Tonic PKA Activity Regulates SK Channel Nanoclustering and Somatodendritic Distribution

Abstract

Small-conductance calcium-activated potassium (SK) channels mediate a potassium conductance in the brain and are involved in synaptic plasticity, learning, and memory. SK channels show a distinct subcellular localization that is crucial for their neuronal functions. However, the mechanisms that control this spatial distribution are unknown. We imaged SK channels labeled with fluorophore-tagged apamin and monitored SK channel nanoclustering at the single molecule level by combining atomic force microscopy and toxin (i.e., apamin) pharmacology. Using these two complementary approaches, we found that native SK channel distribution in pyramidal neurons, across the somatodendritic domain, depends on ongoing cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) levels, strongly limiting SK channel expression at the pyramidal neuron soma. Furthermore, tonic cAMP-PKA levels also controlled whether SK channels were expressed in nanodomains as single entities or as a group of multiple channels. Our study reveals a new level of regulation of SK channels by cAMP-PKA and suggests that ion channel topography and nanoclustering might be under the control of second messenger cascades.

K Ca 2 channel localization and regulation in the axon initial segment

“…5 A ; KT5720: μ = 20 ± 5.5 pN, surface density = 9.14 ± 1.02%, P < 0.01; Fig. 5 B ; Rp‐cAMPS: μ = 20 ± 5.0 pN, surface density = 10.09 ± 1.41%, F (2, 20) = 12.27, P < 0.01]; however, we did not observe any induction of nanoclustering of K Ca 2 channels, which we had previously found (12) and confirmed again here with K Ca 2 channels in the soma [Fig. 5 D; KT5720: μ 1 = 38 ± 5.5 pN, μ 2 = 55 ± 7 pN, μ 3 = 70 ± 8 pN, surface density = 14.01 ± 1.1%, P < 0.001; Fig.…”

“…We found that K Ca 2.3 is the main K Ca 2 channel subtype that is present along the AIS and is uniformly distributed in the proximal and distal AIS. We also demonstrated that basal cAMP‐PKA activity regulates the surface expression of axonal K Ca 2 channels; however, axonal K Ca 2 channels do not appear in nanoclusters in the absence of cAMP‐PKA activity, unlike their somatodendritic K Ca 2 channel counterparts (12). Furthermore, elevated PKA levels continually retrieve K Ca 2 channels from the axonal membrane in a dynamin‐independent manner.…”

“…This could explain the low unbinding forces (μ =226 8 pN) that were obtained between apamin and axonal K Ca 2 channels when we probed neurons with an apamin‐functionalized AFM cantilever. These unbinding forces were lower than those between K Ca 2.2 (short isoform, K Ca 2.2‐S: μ =286 5 pN) and an apamin‐functionalized cantilever, but higher than those between rat K Ca 2.1, which is insensitive to apamin (29), and an apamin‐functionalized cantilever (μ =136 1 pN) (12). The presence of the K Ca 2.3 channel subtype might also explain why axonal K Ca 2 channels did not exhibit any nanoclustering when cAMP‐PKA was inhibited.…”

“…The role of cAMP‐PKA in the surface expression of K Ca 2 channels is well established. In addition to surface expression and spatial distribution, cAMP‐PKA also affects the nanoclustering of K Ca 2 channels in the soma and dendrites (12). Axonal K Ca 2 channels exhibited an increase in surface expression; however, they did not seem to cluster when cAMP‐PKA was inhibited.…”

“…apamin-functionalized cantilever (m = 13 6 1 pN) (12). The presence of the K Ca 2.3 channel subtype might also explain why axonal K Ca 2 channels did not exhibit any nanoclustering when cAMP-PKA was inhibited.…”