Cytoplasmic amino and carboxyl domains form a wide intracellular vestibule in an inwardly rectifying potassium channel.
We have studied the structural components and architecture of the intracellular vestibule of a strongly rectifying channel (Kir2.1) expressed in Xenopus oocytes. Putative vestibule-lining residues were identified by systematically examining covalent modification by sulfhydryl-specific reagents of cysteine residues engineered into two cytoplasmic regions. In a stretch of 33 amino acids in the amino terminus (from C54 to V86) and 22 amino acids in the carboxyl terminus (from R213 to S234), 15 and 11 residues, respectively, were found to be accessible to methanethiosulfonate ethylammonium (MTSEA) or methanethiosulfonate ethyltrimethylammonium (MTSET) and presumably project into the aqueous intracellular vestibule. The pattern of accessibility suggests that both stretches may adopt an extended loop structure. To explore the physical dimension of the intracellular vestibule, we covalently linked a constrained number (one to four) of positively charged moieties of different sizes to the E224 position and found that this vestibule region is sufficiently wide to accommodate four modifying groups with dimensions of 12 A x 10 A x 6 A. These results suggest that regions in both the amino and carboxyl domains of Kir2.1 channel form a long and wide intracellular vestibule that protrudes beyond the membrane into the cytoplasm.
Lu
T
T
Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Zhu
Y G
YG
Yang
J
J
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D003593:Cytoplasm / Q000737:chemistry; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015221:Potassium Channels / Q000737:chemistry* / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying; D013329:Structure-Activity Relationship; D014981:Xenopus
D007202:Indicators and Reagents; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D003545:Cysteine