Novel heme ligand displacement by CO in the soluble hemophore HasA and its proximal ligand mutants: implications for heme uptake and release

HasASM, a hemophore secreted by the Gram-negative bacteria Serratia marcescens, extracts heme from host hemoproteins and shuttles it to HasRSM, a specific hemophore outer membrane receptor. Heme iron in HasASM is in a six-coordinate ferric state. It is linked to the protein by the heretofore uncommon axial ligand set, His32 and Tyr75. A third residue of the heme pocket, His83, plays a crucial role in heme ligation through hydrogen bonding to Tyr75. The vibrational frequencies of coordinated carbon monoxide constitute a sensitive probe of trans ligand field, FeCO structure, and electrostatic landscape of the distal heme pockets of heme proteins. In this study, carbonyl complexes of wild-type (WT) HasASM and its heme pocket mutants His32Ala, Tyr75Ala, and His83Ala were characterized by resonance Raman spectroscopy. The CO complexes of WT HasASM, HasASM(His32Ala), and HasASM(His83Ala) exhibit similar spectral features and fall above the line that correlates nuFe-CO and nuC-O for proteins having a proximal imidazole ligand. This suggests that the proximal ligand field in these CO adducts is weaker than that for heme-CO proteins bearing a histidine axial ligand. In contrast, the CO complex of HasASM(Tyr75Ala) has resonance Raman signatures consistent with ImH-Fe-CO ligation. These results reveal that in WT HasASM, the axial ImH side chain of His32 is displaced by CO. This is in contrast to other heme proteins known to have the His/Tyr axial ligand set, wherein the phenolic side chain of the Tyr ligand dissociates upon CO addition. The displacement of His32 and its stabilization in an unbound state is postulated to be relevant to heme uptake and/or release.     

Biophysical defects in voltage.gated sodium channels associated with long QT and Brugada syndromes

The activity of voltage-gated sodium channels contributes to onset and duration of the cardiac action potential through an intricate balance with the activity of other ion channels. Activation of sodium channels leads to membrane depolarization and Phase 0 of the cardiac action potential. Sodium channel fast inactivation contributes to Phase 1, the initial repolarization. Slow inactivation and closed state fast inactivation determine channel availability and, thus, overall membrane excitability. Defects in any of these biophysical states or transitions between them, imparted by (over 170 reported thus far, including both Long QT3 and Brugada syndromes) mutations in the (over 2000) amino acids that compose the sodium channel protein, can lead to channel dysfunction that manifests as an abnormal cardiac action potential and electrocardiogram. A causal relationship between several such abnormalities and the panoply of sodium channel mutations have led to a greater understanding of the molecular underpinnings of cardiac arrhythmias as well as a deeper appreciation for the intricacies of sodium channel function. Here, we review the literature regarding these causal relationships from a perspective of the biophysical properties of sodium channels.     

Structure and ligand binding properties of myoglobins reconstituted with monodepropionated heme: functional role of each heme propionate side chain

Two heme propionate side chains, which are attached at the 6 and 7 positions of the heme framework, are linked with Arg45 and Ser92, respectively, in sperm whale myoglobin. To evaluate the role of each propionate, two kinds of one-legged hemins, 6-depropionated and 7-depropionated protohemins, were prepared and inserted into the apomyoglobin to yield two reconstituted proteins. Structural data of the reconstituted myoglobins were obtained via an X-ray crystallographic analysis at a resolution of 1.1-1.4 A and resonance Raman spectroscopy. It was found that the lack of the 6-propionate reduces the number of hydrogen bonds in the distal site and clearly changes the position of the Arg45 residue with the disrupting Arg45-Asp60 interaction. In contrast, the removal of the 7-propionate does not cause a significant structural change in the residues of the distal and proximal sites. However, the resonance Raman studies suggested that the coordination bond strength of the His93-Fe bond for the protein with the 7-depropionated protoheme slightly increases compared to that for the protein with the native heme. The O2 and CO ligand binding studies for the reconstituted proteins with the one-legged hemes provide an important insight into the functional role of each propionate. The lack of the 6-propionate accelerates the O2 dissociation by ca. 3-fold compared to those of the other reconstituted and native proteins. The lack of the 7-propionate enhances the CO affinity by 2-fold compared to that of the protein with the native heme. These results indicate that the 6-propionate clearly contributes to the stabilization of the bound O2, whereas the 7-propionate plays an important role in the regulation of the Fe-His bond.     

Replacement of the axial histidine heme ligand with cysteine in nitrophorin 1: spectroscopic and crystallographic characterization

To evaluate the potential of using heme-containing lipocalin nitrophorin 1 (NP1) as a template for protein engineering, we have replaced the native axial heme-coordinating histidine residue with glycine, alanine, and cysteine. We report here the characterization of the cysteine mutant H60C_NP1 by spectroscopic and crystallographic methods. The UV/vis, resonance Raman, and magnetic circular dichroism spectra suggest weak thiolate coordination of the ferric heme in the H60C_NP1 mutant. Reduction to the ferrous state resulted in loss of cysteine coordination, while addition of exogenous imidazole ligands gave coordination changes that varied with the ligand. Depending on the substitution of the imidazole, we could distinguish three heme coordination states: five-coordinate monoimidazole, six-coordinate bisimidazole, and six-coordinate imidazole/thiolate. Ligand binding affinities were measured and found to be generally 2–3 orders of magnitude lower for the H60C mutant relative to NP1. Two crystal structures of the H60C_NP1 in complex with imidazole and histamine were solved to 1.7- and 1.96-Å resolution, respectively. Both structures show that the H60C mutation is well tolerated by the protein scaffold and suggest that heme–thiolate coordination in H60C_NP1 requires some movement of the heme within its binding cavity. This adjustment may be responsible for the ease with which the engineered heme–thiolate coordination can be displaced by exogenous ligands.     

Reaction of hemoglobin with HOCl: mechanism of heme destruction and free iron release

Hypochlorous acid (HOCl) is generated by myeloperoxidase using chloride and hydrogen peroxide as substrates. HOCl and its conjugate base (OCl⁻) bind to the heme moiety of hemoglobin (Hb) and generate a transient ferric species whose formation and decay kinetics indicate it can participate in protein aggregation and heme destruction along with subsequent free iron release. The oxidation of the Hb heme moiety by OCl⁻ was accompanied by marked heme destruction as judged by the decrease in and subsequent flattening of the Soret absorbance peak at 405 nm. HOCl-mediated Hb heme depletion was confirmed by HPLC analysis and in-gel heme staining. Exposure of Hb to increasing concentrations of HOCl produced a number of porphyrin degradation products resulting from oxidative cleavage of one or more of the carbon-methene bridges of the tetrapyrrole ring, as identified by their characteristic HPLC fluorescence and LC-MS. A nonreducing denaturing SDS-PAGE showed several degrees of protein aggregation. Similarly, porphyrin degradation products were identified after exposure of red blood cells to increasing concentrations of HOCl, indicating biological relevance of this finding. This work provides a direct link between Hb heme destruction and subsequent free iron accumulation, as occurs under inflammatory conditions where HOCl is formed in substantial amounts.     

Looking for probes of gated channels: studies of the inhibition of glucose and choline transport in erythrocytes

Two seemingly contradictory sets of observations have been made in studies of biological transport, which are essential for our understanding of the transport mechanism: carriers are integral membrane proteins, which span the membrane and are not free to rotate across the membrane; carriers appear to function like a ferryboat, with a substrate binding site moving back and forth from one side of the membrane to the other. To reconcile these facts, it is necessary to postulated gated channels connecting the substrate site with the two membrane surfaces: the channels are arranged so that as one opens the other closes, with the result that the substrate site is alternately accessible from opposite sides of the membrane. Based on these properties, the following distinguishing features of molecules specifically bound in the channels may be predicted: if sufficiently bulky, they inhibit transport; they bind outside the substrate site (though adjacent to it), they bind asymmetrically either to the outward-facing carrier and on the outer surface of the membrane, or to the inward-facing carrier and on the inner surface of the membrane. The asymmetrical inhibition of the glucose and choline transport systems of erythrocytes by various inhibitors is examined, and the behavior in every case is found to conform with these criteria. From the results it may be concluded that the glucose carrier binds cytochalasin B in the inner gated channel and phloretin and tetrathionate in the outer gated channel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage gated calcium channels in molluscs: classification,Ca2+ dependent inactivation,modulation and functional roles

Molluscan neurons and muscle cells express transient (T-type like) and sustained LVA calcium channels, as well as transient and sustained HVA channels. In addition weakly voltage sensitive calcium channels are observed. In a number of cases toxin or dihydropyridine sensitivity justifies classification of the HVA currents in L, N or P-type categories. In many cases, however, pharmacological characterization is still preliminary. Characterization of novel toxins from molluscivorousConus snails may facilitate classification of molluscan calcium channels. Molluscan preparations have been very useful to study calcium dependent inactivation of calcium channels. Proposed mechanisms explain calcium dependent inactivation through direct interaction of Ca²⁺ with the channel, through dephosphorylation by calcium dependent phosphatases or through calcium dependent disruption of connections with the cytoskeleton. Transmitter modulation operating through various second messenger mediated pathways is well documented. In general, phosphorylation through PKA, cGMP dependent PK or PKC facilitates the calcium channels, while putative direct G-protein action inhibits the channels. Ca²⁺ and cGMP may inhibit the channels through activation of phosphodiesterases or phosphatases. Detailed evidence has been provided on the role of sustained LVA channels in pacemaking and the generation of firing patterns, and on the role of HVA channels in the dynamic changes in action potentials during spiking, the regulation of the release of transmitters and hormones, and the regulation of growth cone behavior and neurite outgrowth. The accessibility of molluscan preparations (e.g. the squid giant synapse for excitation release studies,Helisoma B5 neuron for neurite and synapse formation) and the large body of knowledge on electrophysiological properties and functional connections of identified molluscan neurons (e.g. sensory neurons, R15, egg laying hormone producing cells, etc.) creates valuable opportunities to increase the insight into the functional roles of calcium channels.     

Mechanisms of ATP release in pain: role of pannexin and connexin channels

Pain is a physiological response to bodily damage and serves as a warning of potential threat. Pain can also transform from an acute response to noxious stimuli to a chronic condition with notable emotional and psychological components that requires treatment. Indeed, the management of chronic pain is currently an important unmet societal need. Several reports have implicated the release of the neurotransmitter adenosine triphosphate (ATP) and subsequent activation of purinergic receptors in distinct pain etiologies. Purinergic receptors are broadly expressed in peripheral neurons and the spinal cord; thus, purinergic signaling in sensory neurons or in spinal circuits may be critical for pain processing. Nevertheless, an outstanding question remains: what are the mechanisms of ATP release that initiate nociceptive signaling? Connexin and pannexin channels are established conduits of ATP release and have been suggested to play important roles in a variety of pathologies, including several models of pain. As such, these large-pore channels represent a new and exciting putative pharmacological target for pain treatment. Herein, we will review the current evidence for a role of connexin and pannexin channels in ATP release during nociceptive signaling, such as neuropathic and inflammatory pain. Collectively, these studies provide compelling evidence for an important role of connexins and pannexins in pain processing.     

Time course of increased heme oxygenase activity and expression after experimental intracerebral hemorrhage: correlation with oxidative injury

Heme oxygenase (HO) activity in tissue adjacent to an intracerebral hematoma may modulate cellular vulnerability to heme-mediated oxidative injury. Although HO-1 is induced after experimental intracerebral hemorrhage (ICH), the time course of this induction, its effect on tissue HO activity, and its association with oxidative injury markers has not been defined. We therefore quantified HO activity, HO-1 expression, tissue heme content, and protein carbonylation for 8 days after injection of autologous blood into the mouse striatum. Increased striatal HO-1 protein was observed within 24 h, peaked on day 5 at a level that was 10-fold greater than baseline, and returned to baseline by day 8; HO-2 expression was not altered. HO activity increased by only 1.6-fold at its peak on day 5, and had also returned to baseline by day 8. A significant increase in protein carbonylation was observed at 3–5 days, which also was markedly attenuated by 8 days, concomitant with a return of tissue heme to near-normal levels. These results suggest that the increase in HO activity in tissue surrounding an experimental ICH is considerably less than would be predicted based on an analysis of HO-1 expression per se . As HO-1 expression is temporally associated with increased tissue heme and increased protein carbonylation, it may be more useful as a marker of heme-mediated oxidative stress in ICH models, rather than as an index of HO activity.     

Heat capacity effects in protein folding and ligand binding: a re-evaluation of the role of water in biomolecular thermodynamics

Large "anomalous" heat capacity (DeltaC(p)) effects are a common feature of the thermodynamics of biomolecular interactions in aqueous solution and, as a result of the improved facility for direct calorimetric measurements, there is a growing body of experimental data for such effects in protein folding, protein-protein and protein-ligand interactions. Conventionally such heat capacity effects have been ascribed to hydrophobic interactions, and there are some remarkably convincing demonstrations of the usefulness of this concept. Nonetheless, there is also increasing evidence that hydrophobic interactions are not the only possible source of such effects. Here we re-evaluate the possible contributions of other interactions to the heat capacity changes to be expected for cooperative biomolecular folding and binding processes, with particular reference to the role of hydrogen bonding and solvent water interactions. Simple models based on the hydrogen-bonding propensity of water as a function of temperature give quantitative estimates of DeltaC(p) that compare well with experimental observations for both protein folding and ligand binding. The thermodynamic contribution of bound waters in protein complexes is also estimated. The prediction from simple lattice models is that trapping of water in a complex should give more exothermic binding (DeltaDeltaH-6 to -12 kJ mol(-1)) with lower entropy (DeltaDeltaS(0) approximately -11 J mol(-1) K(-1)) and more negative DeltaC(p) (by about -75 J mol(-1) K(-1)) per water molecule. More generally, it is clear that significant DeltaC(p) effects are to be expected for any macromolecular process involving a multiplicity of cooperative weak interactions of whatever kind.     

Swelling activated Cl- channels in microglia: Biophysics, pharmacology and role in glutamate release

Microglia have a swelling-activated Cl⁻ current (which we call I_Clswell), and while some of its biophysical properties and functional roles have been elucidated, its molecular identity is unknown. To relate this current to cell functions and determine whether it is regulated by mechanisms other than cell swelling, it is important to establish both biophysical and pharmacological fingerprints. Here, we used rat microglia and a cell line derived from them (MLS-9) to study biophysical, regulatory and pharmacological properties of I_Clswell. The whole-cell current was activated in response to a hypo-osmotic bath solution, but not by voltage, and was time-independent during long voltage steps. The halide selectivity sequence was I⁻>Br⁻>Cl⁻ (Eisenman sequence I) and importantly, the excitatory amino acid, glutamate was permeant. Current activation required internal ATP, and was not affected by the guanine nucleotides, GTPγS or GDPβS, or physiological levels of internal Mg²⁺. The same current was activated by a low intracellular ionic strength solution without an osmotic gradient. I_Clswell was reversibly inhibited by known Cl⁻ channel blockers (NPP B, flufenamic acid, glibenclamide, DCPIB), and by the glutamate release inhibitor, riluzole. Cell swelling evoked glutamate release from primary microglia and MLS-9 cells, and this was inhibited by the blockers (above), and by IAA-94, but not by tamoxifen or the Na⁺/K⁺/Cl⁻ symport inhibitor, bumetanide. Together, these results confirm the similarity of I_Clswell in the two cell types, and point to a role for this channel in inflammation-mediated glutamate release in the CNS.Key words: rat microglia, MLS-9 cells, swelling-activated anion channels, VRAC, Cl⁻ channel biophysics, Cl⁻ channel pharmacology, ionic-strength, ATP-dependence, glutamate release     

The spark and its ember: separately gated local components of Ca(2+) release in skeletal muscle

Amplitude, spatial width, and rise time of Ca(2+) sparks were compared in frog fast-twitch muscle, in three conditions that alter activation of release channels by [Ca(2+)]. A total of approximately 17,000 sparks from 30 cells were evaluated. In cells under voltage clamp, caffeine (0.5 or 1 mM) increased average spark width by 28%, rise time by 18%, and amplitude by 7%. Increases in width were significant even among events of the same rise time. Spontaneous events recorded in permeabilized fibers with low internal [Mg(2+)] (0.4 mM), had width and rise times greater than in reference, and not significantly different than those in caffeine. The spark average in reference rides on a continuous fluorescence "ridge" and is continued by an "ember," a prolongation of width approximately 1 microm and amplitude <0.2, vanishing in approximately 100 ms. Ridge and ember were absent in caffeine and in permeabilized cells. Exposure of voltage-clamped cells to high internal [Mg(2+)] (7 mM) had effects opposite to caffeine, reducing spark width by 26% and amplitude by 27%. In high [Mg(2+)], the ember was visible in individual sparks as a prolongation of variable duration and amplitude up to 1.2. Based on simulations and calculation of Ca(2+) release flux from averaged sparks, the increase in spark width caused by caffeine was interpreted as evidence of an increase in radius of the release source-presumably by recruitment of additional channels. Conversely, spark narrowing suggests loss of contributing channels in high Mg(2+). Therefore, these changes in spark width at constant rise times are evidence of a multichannel origin of sparks. Because ridge and ember were reduced by promoters of Ca(2+)-dependent activation (caffeine, low [Mg(2+)]) and became more visible in the presence of its inhibitors, they are probably manifestations of Ca(2+) release directly operated by voltage sensors.     

Fast lidocaine block of cardiac and skeletal muscle sodium channels: one site with two routes of access.

We have studied the block by lidocaine and its quaternary derivative, QX-314, of single, batrachotoxin (BTX)-activated cardiac and skeletal muscle sodium channels incorporated into planar lipid bilayers. Lidocaine and QX-314, applied to the intracellular side, appear to induce incompletely resolved, rapid transitions between the open and the blocked state of BTX-activated sodium channels from both heart and skeletal muscle. We used amplitude distribution analysis (Yellen, G. 1984. J. Gen. Physiol. 84:157-186.) to estimate the rate constants for block and unblock. Block by lidocaine and QX-314 from the cytoplasmic side exhibits rate constants with similar voltage dependence. The blocking rate increases with depolarization, and the unblocking rate increases with hyperpolarization. Fast lidocaine block was virtually identical for sodium channels from skeletal (rat, sheep) and cardiac (beef, sheep) muscle. Lidocaine block from the extracellular side occurred at similar concentrations. However, for externally applied lidocaine, the blocking rate was voltage-independent, and was proportional to concentration of the uncharged, rather than the charged, form of the drug. In contrast, unblocking rates for internally and externally applied lidocaine were identical in magnitude and voltage dependence. Our kinetic data suggest that lidocaine, coming from the acqueous phase on the cytoplasmic side in the charged form, associates and dissociates freely with the fast block effector site, whereas external lidocaine, in the uncharged form, approaches the same site via a direct, hydrophobic path.     

Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore

Release of charged neurotransmitter molecules through a narrow fusion pore requires charge compensation by other ions. It has been proposed that this may occur by ion flow from the cytosol through channels in the vesicle membrane, which would generate a net outward current. This hypothesis was tested in chromaffin cells using cell-attached patch amperometry that simultaneously measured catecholamine release from single vesicles and ionic current across the patch membrane. No detectable current was associated with catecholamine release indicating that <2% of cations, if any, enter the vesicle through its membrane. Instead, we show that flux of catecholamines through the fusion pore, measured as an amperometric foot signal, decreases when the extracellular cation concentration is reduced. The results reveal that the rate of transmitter release through the fusion pore is coupled to net Na+ influx through the fusion pore, as predicted by electrodiffusion theory applied to fusion-pore permeation, and suggest a prefusion rather than postfusion role for vesicular cation channels.     

Pannexin channels in ATP release and beyond: An unexpected rendezvous at the endoplasmic reticulum

The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²⁺, for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression. Moreover, it is intriguing that Panxs may also function at the endoplasmic reticulum (ER) as intracellular Ca²⁺-leak channel and may be involved in ER-related functions. Although the physiological significance and meaning of such Panx-regulated intracellular Ca²⁺ leak requires further exploration, this functional property places Panx at the centre of many physiological and pathophysiological processes, given the fundamental role of intracellular Ca²⁺ homeostasis and dynamics in a plethora of physiological processes. In this review, we therefore want to focus on Panx as channels at the plasma membrane and at the ER membranes with a particular emphasis on the potential implications of the latter in intracellular Ca²⁺ signalling.     

Oxygen binding and redox properties of the heme in soluble guanylate cyclase: implications for the mechanism of ligand discrimination

Soluble guanylate cyclase is an NO-sensing hemoprotein that serves as a NO receptor in NO-mediated signaling pathways. It has been believed that this enzyme displays no measurable affinity for O(2), thereby enabling the selective NO sensing in aerobic environments. Despite the physiological significance, the reactivity of the enzyme-heme for O(2) has not been examined in detail. In this paper we demonstrated that the high spin heme of the ferrous enzyme converted to a low spin oxyheme (Fe(2+)-O(2)) when frozen at 77 K in the presence of O(2). The ligation of O(2) was confirmed by EPR analyses using cobalt-substituted enzyme. The oxy form was produced also under solution conditions at -7 °C, with the extremely low affinity for O(2). The low O(2) affinity was not caused by a distal steric protein effect and by rupture of the Fe(2+)-proximal His bond as revealed by extended x-ray absorption fine structure. The midpoint potential of the enzyme-heme was +187 mV, which is the most positive among high spin protoheme-hemoproteins. This observation implies that the electron density of the ferrous heme iron is relatively low by comparison to those of other hemoproteins, presumably due to the weak Fe(2+)-proximal His bond. Based on our results, we propose that the weak Fe(2+)-proximal His bond is a key determinant for the low O(2) affinity of the heme moiety of soluble guanylate cyclase.     

Genetic loci associated with stem elongation and winter dormancy release in wheat

In winter wheat (Triticum aestivum L.), the stem begins to elongate after the vernalization requirement is satisfied during winter and when favorable temperature and photoperiod conditions are attained in spring. In this study, we precisely measured elongation of the first extended internode on 96 recombinant inbred lines of a population that was generated from a cross between two winter wheat cultivars, Jagger (early stem elongation) and 2174 (late stem elongation). We mapped a major locus for stem elongation to the region where VRN-A1 resides in chromosome 5A. Visible assessment of winter dormancy release was concomitantly associated with this locus. VRN1 was previously cloned based on variation in vernalization requirement between spring wheat carrying a dominant Vrn-1 allele and winter wheat carrying a recessive vrn-1 allele. Both of two winter wheat cultivars in this study carry a recessive vrn-A1 allele; therefore, our results suggest that either VRN-A1 might invoke a new regulatory mechanism or a new gene residing close to VRN-A1 plays a regulatory role in winter wheat development. Phenotypic expression of the vrn-A1a allele of Jagger was more sensitive to the year of measurement of stem elongation than that of the vrn-A1b allele of 2174. In addition to QSte.osu.5A, several loci were also found to have minor effects on initial stem elongation of winter wheat. Seventeen of nineteen locally adapted cultivars in the southern Great Plaints contained the vrn-A1b allele. Hence, breeders in this area have inadvertently selected this allele, contributing to later stem elongation and more conducive developmental patterns for grain production.