title: Recognition force microscopy/spectroscopy of ion channels: applications to the skeletal muscle Ca2+ release channel (RYR1).
abstract: 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.
authors.affiliation: Institute for Biophysics, University of Linz, Austria.
authors.forename: G
authors.initials: G
authors.lastname: Kada
authors.forename: L
authors.initials: L
authors.lastname: Blayney
authors.forename: L H
authors.initials: LH
authors.lastname: Jeyakumar
authors.forename: F
authors.initials: F
authors.lastname: Kienberger
authors.forename: V P
authors.initials: VP
authors.lastname: Pastushenko
authors.forename: S
authors.initials: S
authors.lastname: Fleischer
authors.forename: H
authors.initials: H
authors.lastname: Schindler
authors.forename: F A
authors.initials: FA
authors.lastname: Lai
authors.forename: P
authors.initials: P
authors.lastname: Hinterdorfer
mesh_terms: 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*
chemical_list: D000906:Antibodies; D008024:Ligands; D019837:Ryanodine Receptor Calcium Release Channel