Measuring the influence of the BKCa {beta}1 subunit on Ca2+ binding to the BKCa channel.
The large-conductance Ca(2+)-activated potassium (BK(Ca)) channel of smooth muscle is unusually sensitive to Ca(2+) as compared with the BK(Ca) channels of brain and skeletal muscle. This is due to the tissue-specific expression of the BK(Ca) auxiliary subunit beta1, whose presence dramatically increases both the potency and efficacy of Ca(2+) in promoting channel opening. beta1 contains no Ca(2+) binding sites of its own, and thus the mechanism by which it increases the BK(Ca) channel's Ca(2+) sensitivity has been of some interest. Previously, we demonstrated that beta1 stabilizes voltage sensor activation, such that activation occurs at more negative voltages with beta1 present. This decreases the work that Ca(2+) must do to open the channel and thereby increases the channel's apparent Ca(2+) affinity without altering the real affinities of the channel's Ca(2+) binding sites. To explain the full effect of beta1 on the channel's Ca(2+) sensitivity, however, we also proposed that there must be effects of beta1 on Ca(2+) binding. Here, to test this hypothesis, we have used high-resolution Ca(2+) dose-response curves together with binding site-specific mutations to measure the effects of beta1 on Ca(2+) binding. We find that coexpression of beta1 alters Ca(2+) binding at both of the BK(Ca) channel's two types of high-affinity Ca(2+) binding sites, primarily increasing the affinity of the RCK1 sites when the channel is open and decreasing the affinity of the Ca(2+) bowl sites when the channel is closed. Both of these modifications increase the difference in affinity between open and closed, such that Ca(2+) binding at either site has a larger effect on channel opening when beta1 is present.
Sweet
Tara-Beth
TB
Molecular Cardiology Research Institute, Tufts Medical Center, Department of Neuroscience, Tufts University School of Medicine, Boston, MA 02111, USA.
Cox
Daniel H
DH
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002461:Cell Line, Transformed; D004735:Energy Transfer / Q000502:physiology; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000502:physiology; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D021122:Protein Subunits; D002118:Calcium