SK Channel Distribution on Neuronal Axons Revealed by Single Molecule Atomic Force Microscopy

Abstract

Introduction: The axon initial segment (AIS) is a specialized cellular compartment where action potentials (APs) initiate. Calcium-activated small conductance potassium (SK) channels reduce excitability by mediating the afterhyperpolarizing (AHP) current and thus modulate the intervals between spikes during a burst of action potentials. SK channels have a polarized distribution in neurons with high density in dendrites and lower density in the soma. However, it is not clear if and which SK channels are expressed on the axon and their spatial distribution. Here, we employ single molecule atomic force microscopy (AFM) combined with natural toxins to determine the localization of SK channels on neuronal axons.Methods and Results: We have previously demonstrated that integration of single molecule AFM and toxin pharmacology allows for high resolution mapping of native SK2 channels on soma and dendrites of living neurons. AFM tip functionalized with SK channel blocker-apamin, was used to detect the distribution of SK channels by measuring the unbinding forces between apamin and SK channels. The axon was identified by transfecting rat pyramidal neurons with tau-gfp which localizes at the axon. Apamin-functionalized AFM tips were used to probe 1µm∧2 scan area on the axon of transfected pyramidal neurons. We detected unbinding forces with a mean of µ=20± 9pN and a surface density of 5.9% (n=8). To determine the specificity of the detected unbinding forces, the experiments were repeated with neurons pretreated with apamin. Along with a lower frequency of unbinding events, we found that in neurons pretreated with apamin, there was a decrease in the unbinding forces. The results above indicate that apamin sensitive SK channels reside on the axon of pyramidal neurons.Conclusion: Employing single molecule AFM, we have revealed that SK channels reside on axonal surfaces of neurons.