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1.
2.
1. Hemichannels formed by connexin26 (Cx26) on the horizontal cell dendrites that invaginate cone terminals in the vertebrate retina have been implicated in the feedback mechanism by which horizontal cells regulate transmitter release from cone photoreceptors. However, their membrane properties had not been studied previously, and it was unclear whether they could subserve their purported function at the membrane potentials over which horizontal cells operate. 2. We used the two-electrode voltage clamp technique to record the membrane currents and pharmacological properties of Cx26 hemichannels formed in the Xenopus oocyte expression system. 3. Oocytes expressing Cx26 exhibited large membrane conductances over a broad range of hyperpolarizing and depolarizing membrane potentials, and displayed little evidence of voltage-dependent gating, indicating that the hemichannels are constitutively open. The Cx26-mediated nonjunctional currents were relatively insensitive to quinine, a cinchona alkaloid that opens hemichannels formed by several other connexins. However, the hemichannel currents were blocked by carbenoxolone, a rise in extracellular calcium, or lowering intracellular pH. The currents could also be suppressed by reducing extracellular pH, and by the chloride channel blocker NPPB through its direct interaction with Cx26 hemichannels. 4. These findings provide a basis with which to evaluate the in situ pharmacological studies that attempt to assess the putative role of Cx26 hemichannels in the feedback pathway in the distal retina. 相似文献
3.
Transmitter molecules bind to synaptic acetylcholine receptor channels (AChRs) to promote a global channel-opening conformational change. Although the detailed mechanism that links ligand binding and channel gating is uncertain, the energy changes caused by mutations appear to be more symmetrical between subunits in the transmembrane domain compared with the extracellular domain. The only covalent connection between these domains is the pre-M1 linker, a stretch of five amino acids that joins strand β10 with the M1 helix. In each subunit, this linker has a central Arg (Arg3′), which only in the non-α-subunits is flanked by positively charged residues. Previous studies showed that mutations of Arg3′ in the α-subunit alter the gating equilibrium constant and reduce channel expression. We recorded single-channel currents and estimated the gating rate and equilibrium constants of adult mouse AChRs with mutations at the pre-M1 linker and the nearby residue Glu45 in non-α-subunits. In all subunits, mutations of Arg3′ had similar effects as in the α-subunit. In the ϵ-subunit, mutations of the flanking residues and Glu45 had only small effects, and there was no energy coupling between ϵGlu45 and ϵArg3′. The non-α-subunit Arg3′ residues had Φ-values that were similar to those for the α-subunit. The results suggest that there is a general symmetry between the AChR subunits during gating isomerization in this linker and that the central Arg is involved in expression more so than gating. The energy transfer through the AChR during gating appears to mainly involve Glu45, but only in the α-subunits. 相似文献
4.
Michaela Kudrnac Stanislav Beyl Annette Hohaus Anna Stary Thomas Peterbauer Eugen Timin Steffen Hering 《The Journal of biological chemistry》2009,284(18):12276-12284
Voltage dependence and kinetics of CaV1.2 activation are
affected by structural changes in pore-lining S6 segments of the
α1-subunit. Significant effects are induced by either proline
or threonine substitutions in the lower third of segment IIS6 (“bundle
crossing region”), where S6 segments are likely to seal the channel in
the closed conformation (Hohaus, A., Beyl, S., Kudrnac, M., Berjukow, S.,
Timin, E. N., Marksteiner, R., Maw, M. A., and Hering, S. (2005) J. Biol.
Chem. 280, 38471–38477). Here we report that S435P in IS6 results
in a large shift of the activation curve (-25.9 ± 1.2 mV) and slower
current kinetics. Threonine substitutions at positions Leu-429 and Leu-434
induced a similar kinetic phenotype with shifted activation curves (L429T by
-6.6 ± 1.2 and L434T by -12.1 ± 1.7 mV). Inactivation curves of
all mutants were shifted to comparable extents as the activation curves.
Interdependence of IS6 and IIS6 mutations was analyzed by means of mutant
cycle analysis. Double mutations in segments IS6 and IIS6 induce either
additive (L429T/I781T, -34.1 ± 1.4 mV; L434T/I781T, -40.4 ± 1.3
mV; L429T/L779T, -12.6 ± 1.3 mV; and L434T/L779T, -22.4 ± 1.3
mV) or nonadditive shifts of the activation curves along the voltage axis
(S435P/I781T, -33.8 ± 1.4 mV). Mutant cycle analysis revealed energetic
coupling between residues Ser-435 and Ile-781, whereas other paired mutations
in segments IS6 and IIS6 had independent effects on activation gating.Ca2+ current through CaV1.2 channels initiates muscle
contraction, release of hormones and neurotransmitters, and affects
physiological processes such as vision, hearing, and gene expression
(1). Their pore-forming
α1-subunit is composed of four homologous domains formed by
six transmembrane segments (S1–S6)
(2). The signal of the
voltage-sensing machinery, consisting of multiple charged amino acids (located
in segments S4 and adjacent structures of each domain), is transmitted to the
pore region (3). Conformational
changes in pore lining S6 and adjacent segments finally lead to pore openings
(activation) and closures (inactivation).Our understanding of how CaV1.2 channels open and close is
largely based on extrapolations of structural information from potassium
channels. The crystal structures of the closed conformation of two bacterial
potassium channels (KcsA and MlotiK)
(4,
5) show a gate located at the
intracellular channel mouth formed by tightly packed S6 helices. The crystal
structure of the open conformation of Kv1.2
(6,
7) revealed a bent S6 with the
highly conserved PXP motif apparently acting as a hinge (see
8). The activation mechanism
proposed for MthK channels involves helix bending at a highly conserved
glycine at position 83 (see Ref.
9, “glycine gating
hinge” hypothesis).Compared with potassium channels, the pore of CaV is asymmetric,
and none of the four S6 segments has a putative helix-bending PXP
motif. Furthermore, the conserved glycine (corresponding to position 83 in
MthK, see Ref. 10) is only
present in segments IS6 and IIS6 (for review see Ref.
11). We have shown that
substituting proline for this glycine in IIS6 of CaV1.2 does not
significantly affect gating
(12).Zhen et al. (13)
investigated the pore lining S6 segments of CaV2.1 using the
substituted cysteine accessibility method. The accessibility of cysteines was
changed by opening and closing the channel, consistent with the gate being on
the intracellular side. The general picture of a channel gate close to the
inner channel mouth of CaV1.2 was recently supported by
pharmacological studies
(14).Substitution of hydrophilic residues in the lower third of segment IIS6 of
CaV1.2 (LAIA motif, 779–784, see Ref.
12) induces pronounced changes
in channel gating as follows: a shift in the voltage dependence of activation
accompanied by a slowing of the activation kinetics near the footstep of the
m∞(V) curve and a slowing of deactivation
at all potentials. Interestingly, these changes in channel gating resemble the
effects of proline substitution of Gly-219 in the bacterial sodium channel
from Bacillus halodurans (“Gly-219 gating hinge,” see
Ref. 15).The strongest shifts of the activation curve reported so far were observed
for proline substitutions
(12). As prolines in an
α-helix cause a rigid kink with an angle of about 26°
(16), we hypothesized that
these mutants were causing a kink in helix IIS6 similar to a bend that would
normally occur flexibly during the activation process
(12).Here we extend our previous study by systematically substituting residues
in segment IS6 of CaV1.2 by proline or the small and polar
threonine. Several functional IS6 mutants with shifted activation and
inactivation characteristics were identified (S435P, L429T, and L434T), and
the interdependence of IS6 and IIS6 mutations was analyzed. Mutant cycle
analysis revealed both mutually independent and energetically coupled
contributions of IS6 and IIS6 residues on activation gating. 相似文献
5.
Human HeLa cells transfected with mouse connexin Cx45 were used to examine the conductive and kinetic properties of Cx45 hemichannels. The experiments were carried out on single cells using a voltage-clamp method. Lowering the [Ca2+]o revealed an extra current. Its sensitivity to extracellular Ca2+ and gap junction channel blockers (18α-glycyrrhetinic acid, palmitoleic acid, heptanol), and its absence in non-transfected HeLa cells suggested that it is carried by Cx45 hemichannels. The conductive and kinetic properties of this current, I
hc, were determined adopting a biphasic pulse protocol. I
hc activated at positive V
m and deactivated partially at negative V
m. The analysis of the instantaneous I
hc yielded a linear function g
hc,inst = f(V
m) with a hint of a negative slope (g
hc,inst: instantaneous conductance). The analysis of the steady-state I
hc revealed a sigmoidal function g
hc,ss = f(V
m) best described with the Boltzmann equation: V
m,0 = −1.08 mV, g
hc,min = 0.08 (g
hc,ss: steady-state conductance; V
m,
0:V
m at which g
hc,ss is half-maximally activated; g
hc,min: minimal conductance; major charge carriers: K+ and Cl−). The g
hc was minimal at negative V
m and maximal at positive V
m. This suggests that Cx45 connexons integrated in gap junction channels are gating with negative voltage. I
hc deactivated exponentially with time, giving rise to single time constants, τd. The function τd = f(V
m) was exponential and increased with positive V
m (τd = 7.6 s at V
m
= 0 mV). The activation of I
hc followed the sum of two exponentials giving rise to the time constants, τa1 and τa2. The function τa1 = f(V
m) and τa2 = f(V
m) were bell-shaped and yielded a maximum of ≅ 0.6 s at V
m ≅ −20 mV and ≅ 4.9 s at V
m ≅ 15 mV, respectively. Neither τa1 = f(V
m) nor τa2 = f(V
m) coincided with τd = f(V
m). These findings conflict with the notion that activation and deactivation follow a simple reversible reaction scheme governed by first-order voltage-dependent processes. 相似文献
6.
Brian Skriver Nielsen Daniel Bloch Hansen Bruce R. Ransom Morten Schak Nielsen Nanna MacAulay 《Neurochemical research》2017,42(9):2537-2550
Astrocytes in the mammalian central nervous system are interconnected by gap junctions made from connexins of the subtypes Cx30 and Cx43. These proteins may exist as hemichannels in the plasma membrane in the absence of a ‘docked’ counterpart on the neighboring cell. A variety of stimuli are reported to open the hemichannels and thereby create a permeation pathway through the plasma membrane. Cx30 and Cx43 have, in their hemichannel configuration, been proposed to act as ion channels and membrane pathways for different molecules, such as fluorescent dyes, ATP, prostaglandins, and glutamate. Published studies about astrocyte hemichannel behavior, however, have been highly variable and/or contradictory. The field of connexin hemichannel research has been complicated by great variability in the experimental preparations employed, a lack of highly specific pharmacological inhibitors and by confounding changes associated with genetically modified animal models. This review attempts to critically assess the gating, inhibition and permeability of astrocytic connexin hemichannels and proposes that connexins in their hemichannel configuration act as gated pores with isoform-specific permeant selectivity. We expect that some, or all, of the controversies discussed here will be resolved by future research and sincerely hope that this review serves to motivate such clarifying investigations. 相似文献
7.
Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag+ or Cd2+ on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag+ and Cd2+ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag+ or Cd2+ chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F1 from F0 with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H+ translocation through F0 as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H+ translocation to the cytoplasm. 相似文献
8.
Agnese S. Minazzo 《Biophysical journal》2009,96(2):681-692
In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro79-Pro92 loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu84 resides in the Pro79-Pro92 loop, and Thr121 resides in the turn between the first and second antiparallel β-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu84 nor Thr121 makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro79-Pro92 loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro79-Pro92 loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation. 相似文献
9.
Madhusudan Dey Brian Rick Mann Ashish Anshu M. Amin-ul Mannan 《The Journal of biological chemistry》2014,289(9):5747-5757
Protein kinase R (PKR) functions in a plethora of cellular processes, including viral and cellular stress responses, by phosphorylating the translation initiation factor eIF2α. The minimum requirements for PKR function are homodimerization of its kinase and RNA-binding domains, and autophosphorylation at the residue Thr-446 in a flexible loop called the activation loop. We investigated the interdependence between dimerization and Thr-446 autophosphorylation using the yeast Saccharomyces cerevisiae model system. We showed that an engineered PKR that bypassed the need for Thr-446 autophosphorylation (PKRT446∼P-bypass mutant) could function without a key residue (Asp-266 or Tyr-323) that is essential for PKR dimerization, suggesting that dimerization precedes and stimulates activation loop autophosphorylation. We also showed that the PKRT446∼P-bypass mutant was able to phosphorylate eIF2α even without its RNA-binding domains. These two significant findings reveal that PKR dimerization and activation loop autophosphorylation are mutually exclusive yet interdependent processes. Also, we provide evidence that Thr-446 autophosphorylation during PKR activation occurs in a cis mechanism following dimerization. 相似文献
10.
Krassimira Angelova Hugo de Jonge Joke C. M. Granneman David Puett Jan Bogerd 《The Journal of biological chemistry》2010,285(45):34813-34827
Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys74), 4 (Tyr124, Asn129, and Thr130), and 5 (Asp150 and Asp153) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser78) and 8 (Asp224 and Ser226) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation. 相似文献
11.
《Cell communication & adhesion》2013,20(4-6):265-270
A detailed understanding of the mechanisms regulating cell-to-cell communication in the lens necessitates information about the distribution and density of Cx46 and Cx50 in their native cellular environment. These isoforms constitute the extensive pathway between the lens surface and the interior, helping to maintain its striking optical properties. To identify Cx50 channels and hemichannels in the plasma membrane and to differentiate between them, immuno-freeze-fracture-labeling (FRIL) with immuno-gold particles in used. In equatorial lens fibers, the Cx50-gold complexes label gap junctions at high densities and non-junctional plasma membranes at lower densities. Small depressions in the non-junctional plasma membrane labeled by the gold-complexes most likely represent points of hemichannel insertion. Measurement of the width of the extra-cellular space separating adjacent plasma membranes indicates that the gold complexes in the gap junctions represent Cx50 channels and those in the non-junctional plasma membrane, Cx50 hemichannels. Estimates of their densities indicate that the channels are at least one order of magnitude more numerous than the hemichannels. Therefore, in lens fibers, Cx50 hemichannels are inserted via exocytosis and are rapidly assembled into channels assembled in gap junction plaques. 相似文献
12.
Tianbo Li Youshan Yang Cecilia M. Canessa 《The Journal of biological chemistry》2009,284(7):4689-4694
Acid-sensing ion channels are proton-activated ion channels expressed in
the nervous system. They belong to the family of ENaC/Degenerins whose members
share a conserved structure but are activated by widely diverse stimuli. We
show that interaction of two aromatic residues, Tyr-72, located immediately
after the first transmembrane segment, and Trp-288, located at the tip of a
loop of the extracellular domain directed toward the first transmembrane
segment, is essential for proton activation of the acid-sensing ion channels.
The subdomain containing Trp-288 is a module tethered to the rest of the
extracellular domain by short linkers and intrasubunit interactions between
residues in the putative “proton sensor.” Mutations in these two
areas shift the apparent affinity of protons toward a more acidic range and
change the kinetics of activation and desensitization. These results are
consisting with displacement of the module relative to the rest of the
extracellular domain to allow interaction of Trp-288 with Tyr-72 during
gating. We propose that such interaction may provide functional coupling
between the extracellular domain and the pore domain.The acid-sensing ion channels
(ASICs)2 are
voltage-insensitive sodium channels turned on and off by extracellular
protons. Four ASIC genes in the human genome, ASIC1 to ASIC4, give rise to at
least six isoforms that associate in various combinations to form channels
with different functional properties
(1,
2). The ASICs constitute a
distinct group in the large family of channels known as Degenerins
characterized by a common structure but widely diverse gating stimuli:
mechanical forces (3),
neuropeptides (4,
5), protons
(6), or no stimulus at all,
such as ENaC, which exhibits constitutive activity
(7). The structure shared by
all Degenerins consists of two transmembrane segments, TM1 and TM2, a large
extracellular domain, and short cytoplasmic amino and carboxyl termini. The
recently published crystal structure of a truncated chicken ASIC1 (cASIC1) at
a resolution of 1.9 Å (8)
shows that ASIC1 is a trimer, and it provides detailed structure of the large
extracellular domain that is crucial for understanding the gating mechanism of
the ASICs. A feature revealed by the atomic structure is a cluster of
negatively and one positively charged residue in the interface of subdomain D
(Arg-191), subdomain E (Asp-238 and Glu-239), and subdomain F (Asp-346 and
Asp-350) (see Fig. 1A)
that was hypothesized to constitute the proton sensor. Furthermore, it was
proposed that binding of protons to this site displaces subdomain F toward TM1
to open the pore (8).Open in a separate windowFIGURE 1.Ribbon representation of chicken ASIC1 structure. a, a
single subunit is shown for simplicity with subdomains, A to F, indicated in
different colors. The arrow points to the putative proton sensor with
side chains of charged residues represented as sticks. Amino acids
important for ASIC1 gating that were mutated in this study are also shown. The
image was obtained with the molecular graphics program Chimera. b,
amino acid sequence of subdomain F loop. Residues conserved in all ASIC
proteins are in red.Although the solved atomic structure of cASIC1 provides a valuable tool to
advance the understanding of how external protons activate the ASICs, it
represents only a snapshot of the gating process thereby additional
experimental evidence is needed to elucidate the gating mechanism. The general
idea that conformational changes triggered by binding of the specific agonist
to the extracellular domain of a ligand activated channel need to be
transmitted to the transmembrane domain, where the pore gate is located, draws
attention to the pair of closely located residues, Tyr-72 and Trp-288, as they
provide a potential contact site between the extracellular and the
transmembrane domains.This study examines the functional role of the conserved residues, Tyr-72
and Trp-288, that are located distantly in the primary sequence but are
brought to close proximity (∼3.7 Å) by the folding of the
extracellular domain (ECD). This arrangement could provide a contact site
between the ECD and TM1 whereby a conformation change of the ECD is
transmitted to the pore gate in the transmembrane domain. 相似文献
13.
Annette Ehrhardt W. Joon Chung Louise C. Pyle Wei Wang Krzysztof Nowotarski Cory M. Mulvihill Mohabir Ramjeesingh Jeong Hong Sadanandan E. Velu Hal A. Lewis Shane Atwell Steve Aller Christine E. Bear Gergely L. Lukacs Kevin L. Kirk Eric J. Sorscher 《The Journal of biological chemistry》2016,291(4):1854-1865
In this study, we present data indicating a robust and specific domain interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) first cytosolic loop (CL1) and nucleotide binding domain 1 (NBD1) that allows ion transport to proceed in a regulated fashion. We used co-precipitation and ELISA to establish the molecular contact and showed that binding kinetics were not altered by the common clinical mutation F508del. Both intrinsic ATPase activity and CFTR channel gating were inhibited severely by CL1 peptide, suggesting that NBD1/CL1 binding is a crucial requirement for ATP hydrolysis and channel function. In addition to cystic fibrosis, CFTR dysregulation has been implicated in the pathogenesis of prevalent diseases such as chronic obstructive pulmonary disease, acquired rhinosinusitis, pancreatitis, and lethal secretory diarrhea (e.g. cholera). On the basis of clinical relevance of the CFTR as a therapeutic target, a cell-free drug screen was established to identify modulators of NBD1/CL1 channel activity independent of F508del CFTR and pharmacologic rescue. Our findings support a targetable mechanism of CFTR regulation in which conformational changes in the NBDs cause reorientation of transmembrane domains via interactions with CL1 and result in channel gating. 相似文献
14.
James G. Laing Peter N. Tadros Eileen M. Westphale Eric C. Beyer 《Experimental cell research》1997,236(2)
Intercellular communication may be modulated by the rather rapid turnover and degradation of gap junction proteins, since many connexins have half-lives of 1–3 h. While several morphological studies have suggested that gap junction degradation occurs after endocytosis, our recent biochemical studies have demonstrated involvement of the ubiquitin–proteasome pathway in proteolysis of the connexin43 polypeptide. The present study was designed to reconcile these observations by examining the degradation of connexin43-containing gap junctions in rat heart-derived BWEM cells. After treatment of BWEM cells with Brefeldin A to prevent transport of newly synthesized connexin43 polypeptides to the plasma membrane, quantitative confocal microscopy showed the disappearance of immunoreactive connexin43 from the cell surface with a half-life of 1 h. This loss of connexin43 immunoreactivity was inhibited by cotreatment with proteasomal inhibitors (ALLN, MG132, or lactacystin) or lysosomal inhibitors (leupeptin or E-64). Similar results were seen when connexin43 export was blocked with monensin. After treatment of BWEM cells with either proteasomal or lysosomal inhibitors alone, immunoblots showed accumulation of connexin43 in both whole cell lysates and in a 1% Triton X-100-insoluble fraction. Immunofluorescence studies showed that connexin43 accumulated at the cell surface in lactacystin-treated cells, but in vesicles in BWEM cells treated with lysosomal inhibitors. These results implicate both the proteasome and the lysosome in the degradation of connexin43-containing gap junctions. 相似文献
15.
The Pattern of Disulfide Linkages in the Extracellular Loop Regions of Connexin 32 Suggests a Model for the Docking Interface of Gap Junctions 总被引:5,自引:0,他引:5 下载免费PDF全文
Cynthia I. Foote Lan Zhou Xing Zhu Bruce J. Nicholson 《The Journal of cell biology》1998,140(5):1187-1197
Connexins, like true cell adhesion molecules, have extracellular domains that provide strong and specific homophilic, and in some cases, heterophilic interactions between cells. Though the structure of the binding domains of adhesion proteins have been determined, the extracellular domains of connexins, consisting of two loops of ~34–37 amino acids each, are not easily studied in isolation from the rest of the molecule. As an alternative, we used a novel application of site-directed mutagenesis in which four of the six conserved cysteines in the extracellular loops of connexin 32 were moved individually and in all possible pairwise and some quadruple combinations. This mapping allowed us to deduce that all disulfides form between the two loops of a single connexin, with the first cysteine in one loop connected to the third of the other. Furthermore, the periodicity of movements that produced functional channels indicated that these loops are likely to form antiparallel β sheets. A possible model that could explain how these domains from apposed connexins interact to form a complete channel is discussed. 相似文献
16.
17.
Margaret C. Della Vecchia Anna C. Rued Marcelo D. Carattino 《The Journal of biological chemistry》2013,288(8):5487-5495
Acid-sensing ion channels (ASICs) are trimeric cation-selective proton-gated ion channels expressed in the central and peripheral nervous systems. The pore-forming transmembrane helices in these channels are linked by short loops to the palm domain in the extracellular region. Here, we explore the contribution to proton gating and desensitization of Glu-79 and Glu-416 in the palm domain of ASIC1a. Engineered Cys, Lys, and Gln substitutions at these positions shifted apparent proton affinity toward more acidic values. Double mutant cycle analysis indicated that Glu-79 and Glu-416 cooperatively facilitated pore opening in response to extracellular acidification. Channels bearing Cys at position 79 or 416 were irreversibly modified by thiol-reactive reagents in a state-dependent manner. Glu-79 and Glu-416 are located in β-strands 1 and 12, respectively. The covalent modification by (2-(trimethylammonium)ethyl) methanethiosulfonate bromide of Cys at position 79 impacted conformational changes associated with pore closing during desensitization, whereas the modification of Cys at position 416 affected conformational changes associated with proton gating. These results suggest that β-strands 1 and 12 contribute antagonistically to activation and desensitization of ASIC1a. Site-directed mutagenesis experiments indicated that the lower palm domain contracts in response to extracellular acidification. Taken together, our studies suggest that the lower palm domain mediates conformational movements that drive pore opening and closing events. 相似文献
18.
1. Forskolin acts as an allosteric modulator of muscle-type nicotinic acetylcholine receptors. Receptors from mouse muscle and Torpedo electroplax demonstrate differential sensitivity to inhibition by forskolin. Previous work from this laboratory suggested that the subunit is responsible for this differential sensitivity.2. We have used a series of mouse/Torpedo species-chimeric subunits to further define the site of forskolin interaction with the subunit. Analysis of the patterns of forskolin inhibition of receptors containing mouse/Torpedo chimeric subunits along with the mouse , , and subunits suggests that forskolin interacts with the small extracellular domain that links the M2 and M3 transmembrane domains (the M2–M3 linker).3. We suggest that the M2–M3 linker domain plays an important role in the transduction of ligand binding to the conformational changes that result in channel opening. 相似文献
19.
Daniel M. Collier Zerubbabel J. Peterson Ilya O. Blokhin Christopher J. Benson Peter M. Snyder 《The Journal of biological chemistry》2012,287(49):40907-40914
A growing body of evidence suggests that the extracellular domain of the epithelial Na+ channel (ENaC) functions as a sensor that fine tunes channel activity in response to changes in the extracellular environment. We previously found that acidic pH increases the activity of human ENaC, which results from a decrease in Na+ self-inhibition. In the current work, we identified extracellular domain residues responsible for this regulation. We found that rat ENaC is less sensitive to pH than human ENaC, an effect mediated in part by the γ subunit. We identified a group of seven residues in the extracellular domain of γENaC (Asp-164, Gln-165, Asp-166, Glu-292, Asp-335, His-439, and Glu-455) that, when individually mutated to Ala, decreased proton activation of ENaC. γE455 is conserved in βENaC (Glu-446); mutation of this residue to neutral amino acids (Ala, Cys) reduced ENaC stimulation by acidic pH, whereas reintroduction of a negative charge (by MTSES modification of Cys) restored pH regulation. Combination of the seven γENaC mutations with βE446A generated a channel that was not activated by acidic pH, but inhibition by alkaline pH was intact. Moreover, these mutations reduced the effect of pH on Na+ self-inhibition. Together, the data identify eight extracellular domain residues in human β- and γENaC that are required for regulation by acidic pH. 相似文献
20.
Morgan E. DeSantis Elizabeth A. Sweeny David Snead Eunice H. Leung Michelle S. Go Kushol Gupta Petra Wendler James Shorter 《The Journal of biological chemistry》2014,289(2):848-867
The homologous hexameric AAA+ proteins, Hsp104 from yeast and ClpB from bacteria, collaborate with Hsp70 to dissolve disordered protein aggregates but employ distinct mechanisms of intersubunit collaboration. How Hsp104 and ClpB coordinate polypeptide handover with Hsp70 is not understood. Here, we define conserved distal loop residues between middle domain (MD) helix 1 and 2 that are unexpectedly critical for Hsp104 and ClpB collaboration with Hsp70. Surprisingly, the Hsp104 and ClpB MD distal loop does not contact Hsp70 but makes intrasubunit contacts with nucleotide-binding domain 2 (NBD2). Thus, the MD does not invariably project out into solution as in one structural model of Hsp104 and ClpB hexamers. These intrasubunit contacts as well as those between MD helix 2 and NBD1 are different in Hsp104 and ClpB. NBD2-MD contacts dampen disaggregase activity and must separate for protein disaggregation. We demonstrate that ClpB requires DnaK more stringently than Hsp104 requires Hsp70 for protein disaggregation. Thus, we reveal key differences in how Hsp104 and ClpB coordinate polypeptide handover with Hsp70, which likely reflects differential tuning for yeast and bacterial proteostasis. 相似文献