Activation of pannexin 1 channels by ATP through P2Y receptors and by cytoplasmic calcium

The ability for long-range communication through intercellular calcium waves is inherent to cells of many tissues. A dual propagation mode for these waves includes passage of IP3 through gap junctions as well as an extracellular pathway involving ATP. The wave can be regenerative and include ATP-induced ATP release via an unknown mechanism. Here, we show that pannexin 1 channels can be activated by extracellular ATP acting through purinergic receptors of the P2Y group as well as by cytoplasmic calcium. Based on its properties, including ATP permeability, pannexin 1 may be involved in both initiation and propagation of calcium waves.     

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.     

Regulation of pannexin channels by post-translational modifications

The large-pore channels formed by the pannexin family of proteins have been implicated in many physiological and pathophysiological functions, mainly through their ATP release function. However, a tight regulation of channel opening is necessary to modulate their function in vivo. Post-translational modifications have been postulated as some of the regulating mechanisms for Panx1, while Panx2 and Panx3 have not been as well characterized. Positive regulators include caspase cleavage to open Panx1 channels in apoptotic cells, and activation by Src family kinases via ionotropic receptors in neurons and macrophages. S-nitrosylation of cysteines has been shown to both inhibit and activate the Panx1 channel in different cell types. All three pannexins are N-glycosylated but to different levels of modification. Their diverse glycosylation appears to regulate cellular localization, intermixing, and may restrict their ability to function as inter-cellular channels. It is clear that our understanding of pannexin post-translational modification and their role in channel function regulation is still in its infancy even a decade after their discovery.     

Pore-to-gate coupling of HCN channels revealed by a pore variant that contributes to gating but not permeation

Although ample evidence suggests the presence of an intracellular activation gate in HCN (pacemaker) channels, mutations in the outer pore can alter gating properties. Here we investigated the role of the outer pore residue A354 in HCN1 gating by systematically converting it to the equivalent residues (T, Y, and F) found in K(+)-channels. A354T negatively shifted steady-state activation (DeltaV(1/2) approximately -25 mV), decelerated gating kinetics (by up to 8-fold), and abolished the effects of external ions on gating. A354Y and A354F did not yield functional currents when expressed alone, although immunofluorescence microscopy indicated the presence of these channel proteins on the membrane surface. Currents recorded after co-expressing A354Y with WT HCN1 were reduced in amplitude (relative to WT alone) and had changes in gating similar to those of A354T. We conclude that the pore variant at position 354 contributes to gating but not permeation, and that the HCN outer pore may be involved in gating via a pore-to-gate coupling mechanism.     

Block of endplate channels by permeant cations in frog skeletal muscle

Motor endplates of frog semitendinosus muscles were studied under voltage clamp. Current fluctuations induced by iontophoretic application of acetylcholine were analyzed to give the elementary conductance, gamma , and mean open time, tau , of endplate channels. Total replacement of the external Na+ ion by several other metal ions and by many permeant organic cations changed both gamma and tau . Except with NH4+ ions, the gamma values with foreign test ions were all smaller than expected from the independence relation and their previously measured permeability ratios. The more hydrophobic ions gave the smallest gamma values. Foreign permeant cations also depress gamma when mixed with Na+ ions. These effects could be interpreted in terms of binding of ions to a saturable site within the endplate channel as they pass through. The site for organic ions would have a hydrophobic component. Similar evidence is given for a metal ion binding site on the cytoplasmic end of the channel accessible to internal ions. Most foreign cations also shortened tau when applied externally. The changes of gating did not seem to be correlated with changes in gamma . Thus there is no evidence for control of tau by ions bound within the pore.     

Structural basis of GLUT1 inhibition by cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)-mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231-13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1-ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7-8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1-GLUT4 chimera in which loop 6-7 of GLUT1 is substituted with loop 6-7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6-7.     

A residue at the cytoplasmic entrance of BK-type channels regulating single-channel opening by its hydrophobicity

Single large-conductance calcium-activated K⁺ (BK) channels encoded by the mSlo gene usually have synchronous gating, but a Drosophila dSlo (A2/C2/E2/G5/10) splice variant (dSlo1A) exhibits very flickery openings. To probe this difference in gating, we constructed a mutant I323T. This channel exhibits four subconductance levels similar to those of dSlo1A. Rectification of the single-channel current-voltage relation of I323T decreased as [Ca²⁺ ]_in increased from 10 to 300 μM. Mutagenesis suggests that the hydrophobicity of the residue at the position is important for the wild-type gating; i.e., increasing hydrophobicity prolongs open duration. Molecular dynamics simulation suggests that four hydrophobic pore-lining residues at position 323 of mSlo act cooperatively in a “shutter-like” mechanism gating the permeation of K⁺ ions. Rate-equilibrium free energy relations analysis shows that the four I323 residues in an mSlo channel have a conformation 65% similar to the closed conformation during gating. Based on these observations, we suggest that the appearance of rectification and substates of BK-type channels arise from a reduction of the cooperativity among these four residues and a lower probability of being open.     

Structure and dynamics of the pore of inwardly rectifying K(ATP) channels

Inwardly rectifying K(+) currents are generated by a complex of four Kir (Kir1-6) subunits. Pore properties are conferred by the second transmembrane domain (M2) of each subunit. Using cadmium ions as a cysteine-interacting probe, we examined the accessibility of substituted cysteines in M2 of the Kir6.2 subunit of inwardly rectifying K(ATP) channels. The ability of Cd(2+) ions to inhibit channels was used as the estimate of accessibility. The distribution of Cd(2+) accessibility is consistent with an alpha-helical structure of M2. The apparent surface of reactivity is broad, and the most reactive residues correspond to the solvent-accessible residues in the bacterial KcsA channel crystal structure. In several mutants, single channel measurements indicated that inhibition occurred by a single transition from the open state to a zero-conductance state. Analysis of currents expressed from mixtures of control and L164C mutant subunits indicated that at least three cysteines are required for coordination of the Cd(2+) ion. Application of phosphatidylinositol 4,5-diphosphate to inside-out membrane patches stabilized the open state of all mutants and also reduced cadmium sensitivity. Moreover, the Cd(2+) sensitivity of several mutants was greatly reduced in the presence of inhibitory ATP concentrations. Taken together, these results are consistent with state-dependent accessibility of single Cd(2+) ions to coordination sites within a relatively narrow inner vestibule.     

Pathways regulating the trafficking and turnover of pannexin1 protein and the role of the C-terminal domain

Pannexin1 (Panx1) is an integral membrane protein comprised of three species as follows: an unglycosylated core-Gly0, a high mannose-Gly1, and a complex glycosylated Gly2 species. Although Panx1 channels mediate several cellular responses, the domain regulating its oligomerization and cell surface trafficking and the mechanisms governing its internalization and degradation have not been identified. This study characterizes the role of the Panx1 C-tail domain by truncating the polypeptide at residue 307 and expressing the mutant in BICR-M1R(k) and HEK-293T cells. Enzymatic digestion and immunolabeling assays revealed that the Panx1(T307)-RFP was glycosylated primarily to the high mannose species consistent with its retention in the endoplasmic reticulum. Co-expression of Panx1(T307)-RFP with Panx1 followed by co-immunoprecipitation assays revealed that the mutant and Panx1 could interact, whereas biotinylation assays showed that this interaction inhibited Panx1 from maturing into the Gly2 species and reaching the cell surface. Additional inhibitor studies indicated that the degradation of the mutant was via proteasomes, whereas Panx1 was degraded by lysosomes. Analysis of the pathways important in Panx1 internalization revealed partial co-distribution of Panx1 with many molecular constituents of the endocytic machinery that include clathrin, AP2, dynamin II, caveolin-1, and caveolin-2. However, co-immunoprecipitation assays together with the disruption of lipid rafts by methyl-β-cyclodextrin suggest that Panx1 does not engage this endocytic machinery. Furthermore, dominant-negative and pharmacological studies revealed that Panx1 internalization was dynamin II-independent. Collectively, these results indicate that the oligomerization and trafficking of Panx1 are regulated by the C-terminal domain, whereas internalization of long lived Panx1 channels occurs in a manner that is distinct from classical endocytic pathways.     

ATP inhibition of CLC-1 is controlled by oxidation and reduction

The effect of intracellular adenosine triphosphate (ATP) on the “common gating” of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217–221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780–32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109–116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly—within 2–3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.     

Cytoplasmic ATP inhibition of CLC-1 is enhanced by low pH

The CLC-1 Cl(-) channel is abundantly expressed on the plasma membrane of muscle cells, and the membrane potential of muscle cells is largely controlled by the activity of this Cl(-) channel. Previous studies showed that low intracellular pH increases the overall open probability of recombinant CLC-1 channels in various expression systems. Low intracellular pH, however, is known to inhibit the Cl(-) conductance on the native muscle membrane, contradicting the findings from the recombinant CLC-1 channels in expressed systems. Here we show that in the presence of physiological concentrations of ATP, reduction of the intracellular pH indeed inhibits the expressed CLC-1, mostly by decreasing the open probability of the common gate of the channel.     

Reversible pore block of connexin channels by cyclodextrins

Cyclodextrins (CDs), a series of hollow cyclic glucosaccharides, can reversibly block molecular permeation through channels formed by connexin-32 and/or connexin-26 reconstituted into liposomes. The character of the block changes as a function of the size of the CD relative to the connexin pore diameter, suggesting that the block occurs via entry of the CD into the pore lumen and occlusion of the permeability pathway. The block occurs only when the CD is applied to the side of the pore that is normally cytoplasmic and not from the side that is normally extracellular. The block is potentiated when organic analytes are sequestered in the hydrophobic interior of the CDs. CDs may be useful as molecular tools with which to explore the structure of the connexin pore and to alter molecular movement through connexin channels.     

The effect of permeant buffers on initial ATP synthesis by chloroplasts using rapid mix-quench techniques

The chemiosmotic hypothesis predicts that buffers which permeate chloroplast membranes should delay the formation of the proton gradient at the onset of illumination. If valinomycin and KCl are present to collapse the electrical potential as well, this delay should result in a lag in initial ATP synthesis. Using rapid-mix, acid-quench techniques, we have found that in light-driven ATP synthesis the permeant buffer imidazole does not increase the initial lag caused by the valinomycin-KCl pair. Similar results are obtained under methyl viologen or phenazine methosulfate/ascorbate-mediated photophosphorylation and are independent of the internal volume of the chloroplasts. Furthermore, we have observed that chloroplasts can synthesize significant amounts of ATP in darkness following an illumination period as short as 100 ms. This capacity for ATP synthesis in darkness after short pre-illumination periods is decreased in the presence of imidazole, and this may account for the apparent lags reported in earlier studies which have used rapid flash photophosphorylation in the presence of permeant buffers. The results of the present study argue that in chloroplasts, initial ATP synthesis and post-illumination ATP synthesis are driven by distinct components of the proton motive potential.     

Titration of tertiapin-Q inhibition of ROMK1 channels by extracellular protons

Tertiapin-Q (TPN(Q)), a honey bee toxin derivative, inhibits inward-rectifier K(+) channels by binding to their external vestibule. In the present study we found that TPN(Q) inhibition of the channels is profoundly affected by extracellular pH. This pH dependence mainly reflects titration of histidine residue 12 in TPN(Q) by extracellular protons, since it largely vanishes when the histidine residue is replaced with alanine. Not surprisingly, this alanine derivative of TPN(Q) binds to the channel with much lower affinity. Quantitative thermodynamic cycle analysis shows that deprotonation of the histidine residue reduces the TPN(Q)-ROMK1 binding energy by 1.6 kcal/mol. To eliminate pH sensitivity but retain high affinity, we derivatized TPN(Q) by replacing histidine 12 with lysine. This derivative-denoted tertiapin-KQ (TPN(KQ))-not only is practically insensitive to extracellular pH but also binds to the channel with even higher affinity than TPN(Q) at extracellular pH 7.6.     

The inhibition of hexose transport by permeant and impermeant sulfhydryl agents in rat adipocytes

The importance of exofacial sulfhydryl groups for hexose transport and its regulation was studied by comparing the effects of plasma membrane-permeant maleimide (N-ethylmaleimide) to an impermeant maleimide (glutathione-maleimide I) on 3-O-methylglucose transport into isolated rat adipocytes. The impermeant nature of glutathione-maleimide was confirmed by the finding that after a 15-min incubation, concentrations as high as 10 mM had no effect on intracellular glutathione content, while 1.7 mM N-ethylmaleimide decreased intracellular glutathione by 61%. Although N-ethylmaleimide appeared to be a more potent inhibitor of transport below 5 mM and at incubation times of less than 5 min, neither agent at concentrations which did not cause significant cell breakage inhibited basal transport rates more than 60-70%. The inhibition of transport by both agents was unaffected by extensive washing, suggesting a possible covalent interaction with the carrier. Preincubation with p-chloromercuribenzenesulfonic acid protected against the transport inhibition induced by both agents. However, only the transport inhibition induced by glutathione-maleimide was prevented by preincubation with D-glucose (50 mM) and maltose (50 mM). Transport in cells pretreated with insulin was inhibited by both agents to a similar extent as basal transport. However, treatment of cells with the maleimides before insulin caused a greater degree of inhibition. Thus, the insulin-induced increase in transport was inhibited half-maximally by 1 mM glutathione-maleimide. These results show that exofacial sulfhydryl groups, perhaps on the hexose-binding site of the carrier, are important for both the function and regulation of hexose transport.     

Mechanisms of cation permeation in cardiac sodium channel: description by dynamic pore model.

The selective permeability to monovalent metal cations, as well as the relationship between cation permeation and gating kinetics, was investigated for native tetrodotoxin-insensitive Na-channels in guinea pig ventricular myocytes using the whole-cell patch clamp technique. By the measurement of inward unidirectional currents and biionic reversal potentials, we demonstrate that the cardiac Na-channel is substantially permeable to all of the group Ia and IIIa cations tested, with the selectivity sequence Na(+) >/= Li(+) > Tl(+) > K(+) > Rb(+) > Cs(+). Current kinetics was little affected by the permeant cation species and concentrations tested (相似文献   

State-dependent inhibition of sodium channels by local anesthetics: A 40-year evolution

Knowledge about the mechanism of impulse blockade by local anesthetics has evolved over the past four decades, from the realization that Na⁺ channels were inhibited to affect the impulse blockade to an identification of the amino acid residues within the Na⁺ channel that bind the local anesthetic molecule. Within this period appreciation has grown of the state-dependent nature of channel inhibition, with rapid binding and unbinding at relatively high affinity to the open state, and weaker binding to the closed resting state. Slow binding of high affinity for the inactivated state accounts for the salutary therapeutic as well as the toxic actions of diverse class I anti-arrhythmic agents, but may have little importance for impulse blockade, which requires concentrations high enough to block the resting state. At the molecular level, residues on the S6 transmembrane segments in three of the homologous domains of the channel appear to contribute to the binding of local anesthetics, with some contribution also from parts of the selectivity filter. Binding to the inactivated state, and perhaps the open state, involves some residues that are not identical to those that bind these drugs in the resting state, suggesting spatial flexibility in the “binding site”. Questions remaining include the mechanism that links local anesthetic binding with the inhibition of gating charge movements, and the molecular nature of the theoretical “hydrophobic pathway” that may be critical for determining the recovery rates from blockade of closed channels, and thus account for both therapeutic and cardiotoxic actions.     

ATP and glutamate released via astroglial connexin 43 hemichannels mediate neuronal death through activation of pannexin 1 hemichannels

Inflammation contributes to neurodegeneration in post-ischemic brain, diabetes, and Alzheimer's disease. Participants in this inflammatory response include activation of microglia and astrocytes. We studied the role of microglia treated with amyloid-β peptide (Aβ) on hemichannel activity of astrocytes subjected to hypoxia in high glucose. Reoxygenation after 3?h hypoxia in high glucose induced transient astroglial permeabilization via Cx43 hemichannels and reduction in intercellular communication via Cx43 cell-cell channels. Both responses were greater and longer lasting in astrocytes previously exposed for 24 h to conditioned medium from Aβ-treated microglia (CM-Aβ). The effects of CM-Aβ were mimicked by TNF-α and IL-1β and were abrogated by neutralizing TNF-α with soluble receptor and IL-1β with a receptor antagonist. Astrocytes under basal conditions protected neurons against hypoxia, but exposure to CM-Aβ made them toxic to neurons subjected to a sub-lethal hypoxia/reoxygenation episode, revealing the additive nature of the insults. Astrocytes exposed to CM-Aβ induced permeabilization of cortical neurons through activation of neuronal pannexin 1 (Panx1) hemichannels by ATP and glutamate released through astroglial Cx43 hemichannels. In agreement, inhibition of NMDA or P2X receptors only partially reduced the activation of neuronal Panx1 hemichannels and neuronal mortality, but simultaneous inhibition of both receptors completely prevented the neurotoxic response. Therefore, we suggest that responses to ATP and glutamate converge in activation of neuronal Panx1 hemichannels. Thus, we propose that blocking hemichannels expressed by astrocytes and/or neurons in the inflamed nervous system could represent a novel and alternative strategy to reduce neuronal loss in various pathological states including Alzheimer's disease, diabetes and ischemia.     

Tonoplast ion channels from sugar beet cell suspensions : inhibition by amiloride and its analogs

The properties of the vacuolar membrane (tonoplast) ion channels of sugar beet (Beta vulgaries) cell cultures were studied using the patch-clamp technique. Tonoplast currents displayed inward rectification in the whole vacuole and isolated outside-out patch configurations and permeability ratios P_K+/P_Na+ = 1 and P_K+/P_Cl− = 5. Amiloride and two of its analogs, 5-(N-methyl-N-isobutyl)-amiloride and benzamil, inhibitors of Na⁺ channels in animal systems, blocked inward currents by reducing single-channel openings. Concentrations for 50% inhibition of vacuolar currents of 730 nanomolar, 130 nanomolar, and 1.5 micromolar for amiloride, benzamil, and 5-(N-methyl-N-isobutyl)-amiloride, respectively, were obtained from whole-vacuole recordings. The high inhibitory action (affinity) of amiloride and its analogs for the tonoplast cation channel suggests that these compounds could be used for the isolation and biochemical characterization of this protein.