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1.
Taekyung Kwon Qingxiu Tang Thaddeus A. Bargiello 《The Journal of general physiology》2013,141(2):243-259
Voltage is an important parameter that regulates the open probability of both intercellular channels (gap junctions) and undocked hemichannels formed by members of the connexin gene family. All connexin channels display two distinct voltage-gating processes, termed loop- or slow-gating and Vj- or fast-gating, which are intrinsic hemichannel properties. Previous studies have established that the loop-gate permeability barrier is formed by a large conformational change that reduces pore diameter in a region of the channel pore located at the border of the first transmembrane domain and first extracellular loop (TM1/E1), the parahelix (residues 42–51). Here, we use cadmium metal bridge formation to measure conformational changes reported by substituted cysteines at loci demarcating the intracellular (E109 and L108) and extracellular (Q56) entrance of hemichannels formed by the Cx32 chimera (Cx32*43E1). The results indicate that the intracellular pore entrance narrows from ∼15 Å to ∼10 Å with loop-gate but not apparently with Vj-gate closure. The extracellular entrance does not appear to undergo large conformational changes with either voltage-gating process. The results presented here combined with previous studies suggest that the loop-gate permeability is essentially focal, in that conformational changes in the parahelix but not the intracellular entrance are sufficient to prevent ion flux. 相似文献
2.
Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of Vj- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the Vj (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and Vj gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both Vj- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR219) of the Cx32∗43E1 hemichannel. We conclude that an operational Cx32 Vj (fast) gate does not require CT residues 220–283, as reported previously by others. 相似文献
3.
Connexin hemichannels display two distinct forms of voltage-dependent gating, corresponding to the operation of Vj- or fast gates and loop- or slow gates. The carboxyl terminus (CT) of connexin 32 has been reported to be required for the operation of the Vj (fast) gates, but this conclusion was inferred from the loss of a fast kinetic component in macroscopic currents of CT-truncated intercellular channels elicited by transjunctional voltage. Such inferences are complicated by presence of both fast and slow gates in each hemichannel and the serial head-to-head arrangement of these gates in the intercellular channel. Examination of voltage gating in undocked hemichannels and Vj gate polarity reversal by a negative charge substitution (N2E) in the amino terminal domain allow unequivocal separation of the two gating processes in a Cx32 chimera (Cx32∗43E1). This chimera expresses currents as an undocked hemichannel in Xenopus oocytes and provides a model system to study the molecular determinants and mechanisms of Cx32 voltage gating. Here, we demonstrate that both Vj- and loop gates are operational in a truncation mutation that removes all but the first four CT residues (ACAR219) of the Cx32∗43E1 hemichannel. We conclude that an operational Cx32 Vj (fast) gate does not require CT residues 220–283, as reported previously by others. 相似文献
4.
5.
Nerijus Paulauskas Mindaugas Pranevicius Feliksas F. Bukauskas 《Biophysical journal》2009,96(10):3936-3948
Connexins, a family of membrane proteins, form gap junction (GJ) channels that provide a direct pathway for electrical and metabolic signaling between cells. We developed a stochastic four-state model describing gating properties of homotypic and heterotypic GJ channels each composed of two hemichannels (connexons). GJ channel contain two “fast” gates (one per hemichannel) oriented opposite in respect to applied transjunctional voltage (Vj). The model uses a formal scheme of peace-linear aggregate and accounts for voltage distribution inside the pore of the channel depending on the state, unitary conductances and gating properties of each hemichannel. We assume that each hemichannel can be in the open state with conductance γh,o and in the residual state with conductance γh,res, and that both γh,o and γh,res rectifies. Gates can exhibit the same or different gating polarities. Gating of each hemichannel is determined by the fraction of Vj that falls across the hemichannel, and takes into account contingent gating when gating of one hemichannel depends on the state of apposed hemichannel. At the single-channel level, the model revealed the relationship between unitary conductances of hemichannels and GJ channels and how this relationship is affected by γh,o and γh,res rectification. Simulation of junctions containing up to several thousands of homotypic or heterotypic GJs has been used to reproduce experimentally measured macroscopic junctional current and Vj-dependent gating of GJs formed from different connexin isoforms. Vj-gating was simulated by imitating several frequently used experimental protocols: 1), consecutive Vj steps rising in amplitude, 2), slowly rising Vj ramps, and 3), series of Vj steps of high frequency. The model was used to predict Vj-gating of heterotypic GJs from characteristics of corresponding homotypic channels. The model allowed us to identify the parameters of Vj-gates under which small changes in the difference of holding potentials between cells forming heterotypic junctions effectively modulates cell-to-cell signaling from bidirectional to unidirectional. The proposed model can also be used to simulate gating properties of unapposed hemichannels. 相似文献
6.
It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels. 相似文献
7.
《Cell communication & adhesion》2013,20(4-6):245-249
It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was ~220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of ~75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels. 相似文献
8.
Opposing gates model for voltage gating of gap junction channels 总被引:2,自引:0,他引:2
Chen-Izu Y Moreno AP Spangler RA 《American journal of physiology. Cell physiology》2001,281(5):C1604-C1613
9.
《FEBS letters》2014,588(8):1372-1378
Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca2+]o, membrane depolarization, metabolic inhibition, and some disease-causing gain-of-function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X7 receptor channels as studied in exogenous expression systems including Xenopus oocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X7 expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies. 相似文献
10.
Using an atomic force microscope, we have studied three-dimensional molecular topography and calcium-sensitive conformational changes of individual hemichannels. Full-length (non-truncated) Cx43 hemichannels (connexons), when reconstituted in lipid bilayer, appear as randomly distributed individual particles and clusters. They show a lack of preferential orientation of insertion into lipid membrane; in a single bilayer, connexons with protrusion of either the extracellular face or the large non-truncated cytoplasmic face are observed. Extracellular domains of these undocked hemichannels are structurally different from hemichannels in the docked gap junctional plaques examined after their exposure by force dissection or chemical dissection. Calcium induced a reversible change in the extracellular pore diameter. Hemichannels imaged in a physiological buffer with 1.8 mm Ca(+2) had the pore diameter of approximately 1.8 nm, consistent with the closed channel conformation. Reducing Ca(+2) concentration to approximately 1.4, 1, and 0 mm, which changes hemichannels from the closed to open conformation, increased the pore diameter to approximately 2.5 nm for approximately 27, 74, and 100% of hemichannels, respectively. Thus, open/close probability of the hemichannel appears to be [Ca(2+)]-dependent. Computational analysis of the atomic force microscopy phase mode imaging reveals a significantly higher interfacial energy for open hemichannels that results from the interactions between the atomic force microscope probe and the hydrophobic domains. Thus, hydrophobic extracellular domains of connexins regulate calcium-dependent conformational changes. 相似文献
11.
Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation
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Trexler EB Bukauskas FF Bennett MV Bargiello TA Verselis VK 《The Journal of general physiology》1999,113(5):721-742
pH is a potent modulator of gap junction (GJ) mediated cell-cell communication. Mechanisms proposed for closure of GJ channels by acidification include direct actions of H+ on GJ proteins and indirect actions mediated by soluble intermediates. Here we report on the effects of acidification on connexin (Cx)46 cell-cell channels expressed in Neuro-2a cells and Cx46 hemichannels expressed in Xenopus oocytes. Effects of acidification on hemichannels were examined macroscopically and in excised patches that permitted rapid (<1 ms) and uniform pH changes at the exposed hemichannel face. Both types of Cx46 channel were found to be sensitive to cytoplasmic pH, and two effects were evident. A rapid and reversible closure was reproducibly elicited with short exposures to low pH, and a poorly reversible or irreversible loss occurred with longer exposures. We attribute the former to pH gating and the latter to pH inactivation. Half-maximal reduction of open probability for pH gating in hemichannels occurs at pH 6.4. Hemichannels remained sensitive to cytoplasmic pH when excised and when cytoplasmic [Ca2+] was maintained near resting ( approximately 10(-7) M) levels. Thus, Cx46 hemichannel pH gating does not depend on cytoplasmic intermediates or a rise in [Ca2+]. Rapid application of low pH to the cytoplasmic face of open hemichannels resulted in a minimum latency to closure near zero, indicating that Cx46 hemichannels directly sense pH. Application to closed hemichannels extended their closed time, suggesting that the pH sensor is accessible from the cytoplasmic side of a closed hemichannel. Rapid closure with significantly reduced sensitivity was observed with low pH application to the extracellular face, but could be explained by H+ permeation through the pore to reach an internal site. Closure by pH is voltage dependent and has the same polarity with low pH applied to either side. These data suggest that the pH sensor is located directly on Cx46 near the pore entrance on the cytoplasmic side. 相似文献
12.
Vytas K. Verselis Maria P. Trelles Clio Rubinos Thaddeus A. Bargiello Miduturu Srinivas 《The Journal of biological chemistry》2009,284(7):4484-4493
Unapposed connexin hemichannels exhibit robust closure in response to
membrane hyperpolarization and extracellular calcium. This form of gating,
termed “loop gating,” is largely responsible for regulating
hemichannel opening, thereby preventing cell damage through excessive flux of
ions and metabolites. The molecular components and structural rearrangements
underlying loop gating remain unknown. Here, using cysteine mutagenesis in
Cx50, we demonstrate that residues at the TM1/E1 border undergo movement
during loop gating. Replacement of Phe43 in Cx50 with a cysteine
resulted in small or no appreciable membrane currents. Bath application of
dithiothreitol or TPEN
(N,N,N′,N′-tetrakis(2-pyridylmethyl)
ethylenediamine), reagents that exhibit strong transition metal chelating
activity, led to robust currents indicating that the F43C substitution
impaired hemichannel function, producing “lock-up” in a closed or
poorly functional state due to formation of metal bridges. In support,
Cd2+ at submicromolar concentrations (50–100 nm)
enhanced lock-up of F43C hemichannels. Moreover, lock-up occurred under
conditions that favored closure, indicating that the sulfhydryl groups come
close enough to each other or to other residues to coordinate metal ions with
high affinity. In addition to F43C, metal binding was also found for G46C, and
to a lesser extent, D51C substitutions, positions found to be pore-lining in
the open state using the substituted-cysteine accessibility method, but not
for A40C and A41C substitutions, which were not found to reside in the open
pore. These results indicate that metal ions access the cysteine side chains
through the open pore and that closure of the loop gate involves movement of
the TM1/E1 region that results in local narrowing of the large aqueous
connexin pore.Connexins are a large family of homologous integral membrane proteins that
form gap junction (intercellular) channels that provide a direct communication
pathway between neighboring cells. Gap junctions are formed by the docking of
two hemichannels, which themselves can function in an undocked or unapposed
configuration as ion channels that signal across the plasma membrane. Each
hemichannel is composed of a hexamer of connexin subunits. The accepted
membrane topology of a connexin subunit has four transmembrane domains
(TM1–TM4)3 and
two extracellular loops (E1 and E2) with amino and carboxyl termini located
intracellularly (reviewed in Ref.
1).Connexin cell-cell channels and hemichannels are voltage dependent and two
distinct voltage-sensitive gating mechanisms appear to be built into each
hemichannel (2). One gating
mechanism proposed to be located at the cytoplasmic end of the hemichannel is
termed Vj gating, a name derived from studies of gap junction
(cell-cell) channels describing sensitivity to transjunctional voltage,
Vj, the voltage difference between coupled cells. The other gating
mechanism is putatively ascribed to the extracellular end of the hemichannel
and has been provisionally termed loop gating, because of the resemblance of
gating transitions to those associated with initial opening of newly formed
cell-cell channels (3,
4), a process that conceivably
involves the extracellular loop domains.Loop gating is a robust gating mechanism that together with extracellular
divalent cations, principally Ca2+, is largely responsible for
keeping unapposed hemichannels closed at resting membrane potentials
(5). Reports have suggested
that extracellular divalent cations act as gating particles that enter and
block the pore upon hyperpolarization
(6,
7). An alternative model was
recently proposed whereby extracellular divalent cations act as modulators of
loop gating, an intrinsically voltage-sensitive mechanism, by stabilizing the
closed conformation and shifting activation such that opening occurs at more
positive potentials (8).Although loop gating plausibly involves conformational changes associated
with the extracellular loops, molecular components underlying loop gating as
well as the location of the putative gate remain unknown. A recent study using
chick homologues to the mammalian connexins, Cx46 and Cx50, reported that two
charged residues were important determinants of the different gating
characteristics exhibited by these two connexin hemichannels
(9). The implicated residues
are at position 9 located in the NH2-terminal domain and position
43 in the E1 domain. In Cx46 hemichannels, Glu43 and other flanking
residues at the TM1/E1 border (Ala39, Gly46, and
Asp51) were shown to reside in the aqueous pore in the open state
(10). Because it is likely
that domains involved in permeation and gating of connexin channels are
closely linked (reviewed in Ref.
11), we examined whether these
residues are involved in structural rearrangements associated with loop
gating. In this study, we engineered cysteines at residues in the TM1/E1
border in Cx50 hemichannels and used the ability of sulfhydryl groups to form
disulfide bonds and/or to complex with heavy metal ions to report
conformational changes that occur during gating. 相似文献
13.
Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions. 相似文献
14.
Post-translational modifications of connexins play an important role in the regulation of gap junction and hemichannel permeability. The prerequisite for the formation of functional gap junction channels is the assembly of connexin proteins into hemichannels and their insertion into the membrane. Hemichannels can affect cellular processes by enabling the passage of signaling molecules between the intracellular and extracellular space. For the intercellular communication hemichannels from one cell have to dock to its counterparts on the opposing membrane of an adjacent cell to allow the transmission of signals via gap junctions from one cell to the other. The controlled opening of hemichannels and gating properties of complete gap junctions can be regulated via post-translational modifications of connexins. Not only channel gating, but also connexin trafficking and assembly into hemichannels can be affected by post-translational changes. Recent investigations have shown that connexins can be modified by phosphorylation/dephosphorylation, redox-related changes including effects of nitric oxide (NO), hydrogen sulfide (H2S) or carbon monoxide (CO), acetylation, methylation or ubiquitination. Most of the connexin isoforms are known to be phosphorylated, e.g. Cx43, one of the most studied connexin at all, has 21 reported phosphorylation sites. In this review, we provide an overview about the current knowledge and relevant research of responsible kinases, connexin phosphorylation sites and reported effects on gap junction and hemichannel regulation. Regarding the effects of oxidants we discuss the role of NO in different cell types and tissues and recent studies about modifications of connexins by CO and H2S. 相似文献
15.
Gap junctional hemichannels mediate cell-extracellular communication. A hemichannel is made of six connexin (Cx) subunits; each connexin has four transmembrane domains, two extracellular loops, and cytoplasmic amino- and carboxyl-terminals (CTs). The extracellular domains are arranged differently at non-junctional and junctional (gap junction) regions, although very little is known about their flexibility and conformational energetics. The cytoplasmic tail differs considerably in the size and amino acid sequence for different connexins and is predicted to be involved in the channel open and closed conformations. For large connexins, such as Cx43, the CT makes large cytoplasmic fuzz visible under electron microscopy. If this CT domain controls channel permeability by physical occlusion of the pore mouth, movement of this portion could open or close the channel. We used atomic force microscopy-based single molecule spectroscopy with antibody-modified atomic force microscopy tips and connexin mimetic peptide modified tips to examine the flexibility of extracellular loop and CT domains and to estimate the energetics of their movements. Antibody to the CT portion closer to the membrane stretches the tail to a shorter length, and the antibody to CT tail stretches the tail to a longer length. The stretch length and the energy required for stretching the various portions of the carboxyl tail support the ball and chain model for hemichannel conformational changes. 相似文献
16.
Connexin-based gap junction hemichannels: gating mechanisms 总被引:13,自引:0,他引:13
Sáez JC Retamal MA Basilio D Bukauskas FF Bennett MV 《Biochimica et biophysica acta》2005,1711(2):215-224
Connexins (Cxs) form hemichannels and gap junction channels. Each gap junction channel is composed of two hemichannels, also termed connexons, one from each of the coupled cells. Hemichannels are hexamers assembled in the ER, the Golgi, or a post Golgi compartment. They are transported to the cell surface in vesicles and inserted by vesicle fusion, and then dock with a hemichannel in an apposed membrane to form a cell-cell channel. It was thought that hemichannels should remain closed until docking with another hemichannel because of the leak they would provide if their permeability and conductance were like those of their corresponding cell-cell channels. Now it is clear that hemichannels formed by a number of different connexins can open in at least some cells with a finite if low probability, and that their opening can be modulated under various physiological and pathological conditions. Hemichannels open in different kinds of cells in culture with conductance and permeability properties predictable from those of the corresponding gap junction channels. Cx43 hemichannels are preferentially closed in cultured cells under resting conditions, but their open probability can be increased by the application of positive voltages and by changes in protein phosphorylation and/or redox state. In addition, increased activity can result from the recruitment of hemichannels to the plasma membrane as seen in metabolically inhibited astrocytes. Mutations of connexins that increase hemichannel open probability may explain cellular degeneration in several hereditary diseases. Taken together, the data indicate that hemichannels are gated by multiple mechanisms that independently or cooperatively affect their open probability under physiological as well as pathological conditions. 相似文献
17.
Gating properties of gap junction channels assembled from connexin43 and connexin43 fused with green fluorescent protein. 总被引:4,自引:0,他引:4
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We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (< approximately 2 ms) transitions and a slow gate that fully closes channels via slow transitions (> approximately 10 ms). Here we demonstrate that transjunctional voltage (V(j)) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V(j) gating to a residual state but show slow V(j) gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO(2) and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V(j) gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V(j) gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V(j) gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end. 相似文献
18.
Conductance and permeability of the residual state of connexin43 gap junction channels 总被引:4,自引:0,他引:4
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We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication. 相似文献
19.
《FEBS letters》2014,588(8):1230-1237
Connexin gap junctions comprise assembled channels penetrating two plasma membranes for which gating regulation is associated with a variety of factors, including voltage, pH, Ca2+, and phosphorylation. Functional studies have established that various parts of the connexin peptides are related to channel closure and electrophysiology studies have provided several working models for channel gating. The corresponding structural models supporting these findings, however, are not sufficient because only small numbers of closed connexin structures have been reported. To fully understand the gating mechanisms, the channels should be visualized in both the open and closed states. Electron crystallography and X-ray crystallography studies recently revealed three-dimensional structures of connexin channels in a couple of states in which the main difference is the conformation of the N-terminal domain, which have helped to clarify the structure in regard to channel closure. Here the closure models for connexin gap junction channels inferred from structural and functional studies are described in the context of each domain of the connexin protein associated with gating modulation. 相似文献
20.
Although a functional pore domain is required for connexin 37 (Cx37)–mediated suppression of rat insulinoma (Rin) cell proliferation, it is unknown whether functional hemichannels would be sufficient or if Cx37 gap junction channels are required for growth suppression. To test this possibility, we targeted extracellular loop cysteines for mutation, expecting that the mutated protein would retain hemichannel, but not gap junction channel, functionality. Cysteines at positions 61 and 65 in the first extracellular loop of Cx37 were mutated to alanine and the mutant protein (Cx37-C61,65A) expressed in Rin cells. Although the resulting iRin37-C61,65A cells expressed the mutant protein comparably to Cx37 wild-type (Cx37-WT)–expressing Rin cells (iRin37), Cx37-C61,65A expression did not suppress the proliferation of Rin cells. As expected, iRin37-C61,65A cells did not form functional gap junction channels. However, functional hemichannels also could not be detected in iRin37-C61,65A cells by either dye uptake or electrophysiological approaches. Thus, failure of Cx37-C61,65A to suppress the proliferation of Rin cells is consistent with previous data demonstrating the importance of channel functionality to Cx37’s growth-suppressive function. Moreover, failure of the Cx37-C61,65A hemichannel to function, even in low external calcium, emphasizes the importance of extracellular loop cysteines not only in hemichannel docking but also in determining the ability of the hemichannel to adopt a closed configuration that can open in response to triggers, such as low external calcium, effective at opening Cx37-WT hemichannels. 相似文献