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This Article Full Text Full Text (PDF) All Versions of this Article: 553/1/101    most recent jphysiol.2003.052845v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chang, H.-K. Articles by Shieh, R.-C. Search for Related Content PubMed PubMed Citation Articles by Chang, H.-K. Articles by Shieh, R.-C.

The effects of spermine on the accessibility of residues in the M2 segment of Kir2.1 channels expressed in Xenopus oocytes

Hsueh-Kai Chang, Shih-Hao Yeh and Ru-Chi Shieh

We examined the effects of spermine binding to aspartate at site 172 on the accessibility of internal trimethylammonioethylmethane thiosulphonate (MTSET) to substituted cysteines within the pore of a Kir2.1 channel. Spermine prevented MTSET modification in Q164C and G168C mutants, indicating that sites 164 and 168 are located externally to the spermine binding site. The rates of MTSET modification were significantly reduced by spermine in I176C mutants, indicating that site 176 is located internally to D172 and that the bound spermine hinders the reaction of MTSET with cysteine at site 176. Spermidine, putrescine and Mg²⁺ also decreased MTSET modification at site 176. The order of effect is putrescine > spermidine spermine Mg²⁺. To account for the electrostatic and physical repulsion between MTSET and polyamines, possible locations of polyamines in the pore are discussed. In D172C mutants, the spermine that bound to sites 224 and 299 completely inhibited channels at +40 mV, yet MTSET remained accessible to site 172. In addition, in the D172C mutant, spermine did not affect the exit rate of Ba²⁺ bound to the threonine at the site 141. These results indicate that spermine bound at the cytoplasmic pore induces channel closure at positions 141-172. The effects of spermine on the accessibility of amino acids in the pore may shed light on the structural and functional relationships of the Kir2.1 channels during inward rectification.

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