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1.
The pore-lining residues of gap junction channels determine their permeability to ions and small cellular metabolites. These residues can be identified through systematic cysteine substitution and accessibility analysis, commonly known as SCAM (Substituted Cysteine Accessibility Method). However, application of this technique to intercellular channels is more complicated than for their transmembrane counterparts. We have utilized a novel dual-oocyte perfusion device to apply cysteine reagents to the cytoplasmic face of paired, voltage-clamped Xenopus oocytes. In this configuration, a large and irreversible cysteine reagent MBB (maliemidobutyryl biocytin, mw 537) was shown to readily traverse the gap junction pore and induce conductance changes upon reaction of accessible sites. Of the 11 reactive sites identified, 6 were located in M3, where they span the bilayer. They display a periodicity characteristic of the tilted helix that lines the pore in the gap junction structure of Unger et al. (1999). Access to several of the other sites was attributed to aqueous crevices between transmembrane helices. Reactive sites were slightly different than those identified for gap junction hemichannels (Zhou et al. 1997), suggesting that conformational changes occur upon docking.  相似文献   

2.
Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176-1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.  相似文献   

3.
A Pfahnl  G Dahl 《Biophysical journal》1998,75(5):2323-2331
Cysteine replacement mutagenesis has identified positions in the first transmembrane domain of connexins as contributors to the pore lining of gap junction hemichannels (Zhou et al. 1997. Biophys. J. 72:1946-1953). Oocytes expressing a mutant cx46 with a cysteine in position 35 exhibited a membrane conductance sensitive to the thiol reagent maleimidobutyryl biocytin (MBB). MBB irreversibly reduced the single-channel conductance by 80%. This reactive cysteine was used to probe the localization of a voltage gate that closes cx46 gap junction hemichannels at negative potentials. MBB was applied to the closed channel either from outside (whole cell) or from inside (excised membrane patches). After washout of the thiol reagent the channels were tested at potentials at which the channels open. After extracellular application of MBB to intact oocytes, the membrane conductance was unaffected. In contrast, channels treated with intracellular MBB were blocked. Thus the cysteine in position 35 of cx46 is accessible from inside but not from the outside while the channel is closed. These results suggest that the voltage gate, which may be identical to the "loop gate" (Trexler et al. 1996. Proc. Natl. Acad. Sci. USA. 93:5836-5841), is located extracellular to the 35 position. The voltage gate results in regional closure of the pore rather than closure along the entire pore length.  相似文献   

4.
Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.  相似文献   

5.
Epithelial sodium channels (ENaCs) are composed of three homologous subunits that have regions preceding the second transmembrane domain (also referred as pre-M2) that form part of the channel pore. To identify residues within this region of the beta-subunit that line the pore, we systematically mutated residues Gln(523)-Ile(536) to cysteine. Wild type and mutant mouse ENaCs were expressed in Xenopus oocytes, and a two-electrode voltage clamp was used to examine the properties of mutant channels. Cysteine substitutions of 9 of 13 residues significantly altered Li(+) to Na(+) current ratios, whereas only cysteine replacement of beta Gly(529) resulted in K(+)-permeable channels. Besides beta G525C, large increases in the inhibitory constant of amiloride were observed with mutations at beta Gly(529) and beta Ser(531) within the previously identified 3-residue tract that restricts K(+) permeation. Cysteine substitution preceding (beta Phe(524) and beta Gly(525)), within (beta Gly(530)) or following (beta Leu(533)) this 3-residue tract, resulted in enhanced current inhibition by external MTSEA. External MTSET partially blocked channels with cysteine substitutions at beta Gln(523), beta Phe(524), and beta Trp(527). MTSET did not inhibit alpha beta G525C gamma, although previous studies showed that channels with cysteine substitutions at the corresponding sites within the alpha- and gamma-subunits were blocked by MTSET. Our results, placed in context with previous observations, suggest that pore regions from the three ENaC subunits have an asymmetric organization.  相似文献   

6.
Thermosensation is mediated by ion channels that are highly temperature-sensitive. Several members of the family of transient receptor potential (TRP) ion channels are activated by cold or hot temperatures and have been shown to function as temperature sensors in vivo. The molecular mechanism of temperature-sensitivity of these ion channels is not understood. A number of domains or even single amino acids that regulate temperature-sensitivity have been identified in several TRP channels. However, it is unclear what precise conformational changes occur upon temperature activation. Here, we used the cysteine accessibility method to probe temperature-dependent conformations of single amino acids in TRP channels. We screened over 50 amino acids in the predicted outer pore domains of the heat-activated ion channels TRPV1 and TRPV3. In both ion channels we found residues that have temperature-dependent accessibilities to the extracellular solvent. The identified residues are located within the second predicted extracellular pore loop. These residues are identical or proximal to residues that were shown to be specifically required for temperature-activation, but not chemical activation. Our data precisely locate conformational changes upon temperature-activation within the outer pore domain. Collectively, this suggests that these specific residues and the second predicted pore loop in general are crucial for the temperature-activation mechanism of these heat-activated thermoTRPs.  相似文献   

7.
The ability of certain connexins to form open hemichannels has been exploited to study the pore structure of gap junction (hemi)channels. Cysteine scanning mutagenesis was applied to cx46 and to a chimeric connexin, cx32E(1)43, which both form patent hemichannels when expressed in Xenopus oocytes. The thiol reagent maleimido-butyryl-biocytin was used to probe 12 cysteine replacement mutants in the first transmembrane segment and two in the amino-terminal segment. Maleimido-butyryl-biocytin was found to inhibit channel activity with cysteines in two equivalent positions in both connexins: I33C and M34C in cx32E(1)43 and I34C and L35C in cx46. These two positions in the first transmembrane segment are thus accessible from the extracellular space and consequently appear to contribute to the pore lining. The data also suggest that the pore structure is complex and may involve more than one transmembrane segment.  相似文献   

8.
The cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel is gated by intracellular factors; however, conformational changes in the channel pore associated with channel activation have not been identified. We have used patch clamp recording to investigate the state-dependent accessibility of substituted cysteine residues in the CFTR channel pore to a range of cysteine-reactive reagents applied to the extracellular side of the membrane. Using functional modification of the channel current-voltage relationship as a marker of modification, we find that several positively charged reagents are able to penetrate deeply into the pore from the outside irrespective of whether or not the channels have been activated. In contrast, access of three anionic cysteine-reactive reagents, the methanesulfonate sodium (2-sulfonatoethyl)methanesulfonate, the organic mercurial p-chloromercuriphenylsulfonic acid, and the permeant anion Au(CN)(2)(-), to several different sites in the pore is strictly limited prior to channel activation. This suggests that in nonactivated channels some ion selectivity mechanism exists to exclude anions yet permit cations into the channel pore from the extracellular solution. We suggest that activation of CFTR channels involves a conformational change in the pore that removes a strong selectivity against anion entry from the extracellular solution. We propose further that this conformational change occurs in advance of channel opening, suggesting that multiple distinct closed pore conformations exist.  相似文献   

9.
Epithelial sodium channels (ENaC) are composed of three structurally related subunits (alpha, beta, and gamma). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human alpha-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse alphaENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (alphaGlu(595), alphaGlu(598), and alphaAsp(602)) led to a significant reduction of expressed amiloride-sensitive Na(+) currents. Two mutations (alphaE595C and alphaD602C) resulted in K(+)-permeable channels whereas multiple mutations altered Li(+)/Na(+) current ratios. Channels containing alphaD602K or alphaD602A also conducted K(+) whereas more conservative mutations (alphaD602E and alphaD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to alphaAsp(602) within beta mENaC (betaD544C) did not alter either Li(+)/Na(+) or K(+)/Na(+) current ratios, although mutation of the equivalent site within gamma mENaC (gammaD562C) significantly increased the Li(+)/Na(+) current ratio. Mutants containing introduced cysteine residues at alphaGlu(595), alphaGlu(598), alphaAsp(602), or alphaThr(607) did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of alphaENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (alphaGlu(595) and alphaAsp(602)) may have a role in conferring ion selectivity.  相似文献   

10.
To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37-Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.  相似文献   

11.
Gap junction (GJ) channels provide an important pathway for direct intercellular transmission of signaling molecules. Previously we showed that fixed negative charges in the first extracellular loop domain (E1) strongly influence charge selectivity, conductance, and rectification of channels and hemichannels formed of Cx46. Here, using excised patches containing Cx46 hemichannels, we applied the substituted cysteine accessibility method (SCAM) at the single channel level to residues in E1 to determine if they are pore-lining. We demonstrate residues D51, G46, and E43 at the amino end of E1 are accessible to modification in open hemichannels to positively and negatively charged methanethiosulfonate (MTS) reagents added to cytoplasmic or extracellular sides. Positional effects of modification along the length of the pore and opposing effects of oppositely charged modifying reagents on hemichannel conductance and rectification are consistent with placement in the channel pore and indicate a dominant electrostatic influence of the side chains of accessible residues on ion fluxes. Hemichannels modified by MTS-EA+, MTS-ET+, or MTS-ES- were refractory to further modification and effects of substitutions with positively charged residues that electrostatically mimicked those caused by modification with the positively charged MTS reagents were similar, indicating all six subunits were likely modified. The large reductions in conductance caused by MTS-ET+ were visible as stepwise reductions in single-channel current, indicative of reactions occurring at individual subunits. Extension of single-channel SCAM using MTS-ET+ into the first transmembrane domain, TM1, revealed continued accessibility at the extracellular end at A39 and L35. The topologically complementary region in TM3 showed no evidence of reactivity. Structural models show GJ channels in the extracellular gap to have continuous inner and outer walls of protein. If representative of open channels and hemichannels, these data indicate E1 as constituting a significant portion of this inner, pore-forming wall, and TM1 contributing as pore-lining in the extracellular portion of transmembrane span.  相似文献   

12.
Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET+. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.  相似文献   

13.
KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.  相似文献   

14.
Abstract

The hERG potassium channel is a member of the voltage gated potassium (Kv) channel family, comprising a pore domain and four voltage sensing domains (VSDs). Like other Kv channels, the VSD senses changes in membrane voltage and transmits the signal to gates located in the pore domain; the gates open at positive potentials (activation) and close at negative potentials, thereby controlling the ion flux. hERG, however, differs from other Kv channels in that it is activated slowly but inactivated rapidly – a property that is crucial for the role it plays in the repolarization of the cardiac action potential. Voltage-gating requires movement of gating charges across the membrane electric field, which is accomplished by the transmembrane movement of the fourth transmembrane segment, S4, of the VSD containing the positively charged arginine or lysine residues. Here we ask if the functional differences between hERG and other Kv channels could arise from differences in the transmembrane movement of S4. To address this, we have introduced single cysteine residues into the S4 region of the VSD, expressed the mutant channels in Xenopus oocytes and examined the effect of membrane impermeable para-chloromercuribenzene sulphonate on function by the two-electrode voltage clamp technique. Our results show that depolarization results in the accessibility of seven consecutive S4 residues, including the first two charged residues, K525 and R528, to extracellularly applied reagent. These data indicate that the extent of S4 movement in hERG is similar to other Kv channels, including the archabacterial KvAP and the Shaker channel of Drosophila.  相似文献   

15.
In Schwann cells, connexin 32 (Cx32) can oligomerize to form intracellular gap junction channels facilitating a shorter pathway for metabolite diffusion across the layers of the myelin sheath. The mechanisms of Cx32 intracellular channel regulation have not been clearly defined. However, Ca(2+), pH, and the phosphorylation state can regulate Cx32 gap junction channels, in addition to the direct interaction of protein partners with the carboxyl-terminal (CT) domain. In this study, we used different biophysical methods to determine the structure and characterize the interaction of the Cx32CT domain with the protein partners synapse-associated protein 97 (SAP97) and calmodulin (CaM). Our results revealed that the Cx32CT is an intrinsically disordered protein that becomes α-helical upon binding CaM. We identified the GUK domain as the minimal SAP97 region necessary for the Cx32CT interaction. The Cx32CT residues affected by the binding of CaM and the SAP97 GUK domain were determined as well as the dissociation constants for these interactions. We characterized three Cx32CT Charcot-Marie-Tooth disease mutants (R219H, R230C, and F235C) and identified that whereas they all formed functional channels, they all showed reduced binding affinity for SAP97 and CaM. Additionally, we report that in RT4-D6P2T rat schwannoma cells, Cx32 is differentially phosphorylated and exists in a complex with SAP97 and CaM. Our studies support the importance of protein-protein interactions in the regulation of Cx32 gap junction channels and myelin homeostasis.  相似文献   

16.
Gated gap junction channels are important cellular conduits for establishing and maintaining intercellular communication. The three-dimensional structure of a mutant human connexin 26 (Cx26M34A) by electron cryocrystallography revealed a plug-like density in the channel pore suggesting that physical blockage of the pore may be one mechanism of closure (Oshima et al. 2007, Proc Natl Acad Sci USA 104: 10034-10039). However, it remains to be determined what part of the sequence contributes to the plug. Here, we present the projection structure of an N-terminus deletion of Cx26M34A missing amino acids 2 to 7 (Cx26M34Adel2-7) crystallized in the same two-dimensional crystal form. A 10 A resolution projection map of Cx26M34Adel2-7 revealed that the plug density was dramatically reduced in comparison with that found in full-length Cx26 channel. The difference map between the deletion and full-length Cx26M34A channels strongly suggests that the N-terminus of connexin contributes to the plug for the physical closure of gap junction channels.  相似文献   

17.
Paired intercellular transmembrane channels, termed connexons, comprised of hexameric assemblies of gap junction protein, were isolated and purified from rat liver by exploiting their resistance to either Sarkosyl detergent solubilization or alkali extraction. The secondary structures of the gap junction proteins prepared by these methods were compared by circular dichroism (CD) spectroscopy. Both the spectra and the calculated net secondary structures of the proteins obtained by the two isolation methods were different. The protein isolated by the Sarkosyl treatment was found to be approximately 50% alpha-helical, while protein isolated by alkali extraction had a lower helix content (approximately 40%). In both types of preparations, however, the helical content of the gap junction protein was sufficiently large to be consistent with an all-helical model for the membrane-spanning parts of the structure. CD spectroscopy was also used to examine the effects of proteolytic digestion of the cytoplasmic domain on the net secondary structure of the detergent-treated gap junction protein. The membrane-bound fragments had a slightly higher proportion of their residues that were alpha-helical in nature, suggesting that the transmembrane and/or intra-gap domains are indeed enriched in this type of secondary structure. This information constrains the range of models which can be realistically proposed for the channel structure.  相似文献   

18.
19.
The amino terminal domain (NT) of the connexins consists of their first 22-23 amino acids. Site-directed mutagenesis studies have demonstrated that NT amino acids are determinants of gap junction channel properties including unitary conductance, permeability/selectivity, and gating in response to transjunctional voltage. The importance of this region has also been emphasized by the identification of multiple disease-associated connexin mutants affecting amino acid residues in the NT region. The first part of the NT is α-helical. The structure of the Cx26 gap junction channel shows that the NT α-helix localizes within the channel, and lines the wall of the pore. Interactions of the amino acid residues in the NT with those in the transmembrane helices may be critical for holding the channel open. The predicted sites of these interactions and the applicability of the Cx26 structure to the NT of other connexins are considered. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.  相似文献   

20.
N Zilberberg  N Ilan  S A Goldstein 《Neuron》2001,32(4):635-648
Essential to nerve and muscle function, little is known about how potassium leak channels operate. KCNK? opens and closes in a kinase-dependent fashion. Here, the transition is shown to correspond to changes in the outer aspect of the ion conduction pore. Voltage-gated potassium (VGK) channels open and close via an internal gate; however, they also have an outer pore gate that produces "C-type" inactivation. While KCNK? does not inactivate, KCNK? and VGK channels respond in like manner to outer pore blockers, potassium, mutations, and chemical modifiers. Structural relatedness is confirmed: VGK residues that come close during C-type gating predict KCNK? sites that crosslink (after mutation to cysteine) to yield channels controlled by reduction and oxidization. We conclude that similar outer pore gates mediate KCNK? opening and closing and VGK channel C-type inactivation despite their divergent structures and physiological roles.  相似文献   

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