Recognition Force Microscospy of Single Ryanodine Receptor Type 1 (RyR1)

Abstract

The skeletal muscle Ca2+ release channel (ryanodine receptor, RyR1) plays an important role in the excitation-contraction coupling process. We purified ryanodine receptor type1 from rabbit white muscle [1] and adsorbed it to mica sheets from a solution of 14 μg/ml RyR1 in Na-Pipes buffer, containing 1 mM Ca2+ , at pH 7.1. Single receptors and occasionally some aggregates were observed in contact mode AFM images (Fig. 1). The surface coverage was about 50 %. Specific binding events were detected in force-distance cycles (Fig. 2) with AFM tips to which an antibody (Ab#8) was coupled via a PEG spacer [2], indicating that the cytoplasmic side of the RyR was oriented towards the aqueous phase. The binding probability (i.e. the ratio of observed binding events to all measured force distance cycles, 32% on average) showed significant lateral dependence and allowed for the localization of binding sites. The mean unbinding force at a vertical scan frequency of 1 Hz was 70 pN. Specificity of antibody recognition was proven by blocking the antigenic sites with free Ab#8 which reduced the binding probability to 5%. In addition, variation of the force velocity revealed a linear dependence of the unbinding force in a halflogarithmic plot (an increase in the loading rate leads to an increase in the unbinding force). The method shows potential for the epitope mapping of ligand- or antibody-binding sites on ion channels.