Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy

Eskandari, Sepehr; Wright, Ernest M.; Kreman, Mike; Starace, Dorine M.; Zampighi, Guido A.

doi:10.1073/pnas.95.19.11235

Cited by 164 publications

(196 citation statements)

References 43 publications

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“…It has been shown that large proteins appear in freeze fractures as particles having crosssectional areas that vary linearly with the number of membrane-spanning helices, by a factor of 1.4 nm 2 per helix (Eskandari et al, 1998). In the same study, connexin hemichannels, ion channels, a water channel, and a co-transporter were all visible by freeze fracture.…”

Section: Discussionmentioning

confidence: 61%

Intercellular interactions in the mammalian olfactory nerve

Blinder

Pumplin

Paul

et al. 2003

J of Comparative Neurology

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The small, unmyelinated axons of olfactory sensory neurons project to the olfactory bulb in densely packed fascicles, an arrangement conducive to axo-axonal interactions. We recently demonstrated ephaptic interactions between these axons in the olfactory nerve layer, the layer of the olfactory bulb in which the axon fascicles interweave and rearrange extensively. In the present study, we hypothesized that the axons, which express connexins, may have another mode of communication: gap junctions. Previous transmission electron microscopy (TEM) studies have failed to demonstrate such junctions. However, the definitive method for detecting gap junctions, freeze fracture, has not been used to examine the interaxonal connections of the olfactory nerve layer. Here, we apply a combined approach of TEM and freeze fracture to determine if gap junctions are present between the olfactory axons. Gap junctions involving olfactory axons were not found. However, by freeze fracture, P faces of both the axons and ensheathing cells (glia that surround the axon fascicles) contained distinctive linear arrays of particles, aligned along the small columns of extracellular space. In axons, few intramembranous particles were present outside of these arrays. Multi-helix proteins, including ion channels and connexin hemichannels, have been shown to be visible as particles by freeze fracture. This suggests that the proteins important for signal transmission are confined to the linear arrays. Such an arrangement would facilitate ephaptic transmission, calcium waves, current oscillations, and paracrine communication and may be important for olfactory neural code processing.Keywords freeze fracture; ultrastructure; olfactory bulb; ephaptic; connexins; hemichannels Olfactory receptor neurons project from the olfactory epithelium to the olfactory bulb by dense bundles of thin, unmyelinated olfactory axons (Marin-Padilla and Amieva, 1989;Daston et al., 1990;Mori, 1993). The high packing density of these axons and the absence of intervening glia suggest that olfactory coding may be affected by axoaxonal interactions.We recently demonstrated (Bokil et al., 2001) that neighboring axons are able to influence each other by ephaptic interactions-current spread through the small extracellular space. We have argued (Bokil et al., 2001) that ephaptic interactions, as well as interneuronal interactions mediated by extruded potassium ions (Bliss and Rosenberg, 1979) First, olfactory receptors of mature mammals are constantly regenerated (Graziadei and Graziadei, 1979;Graziadei and Monti Graziadei, 1983), and developing neurons of the central nervous system are often extensively coupled by gap junctions (reviewed in Kandler and Katz, 1995;Naus and Bani-Yaghoub, 1998;Chang and Balice-Gordon, 2000). Second, olfactory receptor neurons express connexins, the proteins that form gap junctions. Olfactory receptor neurons express mRNA for connexins 43 (Zhang et al., 2000), 45 (Zhang andRestrepo, 2002), and 36 (Zhang andRestrepo, 2003). Furthermore, in immun...

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Section: Discussionmentioning

confidence: 61%

Intercellular interactions in the mammalian olfactory nerve

Blinder

Pumplin

Paul

et al. 2003

J of Comparative Neurology

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“…This was explained by a dominant negative effect of the missense mutation on the GLUT2 hexamer. Such an effect might not play a role in sodium-dependent transporters because freeze-fracture electron microscopic studies have suggested that they function as monomers (29).…”

Section: Discussionmentioning

confidence: 99%

Molecular Analysis of the SGLT2 Gene in Patients with Renal Glucosuria

Santer

Kinner

Lassen³

et al. 2003

Journal of the American Society of Nephrology

275

262

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Abstract. The role of SGLT2 (the gene for a renal sodiumdependent glucose transporter) in renal glucosuria was evaluated. Therefore, its genomic sequence and its intron-exon organization were determined, and 23 families with index cases were analyzed for mutations. In 21 families, 21 different SGLT2 mutations were detected. Most of them were private; only a splice mutation was found in 5 families of different ethnic backgrounds, and a 12-bp deletion was found in two German families. Fourteen individuals (including the original patient with 'renal glucosuria type 0') were homozygous or compound heterozygous for an SGLT2 mutation resulting in glucosuria in the range of 14.

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“…SDS) or that the CFTR dimers only represent a small percentage of the total CFTR proteins that could not be detected by the co-immunoprecipitation procedure. A recent study by Eskandari et al (22) established structural evidence for a dimeric complex with the CFTR proteins. These investigators used freeze fracture electron microscopy to investigate the oligomeric assembly of membrane proteins expressed in Xenopus oocytes, and they concluded that the intramembrane structure of CFTR was consistent with a dimeric assembly of 12-transmembrane helix of the CFTR monomers.…”

Section: A-s-l-v-p-r-g-s-g-g-g-g) As Shown Inmentioning

confidence: 99%

A Single Conductance Pore for Chloride Ions Formed by Two Cystic Fibrosis Transmembrane Conductance Regulator Molecules

Zerhusen

Zhao

Xie

et al. 1999

Journal of Biological Chemistry

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase (PKA)-and ATP-regulated chloride channel, whose gating process involves intra-or intermolecular interactions among the cytosolic domains of the CFTR protein.Tandem linkage of two CFTR molecules produces a functional chloride channel with properties that are similar to those of the native CFTR channel, including trafficking to the plasma membrane, ATP-and PKA-dependent gating, and a unitary conductance of 8 picosiemens (pS). A heterodimer, consisting of a wild type and a mutant CFTR, also forms an 8-pS chloride channel with mixed gating properties of the wild type and mutant CFTR channels. The data suggest that two CFTR molecules interact together to form a single conductance pore for chloride ions. CFTR1 is a multi-functional protein, which provides the pore of a linear conductance chloride channel (1-5) and also functions to regulate other membrane proteins (6, 7). Mutations in CFTR leading to defective regulation or transport of chloride ions across the apical surface of epithelial cells are the primary cause of the genetic disease of cystic fibrosis (8 -10). Comprehensive genotype-phenotype studies have indicated possible contribution of protein-protein interactions to the severity of the disease (11), but little is known on the stoichiometry of CFTR as a chloride channel.The native CFTR chloride channel is activated by PKAphosphorylation of serine residues in its regulatory or R domain and then gated by binding and hydrolysis of ATP by the nucleotide binding folds (12). The actual pore of the chloride channel is presumably formed by portions of the two membrane spanning domains of CFTR, each consisting of six transmembrane segments (13), with an ohmic conductance of ϳ8 pS in 200 mM KCl solution (14,15). An early study by Rich et al. (16) showed that deletion of amino acids 708 -835 from the R domain (⌬R) renders the CFTR channel PKA independent. The open probability of ⌬R-CFTR is approximately one-third that of the wt channel and does not increase upon PKA phosphorylation (17,18). Based on these different gating properties of the wt and ⌬R channels, we set out to test the intermolecular interactions of CFTR by constructing tandem cDNAs between the wt-CFTR and the ⌬R-CFTR. Our rationale is as follows. If a monomer of CFTR is sufficient to form an 8-pS chloride channel, the dimeric CFTR molecules would form either a 16-pS chloride channel or two 8-pS chloride channels that may gate independently or together. On the other hand, if two CFTR molecules are required to function as a chloride channel, we expected the tandem construct to form a single 8-pS chloride channel, provided that the linker sequence does not affect the CFTR channel function. Furthermore, we predicted that the wt-⌬R (or ⌬R-wt) channel should exhibit mixed properties of the wt and ⌬R channels. EXPERIMENTAL PROCEDURESSubcloning of CFTR cDNAs-The wild type and ⌬R (708 -835) CFTR cDNAs were cloned into the NheI/XhoI sites of the pCEP4 expression vec...

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Structural analysis of cloned plasma membrane proteins by freeze-fracture electron microscopy

Cited by 164 publications

References 43 publications

Intercellular interactions in the mammalian olfactory nerve

Intercellular interactions in the mammalian olfactory nerve

Molecular Analysis of the SGLT2 Gene in Patients with Renal Glucosuria

A Single Conductance Pore for Chloride Ions Formed by Two Cystic Fibrosis Transmembrane Conductance Regulator Molecules

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