Mapping the binding site on ankyrin for the voltage-dependent sodium channel from brain.
Erythrocyte ankyrin is a member of a family of proteins that mediate the linkage between membrane proteins and the underlying spectrin-actin-based cytoskeleton. Ankyrin has been shown to interact with a variety of integral membrane proteins such as the anion exchanger, the Na+K(+)-ATPase, and the voltage-dependent sodium channel (NaCh) in brain. To understand how ankyrin interacts with these proteins and maintains its specificity and high affinity for the voltage-dependent NaCh, we have mapped the binding site on ankyrin for the NaCh by examining the binding of purified ankyrin subfragments, prepared by proteolytic cleavage, to the purified rat brain NaCh incorporated into liposomes. 125I-Labeled ankyrin and the radiolabeled 89- and 43-kDa fragments of ankyrin bind to the NaCh with high affinities and with Kd values of 34, 22, and 63 nM, respectively, and have stoichiometries of approximately 1 mol/mol NaCh. The 72-kDa spectrin binding domain is inactive and does not bind to the NaCh. Dissection of ankyrin reveals that the 43-kDa domain retains all the binding properties of native ankyrin to the NaCh. Analysis of the primary structure reveals that the NaCh binding site is confined to a domain of ankyrin consisting entirely of the 11 terminal 33-amino acid repeats and is distinct from the ankyrin domains that interact with spectrin and the Na+K(+)-ATPase.
Srinivasan
Y
Y
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.
Lewallen
M
M
Angelides
K J
KJ
D000818:Animals; D017487:Ankyrins; D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013049:Spectrin / Q000378:metabolism
D017487:Ankyrins; D001798:Blood Proteins; D007457:Iodine Radioisotopes; D008081:Liposomes; D008565:Membrane Proteins; D015222:Sodium Channels; D013049:Spectrin; D000254:Sodium-Potassium-Exchanging ATPase