatively with other regulatory factors to modulate Kir channels. Here we review how PIP(2) and co-factors modulate channel activities in each subfamily of the Kir channels.

ATP-sensitive K+ channels: regulation of bursting by the sulphonylurea receptor, PIP2 and regions of Kir6.2.

ATP-sensitive K+ channels composed of the pore-forming protein Kir6.2 and the sulphonylurea receptor SUR1 are inhibited by ATP and activated by Phosphatidylinositol Bisphosphate (PIP2). Residues involved in binding of these ligands to the Kir6.2 cytoplasmic domain have been identified, and it has been hypothesized that gating mechanisms involve conformational changes in the regions of the bundle crossing and/or the selectivity filter of Kir6.2. Regulation of Kir6.2 by SUR1, however, is not well-understood, even though this process is ATP and PIP2 dependent. In this study, we investigated the relationship between channel regulation by SUR1 and PIP2 by comparing a number of single and double mutants known to affect open probability (P(o)), PIP2 affinity, and sulphonylurea and MgADP sensitivity. When coexpressed with SUR1, the Kir6.2 mutant C166A, which is characterized by a P(o) value close to 0.8, exhibits no sulphonylurea or MgADP sensitivity. However, when P(o) was reduced by combining mutations at the PIP2-sensitive residues R176 and R177 with C166A, sulphonylurea and MgADP sensitivities were restored. These effects correlated with a dramatic decrease in PIP2 affinity, as assessed by PIP2-induced channel reactivation and inhibition by neomycin, an antagonist of PIP2 binding. Based on macroscopic and single-channel data, we propose a model in which entry into the high-P(o) bursting state by the C166A mutation or by SUR1 depends on the interaction of PIP2 with R176 and R177 and, to a lesser extent, R54. In conjunction with this PIP2-dependent process, SUR1 also regulates channel activity via a PIP2-independent, but MgADP-dependent process.

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.

ATP sensitivity of ATP-sensitive K+ channels: role of the gamma phosphate group of ATP and the R50 residue of mouse Kir6.2.

ATP-sensitive K (K(ATP)) channels are composed of Kir6, the pore-forming protein, and the sulphonylurea receptor SUR, a regulatory protein. We and others have previously shown that positively charged residues in the C terminus of Kir6.2, including R201 and K185, interact with the alpha and beta phosphate groups of ATP, respectively, to induce channel closure. A positively charged residue in the N terminus, R50, is also important, and has been proposed to interact with either the gamma or beta phosphate group of ATP. To examine this issue, we systematically mutated R50 to residues of different size, charge and hydropathy, and examined the effects on adenine nucleotide sensitivity in the absence and presence of SUR1. In the absence of SUR1, only the size of residue 50 significantly altered ATP sensitivity, with smaller side chains decreasing ATP sensitivity. In the presence of SUR1, however, hydrophathy and charge also played a role. Hydrophilic residues decreased ATP sensitivity more than hydrophobic residues for small size residues, and, surprisingly, negatively charged residues E and D preserved ATP sensitivity and increased ADP sensitivity relative to the wild-type residue R. These observations suggest that a negative charge near position 50, due to either mutation of R50 or the interaction of the gamma phosphate group of ATP with R50, facilitates closure of the ATP-dependent gate. Mutation of the nearby positively charged residue R54, known to be involved in stabilizing channel opening via electrostatic interactions with phosphatidylinositol 4,5-bisphosphate (PIP2), also caused increased ADP sensitivity as compared with ATP, suggesting a loss of function of ATP's gamma phosphate. Based on these results, we propose that a phosphate group or a negative charge at position 50 initiates channel closure by destabilizing the electrostatic interactions between negative phosphate groups of PIP2 and residues such as R54.

Regulation of the ATP-sensitive K channel Kir6.2 by ATP and PIP(2).

ATP-sensitive K (K(ATP)) channels are blocked by ATP and activated by PIP(2). Both negatively-charged ligands are presumed to bind to positively-charged residues on the N-and C-termini of the channel's cytoplasmic domain. Evidence summarized here suggests that the channel's interaction with ATP and PIP(2) is regulated by separate groups of residues, involving both direct charge-charge interactions and allosteric effects. ATP interaction is regulated by R50 in the N-terminus and by K185, R192 and R201 in the C-terminus. R192 and R201 mutations decrease channel sensitivity to ATP, ADP and AMP to a similar extent, implying that they regulate interaction with either the alpha phosphate group, common to all three adenine nucleotides, or the adenosine moiety. K185 mutations, and to a lesser extent R50 mutations, decrease ATP and ADP sensitivity without markedly affecting AMP sensitivity, implying that they regulate interaction with the beta phosphate of ATP and ADP. In addition, when open probability decreases due to rundown, ATP sensitivity increases in R50, K185 and R192, but not in R201 mutants. Combining these observations with recent structural data, we hypothesize the following scenario: 1) the ATP binding site is located at the outside of the channel's cytoplasmic domain away from the pore. 2) When the channel is open, R50 and K185 interact directly with the beta phosphate of ATP, whereas R192, which appears to be removed from the ATP binding site, modulates the initial interaction with ATP allosterically. 3) When the channel closes, R201 is in position to interact with the alpha phosphate of ATP to stabilize the closed state. 4) PIP(2) also interacts with the channel's cytoplasmic domain, but at distinct positively-charged residues located above the ATP binding site and near to the plasma membrane. These residues include R54 in the N-terminus and R176, R177 and R206 in the C-terminus. Thus, the binding domains of ATP and PIP(2) in the N- and C-termini do not appear to overlap.

Regulation of gating by negative charges in the cytoplasmic pore in the Kir2.1 channel.

Inward rectifier K(+) channels commonly exhibit long openings (slow gating) punctuated by rapid open-close transitions (fast gating), suggesting that two separate gates may control channel open-closed transitions. Previous studies have suggested possible gate locations at the selectivity filter and at the 'bundle crossing', where the two transmembrane segments (M1 and M2) cross near the cytoplasmic end of the pore. Wild-type Kir2.1 channels exhibit only slow gating, but mutations in the cytoplasmic pore domain at E224 and E299 have been shown to induce fast flickery gating. Since these mutations also affect polyamine affinity, we conjectured that the fast gating mechanism might affect the kinetics of polyamine block/unblock if located more intracellularly than the polyamine blocking site in the pore. Neutralization of either E224 or E299 induced fast gating and slowed both block and unblock rates by the polyamine diamine 10. The slowing of polyamine block/unblock was partly relieved by raising pH from 7.2 to 9.0, which also slowed fast gating kinetics. These findings indicate that the fast flickery gate is located intracellularly with respect to the polyamine pore-plugging site near D172, thereby excluding the selectivity filter, and implicating the bundle crossing or more intracellular site as the gate. As additional proof, fast gating induced at the selectivity filter by disrupting P loop salt bridges in WT-E138D-E138D-WT tandem had no effect on polyamine block and unblock rates. The pH sensitivity of fast gating in E224 and E299 mutants was attributed to the protonation state of H226, since the double mutant E224Q/H226K induced fast gating which was pH insensitive. Moreover, introducing a negative charge in the 224-226 region was sufficient to prevent fast gating, since the double mutant E224Q/H226E rescued wild-type Kir2.1 slow gating. These observations implicate E224 and E299 as allosteric modulators of a fast gate, located at the bundle crossing or below in Kir2.1 channels. By suppressing fast gating, these negative charges facilitate polyamine block and unblock, which may be their physiologically important role.

Mechanosensitivity of GIRK channels is mediated by protein kinase C-dependent channel-phosphatidylinositol 4,5-bisphosphate interaction.

Gprotein-activated inwardly rectifying K+ channel (GIRK or Kir3) currents are inhibited by mechanical stretch of the cell membrane, but the underlying mechanisms are not understood. In Xenopus oocytes heterologously expressing GIRK channels, membrane stretch induced by 50% reduction of osmotic pressure caused a prompt reduction of GIRK1/4, GIRK1, and GIRK4 currents by 16.6-42.6%. Comparable GIRK current reduction was produced by protein kinase C (PKC) activation (phorbol 12-myristate 13-acetate). The mechanosensitivity of the GIRK4 current was abolished by pretreatment with PKC inhibitors (staurosporine or calphostin C). Neither hypo-osmotic challenge nor PKC activation affected IRK1 currents. GIRK4 chimera (GIRK4-IRK1-(Lys207-Leu245)) and single point mutant (GIRK4(I229L)), in which the phosphatidylinositol 4,5-bisphosphate (PIP2) binding domain or residue was replaced by the corresponding region of IRK1 to strengthen the channel-PIP2 interaction, showed no mechanosensitivity and minimal PKC sensitivity. IRK1 gained mechanosensitivity and PKC sensitivity by reverse double point mutation of the PIP2 binding domain (L222I/R213Q). Overexpression of Gbetagamma, which is known to strengthen the channel-PIP2 interaction, attenuated the mechanosensitivity of GIRK4 channels. In oocytes expressing a pleckstrin homology domain of PLC-delta tagged with green fluorescent protein, hypo-osmotic challenge or PKC activation caused a translocation of the fluorescence signal from the cell membrane to the cytosol, reflecting PIP2 hydrolysis. The translocation was prevented by pretreatment with PKC inhibitors. Involvement of PKC activation in the mechanosensitivity of muscarinic K+ channels was confirmed in native rabbit atrial myocytes. These results suggest that the mechanosensitivity of GIRK channels is mediated primarily by channel-PIP2 interaction, with PKC playing an important role in modulating the interaction probably through PIP2 hydrolysis.

Molecular mechanism for ATP-dependent closure of the K+ channel Kir6.2.

In the ATP-dependent K+ (KATP) channel pore-forming protein Kir6.2, mutation of three positively charged residues, R50, K185 and R201, impairs the ability of ATP to close the channel. The mutations do not change the channel open probability (Po) in the absence of ATP, supporting the involvement of these residues in ATP binding. We recently proposed that at least two of these positively charged residues, K185 and R201, interact with ATP phosphate groups to cause channel closure: the beta phosphate group of ATP interacts with K185 to initiate closure, while the alpha phosphate interacts with R201 to stabilize the channel's closed state. In the present study we replaced these three positive residues with residues of different charge, size and hydropathy. For K185 and R201, we found that charge, more than any other property, controls the interaction of ATP with Kir6.2. At these positions, replacement with another positive residue had minor effects on ATP sensitivity. In contrast, replacement of K185 with a negative residue (K185D/E) decreased ATP sensitivity much more than neutral substitutions, suggesting that an electrostatic interaction between the beta phosphate group of ATP and K185 destabilizes the open state of the channel. At R201, replacement with a negative charge (R201E) had multiple effects, decreasing ATP sensitivity and preventing full channel closure at high concentrations. In contrast, the R50E mutation had a modest effect on ATP sensitivity, and only residues such as proline and glycine that affect protein structure caused major decreases in ATP sensitivity at the R50 position. Based on these results and the recently published structure of Kir3.1 cytoplasmic domain, we propose a scheme where binding of the beta phosphate group of ATP to K185 induces a motion of the surrounding region, which destabilizes the open state, favouring closure of the M2 gate. Binding of the alpha phosphate group of ATP to R201 then stabilizes the closed state. R50 on the N-terminus controls ATP binding by facilitating the interaction of the beta phosphate group of ATP with K185 to destabilize the open state.

Inward rectification by polyamines in mouse Kir2.1 channels: synergy between blocking components.

We recently characterized two distinct mechanisms by which the polyamine spermine blocks Kir2.1 channels: (1) by reduction of negative surface charges in the cytoplasmic pore, thereby reducing single-channel conductance, and (2) by direct open channel transmembrane pore block. The extent to which the surface charge reduction component is mediated by passive surface charge screening versus binding of polyamines to these charges, as well as the extent to which the surface charge reduction and pore block mechanisms are synergistic, versus simply additive, was not established. To address these issues, macroscopic currents were recorded from inside-out giant patches from Xenopus oocytes and from single-channel currents from COS7 cells expressing wild-type and mutant Kir2.1 channels, during exposure to polyamines of varying length and charge. The surface charge reduction component was decreased when polyamine charge (at constant length) was decreased from 4 (spermine) to 2 (diamine 10, DA10). Moreover, the surface charge reduction component of block involved more than passive surface charge screening and required binding of polyamines to the cytoplasmic pore, since it was eliminated when polyamine length was shortened below six alkyl groups. Loss of surface charge reduction also dramatically affected open channel pore block. The latter consisted of two subcomponents with fast and slow kinetics, respectively. The slow subcomponent decreased as blocker length decreased (DA10, DA8 and DA6), whereas the fast subcomponent was sensitive to blocker charge (spermine vs. DA10). Neutralization of E224 and E299, which eliminated the surface charge reduction component of block, also eliminated the fast subcomponent of pore block. Neutralization of D172 had no effect on the surface charge reduction component, but weakened both of the subcomponents of pore block. These findings can be accounted for by a model in which the negative charges at E224, E299 and D172 act in a concerted manner to coordinate the surface charge reduction and open channel components of polyamine block. In this model, the binding of polyamines to surface charges E224 and E299 pre-positions them in the cytoplasmic pore in a manner that directly facilitates their entry and exit from a transmembrane pore-occluding site involving D172. A molecular model using the recently reported 1.8 A resolution structure of the inward-rectifier cytoplasmic pore, adapted to Kir2.1, is consistent with longer polyamines binding at their positively charged ends to the E224 and E299 positions in the same subunit, potentially accommodating four polyamine molecules per channel.

Molecular basis for Kir6.2 channel inhibition by adenine nucleotides.

K(ATP) channels are comprised of a pore-forming protein, Kir6.x, and the sulfonylurea receptor, SURx. Interaction of adenine nucleotides with Kir6.2 positively charged amino acids such as K185 and R201 on the C-terminus causes channel closure. Substitution of these amino acids with other positively charged residues had small effects on inhibition by adenine nucleotide, while substitution with neutral or negative residues had major effects, suggesting electrostatic interactions between Kir6.2 positive charges and adenine nucleotide negative phosphate groups. Furthermore, R201 mutation decreased channel sensitivity to ATP, ADP, and AMP to a similar extent, but K185 mutation decreased primarily ATP and ADP sensitivity, leaving the AMP sensitivity relatively unaffected. Thus, channel inhibition by ATP may involve interaction of the alpha-phosphate with R201 and interaction of the beta-phosphate with K185. In addition, decreased open probability due to rundown or sulfonylureas caused an increase in ATP sensitivity in the K185 mutant, but not in the R201 mutant. Thus, the beta-phosphate may bind in a state-independent fashion to K185 to destabilize channel openings, while R201 interacts with the alpha-phosphate to stabilize a channel closed configuration. Substitution of R192 on the C-terminus and R50 on the N-terminus with different charged residues also affected ATP sensitivity. Based on these results a structural scheme is proposed, which includes features of other recently published models.