Abstract
The Kv2.1 voltage-gated K(+) channel is found both freely diffusing over the plasma membrane and concentrated in micron-sized clusters localized to the soma, proximal dendrites, and axon initial segment of hippocampal neurons. In transfected HEK cells, Kv2.1 channels within cluster microdomains are nonconducting. Using total internal reflection fluorescence microscopy, the number of GFP-tagged Kv2.1 channels on the HEK cell surface was compared with K(+) channel conductance measured by whole-cell voltage clamp of the same cell. This approach indicated that, as channel density increases, nonclustered channels cease conducting. At the highest density observed, only 4% of all channels were conducting. Mutant Kv2.1 channels that fail to cluster also possessed the nonconducting state with 17% conducting K(+) at higher surface densities. The nonconducting state was specific to Kv2.1 as Kv1.4 was always conducting regardless of the cell-surface expression level. Anti-Kv2.1 immunofluorescence intensity, standardized to Kv2.1 surface density in transfected HEK cells, was used to determine the expression levels of endogenous Kv2.1 in cultured rat hippocampal neurons. Endogenous Kv2.1 levels were compared with the number of conducting channels determined by whole-cell voltage clamp. Only 13 and 27% of the endogenous Kv2.1 was conducting in neurons cultured for 14 and 20 d, respectively. Together, these data indicate that the nonconducting state depends primarily on surface density as opposed to cluster location and that this nonconducting state also exists for native Kv2.1 found in cultured hippocampal neurons. This excess of Kv2.1 protein relative to K(+) conductance further supports a nonconducting role for Kv2.1 in excitable tissues.