Substituted Cysteine Accessibility Method (SCAM)

2010 ◽  
pp. 229-250
Author(s):  
George Liapakis ◽  
Jonathan A. Javitch
Keyword(s):  
Substituted Cysteine Accessibility

scholarly journals Application of SCAM (Substituted Cysteine Accessibility Method) to Gap Junction Intercellular Channels

2001 ◽  
Vol 8 (4-6) ◽  
pp. 179-185 ◽  
Author(s):  
I. M. Skerrett ◽  
E. Kasperek ◽  
F. L. Cao ◽  
J. H. Shin ◽  
J. Aronowitz ◽  
...  
Keyword(s):  
Gap Junction ◽  
Substituted Cysteine Accessibility ◽  
Intercellular Channels

scholarly journals Bridging the gap between structure and kinetics of human SGLT1

2012 ◽  
Vol 302 (9) ◽  
pp. C1293-C1305 ◽  
Author(s):  
Monica Sala-Rabanal ◽  
Bruce A. Hirayama ◽  
Donald D. F. Loo ◽  
Vincent Chaptal ◽  
Jeff Abramson ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Sugar Transport ◽  
Gene Families ◽  
Structural Studies ◽  
Sugar Binding ◽  
Substituted Cysteine Accessibility ◽  
Biochemical Assays ◽  
Functional Consequences ◽  
Binding Residues

The Na+-glucose cotransporter hSGLT1 is a member of a class of membrane proteins that harness Na+ electrochemical gradients to drive uphill solute transport. Although hSGLT1 belongs to one gene family (SLC5), recent structural studies of bacterial Na+ cotransporters have shown that Na+ transporters in different gene families have the same structural fold. We have constructed homology models of hSGLT1 in two conformations, the inward-facing occluded (based on vSGLT) and the outward open conformations (based on Mhp1), mutated in turn each of the conserved gates and ligand binding residues, expressed the SGLT1 mutants in Xenopus oocytes, and determined the functional consequences using biophysical and biochemical assays. The results establish that mutating the ligand binding residues produces profound changes in the ligand affinity (the half-saturation concentration, K0.5); e.g., mutating sugar binding residues increases the glucose K0.5 by up to three orders of magnitude. Mutation of the external gate residues increases the Na+ to sugar transport stoichiometry, demonstrating that these residues are critical for efficient cotransport. The changes in phlorizin inhibition constant ( Ki) are proportional to the changes in sugar K0.5, except in the case of F101C, where phlorizin Ki increases by orders of magnitude without a change in glucose K0.5. We conclude that glucose and phlorizin occupy the same binding site and that F101 is involved in binding to the phloretin group of the inhibitor. Substituted-cysteine accessibility methods show that the cysteine residues at the position of the gates and sugar binding site are largely accessible only to external hydrophilic methanethiosulfonate reagents in the presence of external Na+, demonstrating that the external sugar (and phlorizin) binding vestibule is opened by the presence of external Na+ and closes after the binding of sugar and phlorizin. Overall, the present results provide a bridge between kinetics and structural studies of cotransporters.

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

scholarly journals Cysteine Mutagenesis Reveals Novel Structure–Function Features within the Predicted Third Extracellular Loop of the Type Iia Na+/Pi Cotransporter

2001 ◽  
Vol 117 (6) ◽  
pp. 533-546 ◽  
Author(s):  
Georg Lambert ◽  
Ian C. Forster ◽  
Gerti Stange ◽  
Katja Köhler ◽  
Jürg Biber ◽  
...  
Keyword(s):  
Structure Function ◽  
Helical Structure ◽  
Cysteine Residue ◽  
Transport Function ◽  
Extracellular Loop ◽  
Substituted Cysteine Accessibility ◽  
Novel Structure ◽  
Important Region ◽  
Α Helix ◽  
Low Sensitivity

The transport function of the rat type IIa Na+/Pi cotransporter is inhibited after binding the cysteine modifying reagent 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) to a cysteine residue substituted for a serine at position 460 (S460C) in the predicted third extracellular loop. This suggests that Ser-460 lies in a functionally important region of the protein. To establish a “structure–function” profile for the regions that flank Ser-460, the substituted cysteine accessibility method was employed. 18 mutants were constructed in which selected amino acids from Arg-437 through Leu-465 were substituted one by one for a cysteine. Mutants were expressed in Xenopus oocytes and transport function (cotransport and slippage) and kinetics were assayed by electrophysiology with or without prior treatment with cysteine modifying (methanethiosulfonate, MTS) reagents. Except for mutant I447C, mutants with cysteines at sites from Arg-437 through Thr-449, as well as Pro-461, were inactive. Cotransport function of mutants with Cys substitutions at sites Arg-462 through Leu-465 showed low sensitivity to MTS reagents. The preceding mutants (Cys substitution at Thr-451 to Ser-460) showed a periodic accessibility pattern that would be expected for an α-helix motif. Apart from loss of transport function, exposure of mutants A453C and A455C to MTSEA or 2-(triethylammonium)ethyl MTS bromide (MTSET) increased the uncoupled slippage current, which implicated the mutated sites in the leak pathway. Mutants from Ala-453 through Ala-459 showed less pH dependency, but generally stronger voltage dependency compared with the wild type, whereas those flanking this group were more sensitive to pH and showed weaker voltage dependence of cotransport mode kinetics. Our data indicate that parts of the third extracellular loop are involved in the translocation of the fully loaded carrier and show a membrane-associated α-helical structure.

Mechanism of activation of Xenopus CFTR by stimulation of PKC

2004 ◽  
Vol 287 (5) ◽  
pp. C1256-C1263 ◽  
Author(s):  
Yongyue Chen ◽  
Guillermo A. Altenberg ◽  
Luis Reuss
Keyword(s):  
Single Channel ◽  
Membrane Conductance ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Open Probability ◽  
Substituted Cysteine Accessibility ◽  
Maximal Stimulation ◽  
A Minor ◽  
Cystic Fibrosis Transmembrane ◽  
Stimulation Of

PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR ( XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was ∼90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (∼30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.

scholarly journals Topology of AspT, the Aspartate:Alanine Antiporter of Tetragenococcus halophilus, Determined by Site-Directed Fluorescence Labeling

2007 ◽  
Vol 189 (19) ◽  
pp. 7089-7097 ◽  
Author(s):  
Kei Nanatani ◽  
Takashi Fujiki ◽  
Kazuhiko Kanou ◽  
Mayuko Takeda-Shitaka ◽  
Hideaki Umeyama ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Amino Acid Sequences ◽  
Fluorescence Labeling ◽  
Dimensional Structure ◽  
Specific Probe ◽  
Cytoplasmic Loop ◽  
Intact Cells ◽  
Tetragenococcus Halophilus ◽  
Substituted Cysteine Accessibility ◽  
And Function

ABSTRACT The gram-positive lactic acid bacterium Tetragenococcus halophilus catalyzes the decarboxylation of l-aspartate (Asp) with release of l-alanine (Ala) and CO2. The decarboxylation reaction consists of two steps: electrogenic exchange of Asp for Ala catalyzed by an aspartate:alanine antiporter (AspT) and intracellular decarboxylation of the transported Asp catalyzed by an l-aspartate-β-decarboxylase (AspD). AspT belongs to the newly classified aspartate:alanine exchanger family (transporter classification no. 2.A.81) of transporters. In this study, we were interested in the relationship between the structure and function of AspT and thus analyzed the topology by means of the substituted-cysteine accessibility method using the impermeant, fluorescent, thiol-specific probe Oregon Green 488 maleimide (OGM) and the impermeant, nonfluorescent, thiol-specific probe [2-(trimethylammonium)ethyl]methanethiosulfonate bromide. We generated 23 single-cysteine variants from a six-histidine-tagged cysteineless AspT template. A cysteine position was assigned an external location if the corresponding single-cysteine variant reacted with OGM added to intact cells, and a position was assigned an internal location if OGM labeling required cell lysis. The topology analyses revealed that AspT has a unique topology; the protein has 10 transmembrane helices (TMs), a large hydrophilic cytoplasmic loop (about 180 amino acids) between TM5 and TM6, N and C termini that face the periplasm, and a positively charged residue (arginine 76) within TM3. Moreover, the three-dimensional structure constructed by means of the full automatic modeling system indicates that the large hydrophilic cytoplasmic loop of AspT possesses a TrkA_C domain and a TrkA_C-like domain and that the three-dimensional structures of these domains are similar to each other even though their amino acid sequences show low similarity.

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