[14] Reagents for rapid reduction of disulfide bonds

Singh, Rajeeva; Lamoureux, Guy; Lees, Watson J.; Whitesides, George M.

doi:10.1016/0076-6879(95)51119-9

Cited by 48 publications

(32 citation statements)

References 9 publications

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“…Experiments where nonspecific leaks developed during the experiment were simply discarded. The reversibility of covalent bonds was tested with 5 mM BMS (bis(2-mercaptoethylsulfone)) (Calbiochem, San Diego, CA) a water-soluble reagent considered to be a superior reducing agent than dithiothreitol (25,26). The percentage of currents remaining after MTS modification was computed at V m ϭ Ϫ150 mV using [(I wash )/(I ctrl )] ϫ 100 where I wash is the whole cell peak current remaining after MTS application and washout and I ctrl is the whole cell peak current measured before MTS application.…”

Section: Methodsmentioning

confidence: 99%

Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis

Dodier

Banderali

Klein

et al. 2004

Journal of Biological Chemistry

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The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu 515 ) to the beginning of S6 (Ala 560 ). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro 527 and Ile 541 was compatible with the presence of a ␣-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions ؊1, 0, ؉1, ؉2 The TRP ion channels form a large class of cationic channels that are related to the product of the Drosophila TRP gene. TRP channels share a similar predicted topology of six transmembrane segments in which the amino acids that link the fifth and sixth transmembrane domains line the pore region (1). According to the recent IUPHAR classification of ion channels (2), the 21 members of the TRP family can be divided by sequence homology into three subfamilies (3, 4) as short (TRPCx), long or melastatin (TRPMx), and osm-9-like or vanilloid-like (TRPVx) channels. The molecular domains that are mostly conserved among TRP channels include part of the S6 segment, ankyrin repeats in the N terminus, and a "TRP domain" in the C terminus (EWKFAR) (5), the latter being absent from TRPV channels. The TRPC and TRPV proteins have 2-4 N-terminal ankyrin domains suggesting that these proteins are coupled to the spectrin-based membrane cytoskeleton.TRP channels vary significantly in their biophysical properties and gating mechanisms. In contrast to other members of the TRP family, TRPV5 and TRPV6 channels show strong inward rectification, exhibit anomalous mole-fraction effect, are activated by low [Ca 2ϩ ] i and inactivated by higher [Ca 2ϩ ] i (6 -8). TRPV5 and TRPV6 are also highly Ca 2ϩ -selective channels with PCa/PNa Ͼ 100. In particular, ECaC-TRPV5 displays a high Ca 2ϩ affinity with a K d of Ϸ2 M (7) that is comparable to the K d of Ϸ1 M for voltage-dependent Ca V channels (9). A single residue in the S5-S6 linker (Asp 542 ) was found to account for the high Ca 2ϩ affinity of ECaC-TRPV5 (7). The absence of the aspartate residue at the equivalent position in the pore region of TRPV1-4 channels might explain, together with the presence of a lysine residue, the Ϸ20-fold lower Ca 2ϩ selectivity of TRPV1-4 channels (10). TRPV5 and TRPV6 channels can also form homo-and heterotetramers suggesting that they are structurally and functionally related (11).There is currently very little structural data available on the pore architecture of Ca 2ϩ -selective TRP channels. It...

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Section: Methodsmentioning

confidence: 99%

Outer Pore Topology of the ECaC-TRPV5 Channel by Cysteine Scan Mutagenesis

Dodier

Banderali

Klein

et al. 2004

Journal of Biological Chemistry

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“…Although other reducing agents, including dithiothreitol and DMPS, also reversed the effects, the reversal was only partial (data not shown). The ability of BMS to fully reverse the effect may be attributed to its higher accessibility (40).…”

Section: CDmentioning

confidence: 99%

Evidence for Intersubunit Interactions between S4 and S5 Transmembrane Segments of the Shaker Potassium Channel

Neale

Elliott

Hunter

et al. 2003

Journal of Biological Chemistry

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Voltage-gated potassium channels are transmembrane proteins made up of four subunits, each comprising six transmembrane (S1-S6) segments. S1-S4 form the voltage-sensing domain and S5-S6 the pore domain with its central pore. The sensor domain detects membrane depolarization and transmits the signal to the activation gates situated in the pore domain, thereby leading to channel opening. An understanding of the mechanism by which the sensor communicates the signal to the pore requires knowledge of the structure of the interface between the voltage-sensing and pore domains. Toward this end, we have introduced single cysteine mutations into the extracellular end of S4 (positions 356 and 357) in conjunction with a cysteine in S5 (position 418) of the Shaker channel and expressed the mutants in Xenopus oocytes. We then examined the propensity of each pair of engineered cysteines to form a metal bridge or a disulfide bridge, respectively, by examining the effect of Cd 2؉ ions and copper phenanthroline on the K ؉ conductance of a whole oocyte. Both reagents reduced currents through the S357C,E418C double mutant channel, presumably by restricting the movements necessary for coupling the voltage-sensing function to pore opening. This inhibitory effect was seen in the closed state of the channel and with heteromers composed of S357C and E418C single mutant subunits; no effect was seen with homomers of any of the single mutant channels. These data indicate that the extracellular end of S4 lies in close proximity to the extracellular end of the S5 of the neighboring subunit in closed channels.

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“…50 Disulfide is an example of a degradable group that can be cleaved reversibly upon reduction to yield a mixture of the corresponding thiols. Thiols, 414 phosphines, 415,416 metal hydrides, and various metal/acid combinations are often employed as reducing agents. The thiol-disulfide interconversion is widely utilized in nature, e.g., in the regulation of enzyme activity, in protein structure stabilization, and in various metabolic redox processes.…”

Section: (Bio)degradable Polymers With Disulfide Groupsmentioning

confidence: 99%