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Figure 1. . Amino acid sequence alignment of IKCa, rSK2, KcsA, and Shaker showing sequence similarities within the S6 transmembrane segment. The sequence alignment of the Pore + S6 region of IKCa, rSK2, KcsA, and Shaker was based on the conserved GYGD pore motif. Sequence similarities of 100% and 75% are shaded in black and gray, respectively. The S6 segment in IKCa is presented as extending from V266 to the A286.
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Figure 9. . State-dependent action of MTSET. (A) Inside-out recordings showing the state dependency of the MTSET action on IKCa mutants. The accessibility to MTSET of the substituting cysteine was estimated in MTSET protection experiments as discussed in materials and methods. The applied potential was maintained at â60 mV throughout. (B) Bar graph representation of the ratio [<I>(test)/<I>(ctr)] measured with (closed) or without (open) MTSET preconditioning in zero internal Ca2+ conditions. The <I>(test)/<I>(ctr) ratios obtained for the second MTSET application in the perfusion protocol illustrated in A were estimated at 0.02 ± .006 (n = 3) for V275C, 0.26 ± 0.02 (n = 2) for T278C, 0.16 ± 0.06 (n = 3) for V282C, 0.54 ± 0.05 (n = 2) for V284, and 2 ± 1 (n = 3) for V285C, respectively. These values are not significantly different from the <I>(test)/<I>(ctr) ratios measured without an initial MTSET exposure in zero internal Ca2+. In contrast the <I>(test)/<I>(ctr) values estimated for A283C and A286C mutants decreased drastically from 19 ± 5 (n = 5) and 10 ± 0.54 in the absence of MTSET preconditioning in zero external Ca2+, to 1.0 ± 0.05 (n = 5) and 1.0 ±0.02 (n = 3) for the perfusion protocol illustrated in A. These observations support a model whereby A283 and A286 remained accessible to MTSET in conditions where channels were maintained in the closed state.
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Figure 3. . Inside-out recordings illustrating the action of MTSET on IKCa mutants. Inside-out current records measured in symmetrical 200 mM K2SO4 + 3 μM internal Ca2+ conditions. The pipette potential was 60 mV throughout. The effect of the MTSET reagent on channel activity was estimated from the ratio <I>(test)/<I>(ctr) where <I>(ctr) is the mean current measured in 3 μM Ca2+ before drug application (labeled line 1 in WT) and <I>(test), the mean current obtained at the same Ca2+ concentration (labeled line 3 in WT) after the sequential washout of the drug with a Ca2+-containing (3 μM) (labeled line 2 in WT) and a Ca2+-free 200 mM K2SO4 solution. The drug was systematically applied for 5 min with a total washout period of 2 min. This procedure ensures that the observed effects of the MTS reagents are resulting from a covalent binding of the drug to the targeted cysteine, and not from nonspecific channel interactions with the open or closed channel. Strong inhibition (>75%) of channel activity was observed with V275C, T278C, and V282C after exposure to MTSET (5 mM) for 5 min, with a complete inhibition recorded with the V275C and V282C mutants. The V284C channel showed a maximal inhibition of 50% despite a steady-state current value reached after 45 s. Notably, MTSET caused an increase in inward currents when applied on the A283C, A286C, and to a lesser extent V285C mutants. The current increase remained Ca2+ and clotrimazole sensitive, ruling out nonspecific effects of MTSET. There were no significant variations in mean currents with A279C suggesting that this residue may not be MTSET accessible.
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Figure 2. . Characterization of the IKCa channel cloned from HeLa cells. (A) Current/voltage properties of the IKCa channel measured in symmetrical 200 K2SO4 conditions. The unitary conductance for inward and outward currents was estimated at 49 and 20 pS, respectively. (B) Inside-out single channel recordings of the wild-type IKCa channels expressed in Xenopus laevis oocytes for internal Ca2+ concentrations ranging from 0.1 to 1.0 μM. Experiments performed in symmetrical 200 mM K2SO4, pH 7.4 conditions. The pipette potential was maintained at 60 mV throughout. Current traces were filtered at 500 Hz and the letter c refers to the zero current level. (C) Normalized mean current at a constant Vp of 60 mV from four different experiments plotted as a function of the internal Ca2+ concentration. The sigmoide curve was computed according to a Hill equation with [Ca]1/2 = 1.2 ± 0.1 μM and Hill coefficient of 4.3 ± 0.4 (n = 4).
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Figure 4. . Effects of MTSET on IKCa channel mutants. (A) Histogram representation of the mean current ratio <I>(test)/<I>(ctr) obtained with MTSET for the amino acids spanning the V275-A286 domain. WT refers to the wild-type form of IKCa. Experiments performed in 3 μM internal Ca2+ (open channel). A ratio of 1 indicates a total absence of MTS-dependent effects on channel activity. C276 and C277 refer to endogenous cysteine and are labeled as *. Similarly, experiments could not be performed on the L280C and L281C mutants due to the absence of detectable single channel events in these cases. (B) Column representation of the modification rate for the MTSET-dependent channel inhibition and/or activation expressed in Mâ1sâ1. With the exception of V285C (2.0 ± 0.5 sâ1Mâ1; n = 5), modification rates for MTSET interactions were higher for the residues within the A283âA286 S6 region with values of 17 ± 0.5 sâ1Mâ1 (n = 2), 17 ± 0.4 sâ1Mâ1 (n = 5), and 54 ± 0.1 sâ1Mâ1 (n = 2) for A283C, V284C, and A286C, respectively, as compared with 7 ± 0.8 sâ1Mâ1 (n = 4) for V275C, 2.5 ± 1.5 sâ1Mâ1 (n = 6) for T278C, and 2.9 ± 2 sâ1Mâ1 (n = 4) for V282C. These results show that A286 is the residue most accessible to MTSET within the S6 segment extending from V275 to A286.
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Figure 5. . Nonstationary noise analysis of the interacting MTSET-IKCa mutants. Relationship between the current variance Ï2 and the mean current <I> during MTSET application illustrated for the V275C, A283C, V284C, and V285C mutants. <I> and Ï2 were measured on successive time periods of 1 s for A283C, V284C, and V285C and 0.5 s for V275C. The V275C mutant displayed a constant Ï2/<I> (left panel right scale) ratio despite an important decrease in <I> (left panel left scale), indicating an important inhibition of the channel unitary current. This proposal is also supported by the fact that the ratio Ï2/<I>2 (right panel) increases in this case with a time constant equal to Ïi, the inhibition time constant measured for <I>. A similar noise pattern was also observed with the V284C mutant, although in this case the variations in Ï2/<I> and Ï2/<I>2 can be accounted for a partial inhibition of the channel unitary current plus a decrease in channel open probability. A different noise behavior is, however, observed with the A283C and V285C channels. The decrease in Ï2/<I>2 observed with A283 correlates the increase in mean current with the Ï2/<I> ratio remaining constant for time >7.5 s. This noise pattern would be compatible with a system where PB > PO with PO and PB << 1, thus supporting a model whereby the action of MTSET consists either to increase the channel open probability or recruit silent A283C mutants. The results obtained with V285C follow a similar pattern, although in this case the fact that the measured variation in Ï2/<I>2 is more important than the mean current increase favors a system where PB > PO with PO << 1.
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Figure 6. . Single channel analysis of the effect of MTSET on IKCa channel mutants. (A) Inside-out single channel recordings obtained from IKCa mutants under low Ca2+ conditions at an internal MTSET concentration of 5 mM. The applied voltage âVp corresponded to â140 mV throughout. Large negative potentials were used in these experiments to optimize the signal to noise ratio, the current jump amplitude at â60 mV being barely detectable (<0.3 pA) after MTSET treatment except for the A286C mutant. The action of MTSET on 283C and 286C is seen to result in a substantial increase in open channel probability in accordance with the increase in mean current observed with the A283C and A286C mutants. (B) Bar graph representation of the effect of MTSET (5 mM) and MTSES (5mM) on the unitary current amplitude of IKCa mutants. I(MTSET) and I(ctr) correspond to the unitary current amplitude measured at â140 mV in the presence or absence of MTSET or MTSES (5 mM) respectively. * refers to endogenous cysteine or nonfunctional mutants as mentioned in Fig. 4. A ratio of 1 indicates an absence of MTSET- or MTSES-based effect on the channel unitary conductance. As seen, internal application of MTSET caused a near inhibition of the unitary current amplitude for the V275C, T278C, and V282C mutant channels. A partial inhibition ranging from 40% (V284C) to 70% (A283C) was observed with mutants obtained by cysteines substitution of the residues in the COOH-terminal region of S6 (A283âA286). In contrast, the application of the negatively charged MTSES reagent on A286C resulted in an increased unitary current amplitude. These observations suggest that the presence of charged groups at the cytoplasmic entrance of the pore can modulate the exit rate of K+ from the channel cavity into the cytosolic solution.
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Figure 7. . Protection by MTSET of TEA block for the A283C and A286C mutants. (A) Inside-out current recording illustrating the blocking action of TEA (30 mM) on the wild-type IKCa channel. (B) Inside-out recording demonstrating the lack of TEA-dependent block with the A283C mutant after application of MTSET. (C) Inside-out recording illustrating the reduced effectiveness of TEA on the A286C mutant stimulated by MTSET. (D) Histogram summarizing the effects of TEA on the wild-type IKCa channel (WT), and on the A283C and A286C mutants activated by MTSET. WT channel was blocked at 79 ± 4% (n = 6), whereas the blocking effect of TEA was reduced to 6.5 ± 4.6% (n = 3) for the A283C + MTSET mutant and to 27 ± 7% (n = 3) for the A286 mutant stimulated by MTSET.
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Figure 8. . Effects of MTSES on IKCa mutants. Inside-out current records measured in symmetrical 200 mM K2SO4 + 3 μM internal Ca2+ conditions. The pipette potential was 60 mV throughout. In contrast to MTSET, MTSES failed to cause over a 5 min period a significant decrease of the V275C-induced currents. As observed with MTSET, however, MTSES succeeded in strongly inhibiting the V284C mutant and to stimulate the inward currents generated by A283C and A286C. Also illustrated are MTSES protection experiments performed on V275C and A286C, where MTSES (5 mM) was applied prior MTSET (5 mM). These experiments confirmed that a cysteine at position 275 is not accessible to MTSES and that the binding of MTSET to 286C prevents the interaction of MTSESâA286C.
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Figure 10. . Homology modeling of the IKCa. Computer modeling of the IKCa channel using either the closed or open KcsA channel structure as template. Surface and ribbon representations of the open IKCa S5-P-S6 region are illustrated in B and C, respectively. B also includes a surface representation of a single S6 segment for the open channel. The molecular surface computed for the closed IKCa S5-P-S6 region is presented in A. According to the proposed models, the residues V275, T278, and V282 are lining the pore lumen resulting in a cavity 10 Ã
wide at the level of V275. Also, the endogenous cysteines (yellow in Fig. 9 C) at positions 269, 276, and 277 are presented as facing opposite to the pore lumen, whereas the C267 residue is predicted to be oriented toward the selectivity filter. A color representation of the MTSET modification rate for channel inhibition and/or activation is superimposed on the closed and open IKCa channel structures. Residues with slow (<5 Mâ1sâ1), intermediate (5 Mâ1sâ1 to 20 Mâ1sâ1), and fast (>50 Mâ1sâ1) rates of modification are colored in blue, orange, and red respectively. The pore lining residue A286 is seen as being the most accessible (red), whereas residues with the slowest rates of modification (blue) turned out to be located either inside (T278) or opposite to the pore lumen (V285). B also illustrates that all the residues from A283 to A286 are accessible to MTSET irrespective of their orientation relative to the pore central axis. (D) Cross-section area for residues 264â292 computed for the open and closed IKCa structures. Z refers to the channel axial distance with Z = 0 at V275. The closed channel structure predicts a central cavity extending from residues 272â278. The cross-section area for residues spanning the 282â286 region appeared under these conditions too small to accommodate an MTSET molecule of 5.8 Ã
diameter assuming a rigid structure for IKCa. A flexible structure extending below V282 can, however, account for the SCAM results.
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