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Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channel.

Simoes M, Garneau L, Klein H, Banderali U, Hobeila F, Roux B, Parent L, Sauvé R - J. Gen. Physiol. (2002)

Bottom Line: In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore.However, the pore dimensions derived for the A283-A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants.Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone.

View Article: PubMed Central - PubMed

Affiliation: Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7.

ABSTRACT
Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K(+) channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca(2+) conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca(2+)-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283-A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.

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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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fig7: 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.

Mentions: MTSET protection experiments were also conducted in which the pore structure of the MTSET-activated A283C and A286C mutant channels were investigated using the hydrophilic blocking agent TEA as probe. The control inside-out recordings presented in Fig. 7 show that the internal application of 30 mM TEA caused a near total block (>79%) of the wild-type IKCa currents. These results confirmed previous observations reported on the effect of internal TEA on the IKCa channels present in human red blood cells (Dunn, 1998). In contrast, there was no TEA-dependent block of the A283C and A286C currents following stimulation by internal MTSET. In fact, the percentage of TEA-related inhibition decreased from >79% ± 4 (n = 6) for the wild-type IKCa channel to <6.5% ± 4.6 (n = 3) for the MTSET-stimulated A283C mutant (Fig. 7 D). These observations strongly suggest that, in addition to an important effect on channel gating, the binding of MTSET to the cysteine engineered at position 283 or 286 leads to a narrowing of the pore such that TEA can no longer reach its blocking site.


Cysteine mutagenesis and computer modeling of the S6 region of an intermediate conductance IKCa channel.

Simoes M, Garneau L, Klein H, Banderali U, Hobeila F, Roux B, Parent L, Sauvé R - J. Gen. Physiol. (2002)

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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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2311397&req=5

fig7: 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.
Mentions: MTSET protection experiments were also conducted in which the pore structure of the MTSET-activated A283C and A286C mutant channels were investigated using the hydrophilic blocking agent TEA as probe. The control inside-out recordings presented in Fig. 7 show that the internal application of 30 mM TEA caused a near total block (>79%) of the wild-type IKCa currents. These results confirmed previous observations reported on the effect of internal TEA on the IKCa channels present in human red blood cells (Dunn, 1998). In contrast, there was no TEA-dependent block of the A283C and A286C currents following stimulation by internal MTSET. In fact, the percentage of TEA-related inhibition decreased from >79% ± 4 (n = 6) for the wild-type IKCa channel to <6.5% ± 4.6 (n = 3) for the MTSET-stimulated A283C mutant (Fig. 7 D). These observations strongly suggest that, in addition to an important effect on channel gating, the binding of MTSET to the cysteine engineered at position 283 or 286 leads to a narrowing of the pore such that TEA can no longer reach its blocking site.

Bottom Line: In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore.However, the pore dimensions derived for the A283-A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants.Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone.

View Article: PubMed Central - PubMed

Affiliation: Département de Physiologie, Groupe de Recherche en Transport Membranaire Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada H3C 3J7.

ABSTRACT
Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K(+) channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca(2+) conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca(2+)-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET-stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283-A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.

Show MeSH
Related in: MedlinePlus