Recognition force microscopy/spectroscopy of ion channels: applications to the skeletal muscle Ca2+ release channel (RYR1).
The skeletal muscle Ca2+ release channel (ryanodine receptor 1, RYR1) plays an important role in the excitation-contraction coupling process. We purified ryanodine receptor type 1 from rabbit white muscle and adsorbed it to mica sheets with the cytoplasmic side facing up. Single receptors of uniformly distributed size and shape of 10-12 nm height and 40-50 nm width, and occasionally some aggregates were seen in contact mode AFM images. These immobilized RYR1 were specifically recognized by rabbit anti-RYR1 (antibody#8) with at least 30% efficiency, as measured by an enzyme immunoassay with goat-anti-rabbit. Single specific antibody-antigen recognition events were detected with AFM tips to which an antibody#8 was tethered. In linear scans, the occurrence of antibody-antigen binding showed significant lateral dependence, which allowed for the localization of binding sites with nm resolution. Variation of the loading rate in force spectroscopy experiments revealed a logarithmic dependence of the unbinding forces, ranging from 42 to 73 pN. From this dependence, a bond width of the binding pocket of L = 0.2 nm and a kinetic off-rate of koff = 12.7s(-1) was determined.
Kada
G
G
Institute for Biophysics, University of Linz, Austria.
Blayney
L
L
Jeyakumar
L H
LH
Kienberger
F
F
Pastushenko
V P
VP
Fleischer
S
S
Schindler
H
H
Lai
F A
FA
Hinterdorfer
P
P
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D007124:Immunoenzyme Techniques; D008024:Ligands; D018625:Microscopy, Atomic Force / Q000379:methods*; D018482:Muscle, Skeletal / Q000502:physiology*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000302:isolation & purification / Q000502:physiology*
D000906:Antibodies; D008024:Ligands; D019837:Ryanodine Receptor Calcium Release Channel