Dataset Information

Homology modeling identifies C-terminal residues that contribute to the Ca2+ sensitivity of a BKCa channel.

ABSTRACT: Activation of BK(Ca) channels by direct Ca(2+) binding and membrane depolarization occur via independent and additive molecular processes. The "calcium bowl" domain is critically involved in Ca(2+)-dependent gating, and we have hypothesized that a sequence within this domain may resemble an EF hand motif. Using a homology modeling strategy, it was observed that a single Ca(2+) ion may be coordinated by the oxygen-containing side chains of residues within the calcium bowl (i.e., (912)ELVNDTNVQFLD(923)). To examine these predictions directly, alanine-substituted BK(Ca) channel mutants were expressed in HEK 293 cells and the voltage and Ca(2+) dependence of macroscopic currents were examined in inside-out membrane patches. Over the range of 1-10 microM free Ca(2+), single point mutations (i.e., E912A and D923A) produced rightward shifts in the steady-state conductance-voltage relations, whereas the mutants N918A or Q920A had no effect on Ca(2+)-dependent gating. The double mutant E912A/D923A displayed a synergistic shift in Ca(2+)-sensitive gating, as well as altered kinetics of current activation/deactivation. In the presence of 1, 10, and 80 mM cytosolic Mg(2+), this double mutation significantly reduced the Ca(2+)-induced free energy change associated with channel activation. Finally, mutations that altered sensitivity of the holo-channel to Ca(2+) also reduced direct (45)Ca binding to the calcium bowl domain expressed as a bacterial fusion protein. These findings, along with other recent data, are considered in the context of the calcium bowl's high affinity Ca(2+) sensor and the known properties of EF hands.

SUBMITTER: Sheng JZ

PROVIDER: S-EPMC1366805 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Homology modeling identifies C-terminal residues that contribute to the Ca2+ sensitivity of a BKCa channel.

Sheng Jian-Zhong JZ Weljie Aalim A Sy Lusia L Ling Shizhang S Vogel Hans J HJ Braun Andrew P AP

Biophysical journal 20050812 5

Activation of BK(Ca) channels by direct Ca(2+) binding and membrane depolarization occur via independent and additive molecular processes. The "calcium bowl" domain is critically involved in Ca(2+)-dependent gating, and we have hypothesized that a sequence within this domain may resemble an EF hand motif. Using a homology modeling strategy, it was observed that a single Ca(2+) ion may be coordinated by the oxygen-containing side chains of residues within the calcium bowl (i.e., (912)ELVNDTNVQFLD ...[more]

PMID: 16100257

Similar Datasets

Project description:Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming ?-subunit. BKCa variants in the intracellular C terminus of the ?-subunit, and their relative expression to total ?-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two ?-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9±4.2% of total ?-BKCa) compared with cerebral vessels (16.5±0.9%). Further, a low level of BKCa SS4 ?-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of ?1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of ?-BKCa subunits, whereas in the absence of ?-BKCa, a significant proportion of ?1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of ?-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total ?-BKCa expression, regardless of differences in splice variant expression.

Dataset Information

Homology modeling identifies C-terminal residues that contribute to the Ca2+ sensitivity of a BKCa channel.

Publications

Homology modeling identifies C-terminal residues that contribute to the Ca2+ sensitivity of a BKCa channel.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure