scholarly journals Probing the Structure of the Diphtheria Toxin Channel

1997 ◽  
Vol 110 (3) ◽  
pp. 229-242 ◽  
Author(s):  
Paul D. Huynh ◽  
Can Cui ◽  
Hangjun Zhan ◽  
Kyoung Joon Oh ◽  
R. John Collier ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Single Channel ◽  
Sudden Change ◽  
Water Soluble ◽  
Soluble Form ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Conducting Pathway ◽  
Ion Conducting ◽  
T Domain

Previous work has established that the 61 amino acid stretch from residue 322 to 382 in the T-domain of diphtheria toxin forms channels indistinguishable in ion-conducting properties from those formed by the entire T-domain. In the crystal structure of the toxin's water-soluble form, the bulk of this stretch is an α-helical hairpin, designated TH8-9. The present study was directed at determining which residues in TH8-9 line the ion-conducting pathway of the channel; i.e., its lumen or entrances. To this end, we singly mutated 49 of TH8-9's 51 residues (328–376) to cysteines, formed channels with the mutant T-domain proteins in planar lipid bilayers, and then determined whether they reacted with small, charged, lipid-insoluble, sulfhydryl-specific methanethiosulfonate (MTS) derivatives added to the bathing solutions. The indication of a reaction, and that the residue lined the ion-conducting pathway, was a sudden change in single-channel conductance and/or flickering behavior. The results of this study were surprising in two respects. First, of the 49 cysteine-substituted residues in TH8-9 tested, 23 reacted with MTS derivatives in a most unusual pattern consisting of two segments: one extending from 329 to 341 (11 of 13 reacted), and the other from 347 to 359 (12 of 13 reacted); none of the residues outside of these two segments appeared to react. Second, in every cysteine mutant channel manifesting an MTS effect, only one transition in single-channel conductance (or flickering behavior) occurred, not the several expected for a multimeric channel. Our results are not consistent with an α-helical or β-strand model for the channel, but instead suggest an open, flexible structure. Moreover, contrary to common sense, they indicate that the channel is not multimeric but is formed from only one TH8-9 unit of the T-domain.

scholarly journals Topography of Diphtheria Toxin's T Domain in the Open Channel State

2000 ◽  
Vol 115 (4) ◽  
pp. 421-434 ◽  
Author(s):  
Lisa Senzel ◽  
Michael Gordon ◽  
Robert O. Blaustein ◽  
K. Joon Oh ◽  
R. John Collier ◽  
...  
Keyword(s):  
Diphtheria Toxin ◽  
Open Channel ◽  
Catalytic Domain ◽  
Water Soluble ◽  
Soluble Form ◽  
Crystallographic Structure ◽  
Channel State ◽  
Amino Terminal ◽  
T Domain ◽  
Membrane Spanning

When diphtheria toxin encounters a low pH environment, the channel-forming T domain undergoes a poorly understood conformational change that allows for both its own membrane insertion and the translocation of the toxin's catalytic domain across the membrane. From the crystallographic structure of the water-soluble form of diphtheria toxin, a “double dagger” model was proposed in which two transmembrane helical hairpins, TH5-7 and TH8-9, anchor the T domain in the membrane. In this paper, we report the topography of the T domain in the open channel state. This topography was derived from experiments in which either a hexahistidine (H6) tag or biotin moiety was attached at residues that were mutated to cysteines. From the sign of the voltage gating induced by the H6 tag and the accessibility of the biotinylated residues to streptavidin added to the cis or trans side of the membrane, we determined which segments of the T domain are on the cis or trans side of the membrane and, consequently, which segments span the membrane. We find that there are three membrane-spanning segments. Two of them are in the channel-forming piece of the T domain, near its carboxy terminal end, and correspond to one of the proposed “daggers,” TH8-9. The other membrane-spanning segment roughly corresponds to only TH5 of the TH5-7 dagger, with the rest of that region lying on or near the cis surface. We also find that, in association with channel formation, the amino terminal third of the T domain, a hydrophilic stretch of ∼70 residues, is translocated across the membrane to the trans side.

scholarly journals Mapping the membrane topography of the TH6–TH7 segment of the diphtheria toxin T-domain channel

2015 ◽  
Vol 145 (2) ◽  
pp. 107-125 ◽  
Author(s):  
Paul K. Kienker ◽  
Zhengyan Wu ◽  
Alan Finkelstein
Keyword(s):  
Lipid Bilayers ◽  
Diphtheria Toxin ◽  
Catalytic Domain ◽  
Single Channel ◽  
Reaction Rates ◽  
Cysteine Residue ◽  
Channel Conductance ◽  
Amino Terminal ◽  
Substituted Cysteine Accessibility ◽  
T Domain

Low pH triggers the translocation domain of diphtheria toxin (T-domain), which contains 10 α helices, to insert into a planar lipid bilayer membrane, form a transmembrane channel, and translocate the attached catalytic domain across the membrane. Three T-domain helices, corresponding to TH5, TH8, and TH9 in the aqueous crystal structure, form transmembrane segments in the open-channel state; the amino-terminal region, TH1–TH4, translocates across the membrane to the trans side. Residues near either end of the TH6–TH7 segment are not translocated, remaining on the cis side of the membrane; because the intervening 25-residue sequence is too short to form a transmembrane α-helical hairpin, it was concluded that the TH6–TH7 segment resides at the cis interface. Now we have examined this segment further, using the substituted-cysteine accessibility method. We constructed a series of 18 mutant T-domains with single cysteine residues at positions in TH6–TH7, monitored their channel formation in planar lipid bilayers, and probed for an effect of thiol-specific reagents on the channel conductance. For 10 of the mutants, the reagent caused a change in the single-channel conductance, indicating that the introduced cysteine residue was exposed within the channel lumen. For several of these mutants, we verified that the reactions occurred primarily in the open state, rather than in the flicker-closed state. We also established that blocking of the channel by an amino-terminal hexahistidine tag could protect mutants from reaction. Finally, we compared the reaction rates of reagent added to the cis and trans sides to quantify the residue’s accessibility from either side. This analysis revealed abrupt changes in cis- versus trans-side accessibility, suggesting that the TH6–TH7 segment forms a constriction that occupies a small portion of the total channel length. We also determined that this constriction is located near the middle of the TH8 helix.

scholarly journals Lack of negative slope in I-V plots for BK channels at positive potentials in the absence of intracellular blockers

2013 ◽  
Vol 141 (4) ◽  
pp. 493-497 ◽  
Author(s):  
Yanyan Geng ◽  
Xiaoyu Wang ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Negative Slope ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Linear Current ◽  
Joint Application ◽  
Current Voltage ◽  
Α Subunits

Large-conductance, voltage- and Ca2+-activated K+ (BK) channels display near linear current–voltage (I-V) plots for voltages between −100 and +100 mV, with an increasing sublinearity for more positive potentials. As is the case for many types of channels, BK channels are blocked at positive potentials by intracellular Ca2+ and Mg2+. This fast block progressively reduces single-channel conductance with increasing voltage, giving rise to a negative slope in the I-V plots beyond about +120 mV, depending on the concentration of the blockers. In contrast to these observations of pronounced differences in the magnitudes and shapes of I-V plots in the absence and presence of intracellular blockers, Schroeder and Hansen (2007. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.200709802) have reported identical I-V plots in the absence and presence of blockers for BK channels, with both plots having reduced conductance and negative slopes, as expected for blockers. Schroeder and Hansen included both Ca2+ and Mg2+ in the intracellular solution rather than a single blocker, and they also studied BK channels expressed from α plus β1 subunits, whereas most previous studies used only α subunits. Although it seems unlikely that these experimental differences would account for the differences in findings between previous studies and those of Schroeder and Hansen, we repeated the experiments using BK channels comprised of α plus β1 subunits with joint application of 2.5 mM Ca2+ plus 2.5 mM Mg2+, as Schroeder and Hansen did. In contrast to the findings of Schroeder and Hansen of identical I-V plots, we found marked differences in the single-channel I-V plots in the absence and presence of blockers. Consistent with previous studies, we found near linear I-V plots in the absence of blockers and greatly reduced currents and negative slopes in the presence of blockers. Hence, studies of conductance mechanisms for BK channels should exclude intracellular Ca2+/Mg2+, as they can reduce conductance and induce negative slopes.

ATP-sensitive K(+)-selective channels of inner medullary collecting duct cells

1994 ◽  
Vol 267 (3) ◽  
pp. F489-F496 ◽  
Author(s):  
S. C. Sansom ◽  
T. Mougouris ◽  
S. Ono ◽  
T. D. DuBose
Keyword(s):  
Single Channel ◽  
Collecting Duct ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Inner Medullary Collecting Duct ◽  
Outward Currents ◽  
Inward Currents ◽  
Excised Patches ◽  
Medullary Collecting Duct

The inner medullary collecting duct (IMCD) in vivo has the capacity to either secrete or reabsorb K+. However, a selective K+ conductance has not been described previously in the IMCD. In the present study, the patch-clamp method was used to determine the presence and properties of K(+)-selective channels in the apical membrane of the inner medullary collecting duct cell line, mIMCD-3. Two types of K(+)-selective channels were observed in both cell-attached and excised patches. The most predominant K+ channel, a smaller conductance K+ channel (SK), was present in cell-attached patches with 140 mM KCl (high bath K+) but not with 135 mM NaCl plus 5 mM KCl (low bath K+) in the bathing solution. The single-channel conductance of SK was 36 pS with inward currents and 29 pS with outward currents in symmetrical 140 mM KCl. SK was insensitive to both voltage and Ca2+. However, SK was inhibited significantly by millimolar concentrations of ATP in excised patches. A second K(+)-selective channel [a larger K+ channel (BK)] displayed a single-channel conductance equal to 132 pS with inward currents and 90 pS with outward currents in symmetrical 140 mM KCl solutions. BK was intermittently activated in excised inside-out patches by Mg(2+)-ATP in concentrations from 1 to 5 mM. With complete removal of Mg2+, BK was insensitive to ATP. BK was also insensitive to potential and Ca2+ and was observed in cell-attached patches with 140 mM KCl in the bath solution. Both channels were blocked reversibly by 1 mM Ba2+ from the intracellular surface but not by external Ba2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Basolateral potassium channels in renal proximal tubule

1987 ◽  
Vol 253 (3) ◽  
pp. F476-F487 ◽  
Author(s):  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
K Channel ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
K Channels ◽  
Open Time ◽  
Excised Patches ◽  
The Mean ◽  
Inside Out

Potassium (K+) channels in the basolateral membrane of unperfused Necturus proximal tubules were studied in both cell-attached and excised patches, after removal of the tubule basement membrane by manual dissection without collagenase. Two different K+ channels were identified on the basis of their kinetics: a short open-time K+ channel, with a mean open time less than 1 ms, and a long open-time K+ channel with a mean open time greater than 20 ms. The short open-time channel occurred more frequently than the longer channel, especially in excised patches. For inside-out excised patches with Cl- replaced by gluconate, the current-voltage relation of the short open-time K+ channel was linear over +/- 60 mV, with a K+-Na+ selectivity of 12 +/- 2 (n = 12), as calculated from the reversal potential with oppositely directed Na+ and K+ gradients. With K-Ringer in the patch pipette and Na-Ringer in the bath, the conductance of the short open-time channel was 47 +/- 2 pS (n = 15) for cell-attached patches, 26 +/- 2 pS (n = 15) for patches excised (inside out) into Na-Ringer, and 36 +/- 6 pS (n = 3) for excised patches with K-Ringer on both sides. These different conductances can be partially explained by a dependence of single-channel conductance on the K+ concentration on the interior side of the membrane. In experiments with a constant K+ gradient across excised patches, large changes in Na+ at the interior side of the membrane produced no change in single-channel conductance, arguing against a direct block of the K+ channel by Na+. Finally, the activity of the short open-time channel was voltage gated, where the mean number of open channels decreased as a linear function of basolateral membrane depolarization for potentials between -60 and 0 mV. Depolarization from -60 to -40 mV decreased the mean number of open K+ channels by 28 +/- 8% (n = 6).

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