Unoprostone activation of BK (KCa1.1) channel splice variants.
This investigation was conducted to study the relationship between intracellular Ca(2+) and activation of large conductance Ca(2+)-activated K(+) (BK) currents by unoprostone, the first synthetic docosanoid. We used HEK293 cells stably transfected with two BK channel splice variants, one sensitive to unoprostone and the other insensitive. We examined the effects of unoprostone on channel activity in excised inside-out patches and cell-attached patches. The half-maximal stimulation of the sensitive BK channels by Ca(2+) was shifted from 3.4±0.017 nM to 0.81±.0058 nM in the presence of 10 nM unoprostone. There was no effect on insensitive channels even at unoprostone concentrations as high as 1000 nM. There was no effect of unoprostone on the voltage dependence of the BK channels. Changes in open probability and effects of Ca(2+) and unoprostone were best described by a synergistic binding model. These data would suggest that Ca(2+) and unoprostone were binding to sites close to one another on the channel protein and that unoprostone binding causes the affinity of the calcium binding site to increase. This idea is consistent with three dimensional models of the Ca(2+) binding site and a putative unoprostone binding domain. Our results have important implications for the clinical use of unoprostone to activate BK channels. Channel activation will be limited if intracellular Ca(2+) is not elevated.
College of Resources and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China.
Ling
L
Yu
Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine Atlanta, GA 30322, United States.
Amity F
AF
Eaton
Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine Atlanta, GA 30322, United States.
Qiang
Q
Yue
Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine Atlanta, GA 30322, United States.
Hui-Fang
HF
Bao
Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine Atlanta, GA 30322, United States.
He-Ping
HP
Ma
Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH 45267, USA.
John
J
Cuppoletti
Department of Physiology and the Center for Cell and Molecular Signaling, Emory University School of Medicine Atlanta, GA 30322, United States. Electronic address: deaton@emory.edu.
Douglas C
DC
Eaton
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015237:Dinoprost / Q000031:analogs & derivatives* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D057809:HEK293 Cells; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D018408:Patch-Clamp Techniques; D020033:Protein Isoforms / Q000235:genetics / Q000502:physiology; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014162:Transfection
C494842:Kcnma1 protein, rat; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D020033:Protein Isoforms; C072630:isopropyl unoprostone; D015237:Dinoprost; D002118:Calcium
BK channels; Ca(2+)-dependence; KCNMA1; Rescula®; Single channels; Unoprostone