Tsushima RG , Li RA , Backx PH .

	Figure 2. (A) Raw current traces of wild-type, Y401C, W402C, and E403C channels recorded in oocytes following depolarization to −10 mV from a holding potential of −120 mV with (broken line) and without (solid line) 100 μM extracellular Cd2+. Clearly, the amount of current reduction is much greater for the mutant channels (Y401C, W402C, and E403C) than for the wild-type channels. The currents amplitudes have been scaled for ease of comparison. Peak currents in μA were: 3.8 (WT), 3.5 (Y401C), 3.5 (W402C), and 2.0 (E403C). (B) Plots of the fraction of peak current remaining as a function of the extracellular [Cd2+] for the same channels shown in A. Curve fits allow estimation of the dissociation constants (i.e., KD) for Cd2+ binding to the channel pore. (C) Summary of the Cd2+ block in single-cysteine mutants. The ratio of the estimated KD for Cd2+ block of current in single-cysteine mutants divided by the wild-type channels (i.e., KD,mut/KD,WT) for control conditions (filled bars) and following the application of 1.0 mM methane-thiosulfate-ethylammonium (MTSEA) to oxidize the inserted free sulfhydryls (open bars). For each mutant, except W756C, KD,mut was significantly reduced (p < 0.001) more than 3-fold, which was abolished by the application of MTSEA.
	Figure 3. (A) Na+ current tracings of Y401C, E758C, and Y401C/E758C mutant channels in response to depolarization to −10 mV from a holding potential of −120 mV before (solid traces) and after (broken traces) the addition of 100 μM Cd2+. Na+ currents for Y401C/E758C channels are shown before and after the addition of DTT. The currents have been scaled for ease of comparison. The current amplitudes in the absence of Cd2+ were (μA): 3.5 (Y401C), 2.7 (E758C), 2.2 (Y401C/E758C −DTT), and 4.6 (Y401C/ E758C + DTT). (B) Dose-response curves of the normalized peak Na+ current as a function of the [Cd2+]. The channels become about 1,200-fold more sensitive to Cd2+ (KD changes from 1,353 ± 382 μM to 1.1 ± 0.2 μM) following the addition of 2 mM DTT.
	Figure 4. Ratios of the predicted dissociation constant, KD,pre, to the experimentally observed dissociation constant, KD,ob for all the double mutants studied before (filled bars) and after (open bars) reduction by DTT. KD,pre is estimated from the dissociation constants recorded for the single-cysteine mutant channels assuming independent binding of Cd2+ to the inserted cysteines in double-cysteine mutants (see methods, Eq. 1). For the mutants with ratios of KD,ob/KD,pre around 1, we assume that the two inserted cysteines are binding Cd2+ independently. Values of KD,ob/KD,pre statistically different from 1 are identified by asterisks (*). The mutant channels with KD,ob/KD,pre below 0.37 (i.e., with stabilization energies of 1 kT) are identified by open triangles.
	Figure 5. Single-channel recordings of Y401C, E758C, and reduced Y401C/E758C mutant channels following fenvalerate application at −80 mV. (A) Currents recorded in Y401C, E758C, and Y401C/E758C channels in the presence of 10 μM fenvalerate which is added to maintain the channels in the open state for tens to hundreds of milliseconds. (B) Currents recorded in Y401C and reduced Y401C/ E758C mutants channels with 5 μM extracellular Cd2+ and in E758C channels with 400 μM Cd2+. Cd2+ caused full closures of the Y401C and Y401C/E758C channels while blocking E758C channels to a sub-conductance level (closed level indicated by the broken line). In the presence of 5 μM Cd2+ the double-mutant channel Y401C/E758C displayed bursts of short-lived blocking events separated by long-lived blocking events not seen in either single-mutant. These long-lived blockages likely represent the “trapping” of the Cd2+ ion as a result of simultaneous interactions with the two free sulfhydryl groups. (C) The mean block-time histograms are shown for the channels illustrated in A for Y401C, E758C, and Y401C/E758C channels. The blocked-time histograms could be adequately fit using a mono-exponential equation for Y401C and E758C channels, while a bi-exponential function was required for Y401C/E758C channels. Note: the time axes are different in the different panels. See text for further details. (D) The mean unblocked-time (i.e., open-time) histogram is shown for the same channels illustrated in B. For Y401C, E758C, and Y401C/E758C channels the unblocked-time histogram could be adequately fit using a mono-exponential function.
	Figure 6. Effect of cross-linking on conductance and selectivity properties of double-cysteine mutants. (A) Raw current traces following depolarization to −10 mV from a holding potential of −120 mV for E403C/D1532C channels expressed in oocytes before (□) and after (▪) reduction with DTT. Note the nearly twofold increase in current after reduction at this voltage. (B) The corresponding current-voltage relationships for E403C/D1532C. In this particular mutant, there is little change in the channel's conductance (161 μS before and 156 μS after DTT), estimated from the slope of the current-voltage curve at voltages above 0 mV, but the reversal potential is shifted by 19 mV to the right following reduction with DTT. (C) Raw traces for E403C/ A1529C before (○) and after (•) the application of DTT. The current increased more than sixfold following reduction with DTT. (D) The current-voltage relationship for E403C/A1529C mutants shows a fivefold increase in slope at voltages above 0 mV (22 μS before and 97 μS after DTT), whereas the reversal potential is only slightly shifted rightward (7 mV) by reduction with DTT.

Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed, Xenbase

Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed , Xenbase
Arnold, Investigation of domain motions in bacteriophage T4 lysozyme. 1994, Pubmed
Backx, Molecular localization of an ion-binding site within the pore of mammalian sodium channels. 1992, Pubmed
Balaji, Modification of protein stability by introduction of disulfide bridges and prolines: geometric criteria for mutation sites. 1989, Pubmed
Bénitah, Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis. 1996, Pubmed , Xenbase
Careaga, Structure and dynamics of Escherichia coli chemosensory receptors. Engineered sulfhydryl studies. 1992, Pubmed
Catterall, Structure and function of voltage-gated ion channels. 1995, Pubmed
Chiamvimonvat, Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis. 1996, Pubmed , Xenbase
Cottrell, The sole lysine residue in porcine pepsin works as a key residue for catalysis and conformational flexibility. 1995, Pubmed
Eisenberg, Channels as enzymes. 1990, Pubmed
Eisenman, Ionic selectivity revisited: the role of kinetic and equilibrium processes in ion permeation through channels. 1983, Pubmed
Ellinor, Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions. 1995, Pubmed , Xenbase
Gross, Agitoxin footprinting the shaker potassium channel pore. 1996, Pubmed , Xenbase
Heginbotham, A functional connection between the pores of distantly related ion channels as revealed by mutant K+ channels. 1992, Pubmed , Xenbase
Heinemann, Calcium channel characteristics conferred on the sodium channel by single mutations. 1992, Pubmed , Xenbase
Hidalgo, Revealing the architecture of a K+ channel pore through mutant cycles with a peptide inhibitor. 1995, Pubmed , Xenbase
Isacoff, Putative receptor for the cytoplasmic inactivation gate in the Shaker K+ channel. 1991, Pubmed , Xenbase
Krovetz, Atomic distance estimates from disulfides and high-affinity metal-binding sites in a K+ channel pore. 1997, Pubmed
Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection. 1985, Pubmed
Kürz, Side-chain accessibilities in the pore of a K+ channel probed by sulfhydryl-specific reagents after cysteine-scanning mutagenesis. 1995, Pubmed , Xenbase
Lan, Evidence for a (triosephosphate isomerase-like) "catalytic loop" near the active site of glyoxalase I. 1995, Pubmed
Larson, Mechanistic insights provided by deletion of a flexible loop at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase. 1995, Pubmed
Läuger, Dynamics of ion transport systems in membranes. 1987, Pubmed
Lipkind, A structural model of the tetrodotoxin and saxitoxin binding site of the Na+ channel. 1994, Pubmed
Liu, Dynamic rearrangement of the outer mouth of a K+ channel during gating. 1996, Pubmed
Lopez, Evidence that the S6 segment of the Shaker voltage-gated K+ channel comprises part of the pore. 1994, Pubmed , Xenbase
MacKinnon, Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor. 1989, Pubmed , Xenbase
MacKinnon, Determination of the subunit stoichiometry of a voltage-activated potassium channel. 1991, Pubmed , Xenbase
Miller, Ion channel structure and function. 1992, Pubmed
Naranjo, A strongly interacting pair of residues on the contact surface of charybdotoxin and a Shaker K+ channel. 1996, Pubmed , Xenbase
Nicholson, Flexibility and function in HIV-1 protease. 1995, Pubmed
Noda, Primary structure of Electrophorus electricus sodium channel deduced from cDNA sequence. , Pubmed
Pascual, K+ pore structure revealed by reporter cysteines at inner and outer surfaces. 1995, Pubmed , Xenbase
Pérez-García, Structure of the sodium channel pore revealed by serial cysteine mutagenesis. 1996, Pubmed , Xenbase
Pompliano, Stabilization of a reaction intermediate as a catalytic device: definition of the functional role of the flexible loop in triosephosphate isomerase. 1990, Pubmed
Ranganathan, Spatial localization of the K+ channel selectivity filter by mutant cycle-based structure analysis. 1996, Pubmed
Satin, A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. 1992, Pubmed , Xenbase
Soman, Secondary structure prediction of the H5 pore of potassium channels. 1995, Pubmed
Stone, Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements. 1992, Pubmed
Sun, Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating. 1996, Pubmed
Tanaka, Flexibility impaired by mutations revealed the multifunctional roles of the loop in glutathione synthetase. 1993, Pubmed
Terlau, Mapping the site of block by tetrodotoxin and saxitoxin of sodium channel II. 1991, Pubmed , Xenbase
Tomaselli, A mutation in the pore of the sodium channel alters gating. 1995, Pubmed , Xenbase
Trimmer, Primary structure and functional expression of a mammalian skeletal muscle sodium channel. 1989, Pubmed , Xenbase
Tsushima, Altered ionic selectivity of the sodium channel revealed by cysteine mutations within the pore. 1997, Pubmed , Xenbase
Vallee, The biochemical basis of zinc physiology. 1993, Pubmed
Wade, Gating of the active site of triose phosphate isomerase: Brownian dynamics simulations of flexible peptide loops in the enzyme. 1993, Pubmed
Welch, The role of protein fluctuations in enzyme action: a review. 1982, Pubmed
Yellen, An engineered cysteine in the external mouth of a K+ channel allows inactivation to be modulated by metal binding. 1994, Pubmed
Yellen, Mutations affecting internal TEA blockade identify the probable pore-forming region of a K+ channel. 1991, Pubmed