Dataset Information

Protein-protein interaction between cPLA2 and splice variants of alpha-subunit of BK channels.

ABSTRACT: Altering the splice variant composition of large-conductance Ca(2+)-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca(2+) and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK alpha-subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A(2) (cPLA(2)). Here we used the Mammalian Matchmaker two-hybrid assay and two BK alpha-subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA(2) associates with one construct [rSlo(27)] and not the other. We hypothesized that differential association of cPLA(2) might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA(2) is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA(2) to the COOH terminus of the rSlo(0) alpha-subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.

SUBMITTER: Li J

PROVIDER: S-EPMC3774341 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Protein-protein interaction between cPLA2 and splice variants of alpha-subunit of BK channels.

Li Juan J Al-Khalili Otor O Ramosevac Semra S Eaton Douglas C DC Denson Donald D DD

American journal of physiology. Cell physiology 20091125 2

Altering the splice variant composition of large-conductance Ca(2+)-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca(2+) and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest differenc ...[more]

PMID: 19940072

Similar Datasets

Project description:Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.

Dataset Information

Protein-protein interaction between cPLA2 and splice variants of alpha-subunit of BK channels.

Publications

Protein-protein interaction between cPLA2 and splice variants of alpha-subunit of BK channels.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets