Voltage-dependent conformational changes in human Ca(2+)- and voltage-activated K(+) channel, revealed by voltage-clamp fluorometry.
Large conductance voltage- and Ca(2+)-activated K(+) (BK(Ca)) channels regulate important physiological processes such as neurotransmitter release and vascular tone. BK(Ca) channels possess a voltage sensor mainly represented by the S4 transmembrane domain. Changes in membrane potential displace the voltage sensor, producing a conformational change that leads to channel opening. By site-directed fluorescent labeling of residues in the S3-S4 region and by using voltage clamp fluorometry, we have resolved the conformational changes the channel undergoes during activation. The voltage dependence of these conformational changes (detected as changes in fluorescence emission, fluorescence vs. voltage curves) always preceded the channel activation curves, as expected for protein rearrangements associated to the movement of the voltage sensor. Extremely slow conformational changes were revealed by fluorescent labeling of position 202, elicited by a mutual interaction of the fluorophore with the adjacent tryptophan 203.
Savalli
Nicoletta
N
Departments of Anesthesiology-Division of Molecular Medicine, Brain Research Institute, David Geffen School of Medicine at University of California, Los Angeles, CA 90095-7115, USA.
Kondratiev
Andrei
A
Toro
Ligia
L
Olcese
Riccardo
R
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D015640:Ion Channel Gating; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D015221:Potassium Channels; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014364:Tryptophan / Q000378:metabolism; D014982:Xenopus laevis
D005456:Fluorescent Dyes; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D015221:Potassium Channels; D020033:Protein Isoforms; D021122:Protein Subunits; D014364:Tryptophan; D002118:Calcium