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Tonic PKA activity regulates the expression of native SK channels in living neurons

Krithika Abiraman (University of Connecticut), Anastasios Tzingounis (University of Connecticut), George Lykotrafitis (University of Connecticut)

Mechanics and Physics of Biological Cells

Mon 10:45 - 12:15

Barus-Holley 166

Modulation of small conductance calcium activated potassium channels (SK) affects neuron plasticity, learning and memory. We have previously shown that forskolin (FSK), a protein kinase A (PKA) activator, decreases the expression of SK channels. In the present study, we demonstrate that FSK acts via the PKA pathway and that there is tonic PKA which regulates the surface expression of these channels. This tonic PKA also caused differential clustering of the two subtypes of the SK2 channel- SK2-S and SK2-L, suggesting potential role of tonic PKA in nanoclustering of SK channels. The surface expression of SK channels in living neurons was determined by combining single molecule AFM and toxin pharmacology. The AFM tip was functionalized with the bee venom toxin apamin, a specific high-affinity (8-10 pM) SK channel blocker which binds to the channel with 1:1 stoichiometry. The direct interactions between the apamin-functionalized probe and cell surface SK channels were recorded as force-distance curves. The sensitivity of apamin functionalized tips to SK channels was determined by carrying out experiments with functionalized probe on untransfected HEK293T cells and non functionalized probes on SK2-S-transfected cells. The specificity of apamin-functionalized probe to the SK2- transfected cells was confirmed by performing experiments using HEK cells transfected with SK1 subtype. Binding forces between apamin and HEK cells transfected with SK2-S and SK2-L isoform were obtained (26pN and 35pN respectively). However, incubation of the cells with the PKA inhibitor KT 5720 (1 µM for 30 minutes), caused a drastic increase in the mean binding forces compared to baseline and the histogram of the forces was best fit as a mixture of two Gaussian distributions (SK2-S-3µg, µ=34pN and µ=51pN ; SK2-L-3µg, µ=46 pN and µ=62 pN) . These results suggest that there is tonic PKA activity which regulates the expression of SK2 channels.