CaM1234 reduces enrichment of HA-KCNQ3/KCNQ2 channels at the axonal surface.

<p>(A) Surface expression of HA-KCNQ3/KCNQ2 channels in hippocampal neurons (DIV 7–8) cotransfected with empty vector (pcDNA3), CaM wild-type (WT) or Ca²⁺-insensitive mutant CaM (CaM1234). Endogenous and transfected CaM proteins were immunostained with anti-CaM antibodies (CaM, lower inverted images). HA-KCNQ3/KCNQ2 channels were enriched at the axonal surface in the presence of CaM WT or CaM1234. Arrows mark the main axon. Scale bars are 20 µm. (B) Overexpression of CaM WT or CaM1234 did not grossly affect neuronal polarity as indicated by immunostaining of the somatodendritic marker MAP2 and the AIS marker ankryin-G. Arrows indicate the AIS. Scale bars are 20 µm. (C) The surface “Axon/Dendrite” ratio was reduced by 33% by coexpression with CaM1234 (n = 21) compared to coexpression with CaM WT (n = 33) or empty vector pcDNA3 (n = 43). The surface “AIS/Axon” ratio of CD4-Q2C was increased by coexpression with CaM1234 compared to CaM WT but not pcDNA3. (D) Background subtracted, mean intensity of the CaM fluorescence in the soma of transfected and untransfected neurons (n = 23). (E) Background subtracted, mean intensity of surface and total (surface and intracellular) HA fluorescence in transfected and untransfected neurons. CaM1234 modestly decreased surface expression of HA-KCNQ3/KCNQ2 channels at the AIS and axon. (D, E) AU, arbitrary unit. Ave ± SEM (*p<0.05, **p<0.01, ***p<0.001). (F) Model by which apoCaM interaction with helices A and B of KCNQ2 and KCNQ3 is critical for trafficking of KCNQ2/KCNQ3 channels from the ER to the axonal surface.