Photolabile derivatives of 125I-apamin: defining the structural criteria required for labeling high and low molecular mass polypeptides associated with small conductance Ca(2+)-activated K+ channels.
The structure of apamin-sensitive Ca(2+)-activated K+ channels has been investigated using high-affinity, photolabile azidoaryl derivatives of 125I-[alpha-formyl-Cys1]apamin and 125I-[epsilon-formyl-Lys4]-apamin. Labeling patterns suggest that similar structural constraints are required for labeling analogous polypeptides associated with distinct channel subtypes. When photoprobes are coupled at the epsilon-amino-Lys4 position of apamin, comparable low molecular mass (approximately 30 kDa) polypeptides are efficiently labeled on either brain or liver plasma membranes, irrespective of the structure of the photoprobe. However, when photoprobes are coupled at the alpha-amino-Cys1 position of apamin, the pattern of labeling on both brain and liver plasma membranes varies, depending upon the length of the spacer arm incorporated into the photoprobe. Spacer arms of approximately 8-9 A efficiently label only high molecular mass polypeptides (86, 59 kDa), accompanied by weak, variable labeling of a 44-kDa component. A shorter spacer arm (5.7 A) results in feeble labeling of 86- and 59-kDa polypeptides and barely detectable labeling of 44- and approximately 30-kDa polypeptides. In contrast, a long spacer arm (12.8 A) efficiently labels only approximately 30-kDa polypeptides. These findings point to close similarities in the topography of the 125I-apamin binding site present on pharmacologically distinct subtypes of apamin-sensitive Ca2+-activated K+ channels and indicates that heterooligomeric association of high and low molecular mass polypeptide subunits may be a general structural feature of members belonging to this family of K+ channels.
Wadsworth
J D
JD
Department of Paediatrics and Neonatal Medicine, Royal Postgraduate Medical School, Hammersmith Hospital, London, U.K.
Doorty
K B
KB
Ganellin
C R
CR
Strong
P N
PN
D000818:Animals; D001030:Apamin / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism*; D001345:Autoradiography; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D007700:Kinetics; D008099:Liver / Q000378:metabolism; D046911:Macromolecular Substances; D010782:Photolysis; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D011817:Rabbits; D011869:Radioligand Assay; D051381:Rats; D013570:Synaptic Membranes / Q000378:metabolism
D003432:Cross-Linking Reagents; D007457:Iodine Radioisotopes; D046911:Macromolecular Substances; D015221:Potassium Channels; D001030:Apamin