Unanticipated region- and cell-specific downregulation of individual KChIP auxiliary subunit isotypes in Kv4.2 knock-out mouse brain

Abstract

Kv4 family voltage-gated potassium channel alpha subunits and Kv channel-interacting protein (KChIP) and dipeptidyl aminopeptidase-like protein subunits comprise somatodendritic A-type channels in mammalian neurons. Recently, a mouse was generated with a targeted deletion of Kv4.2, a Kv4 alpha subunit expressed in many but not all mammalian brain neurons. Kv4.2-/- mice are grossly indistinguishable from wild-type (WT) littermates. Here we used immunohistochemistry to analyze expression of component Kv4 and KChIP subunits of A-type channels in WT and Kv4.2-/- brains. We found that the expression level, and cellular and subcellular distribution of the other prominent brain Kv4 family member Kv4.3, was indistinguishable between WT and Kv4.2-/- samples. However, we found unanticipated regional and cell-specific decreases in expression of KChIPs. The degree of altered expression of individual KChIP isoforms in different regions and neurons precisely follows the level of Kv4.2 normally found at those sites and presumably their extent of association of these KChIPs with Kv4.2. The dramatic effects of Kv4.2 deletion on KChIP expression suggest that, in addition to previously characterized effects of KChIPs on the functional properties, trafficking, and turnover rate of Kv4 channels, Kv4:KChIP association may confer reciprocal Kv4.2-dependent effects on KChIPs. The impact of Kv4.2 deletion on KChIP expression also supports the major role of KChIPs as auxiliary subunits of Kv4 channels.

Figure 1.

Expression of Kv4 and KChIP subunits in WT and Kv4.2^−/− hippocampi. WT and Kv4.2^−/− brain sections immunostained for potassium channel subunits as indicated. Scale bar, 200 μm.

Figure 2.

Quantitative analysis of Kv4 and KChIP subunits in WT and Kv4.2^−/− hippocampi. A, Fluorescence intensity measurements from a 60 × 60 μm area in stratum radiatum (rad) of CA1 adjacent to the pyramidal cell layer and in the molecular layer of dentate gyrus (dg) adjacent to the hippocampal fissure in five WT (white bars) and five Kv4.2^−/− (black bars) mice. We set the highest mean value for a given subunit at 100% and subsequently normalized the remaining three mean values. Values are mean ± SE. Asterisks mark values obtained from Kv4.2^−/− sections that are significantly different (p < 0.05; n = 5) from WT values. B, C, Higher-magnification views of WT and Kv4.2^−/− brain areas used for quantification of fluorescence intensity for KChIP2 (B) and KChIP3 (C). Scale bar, 100 μm.

Figure 3.

Expression of Kv4 and KChIP subunits in WT and Kv4.2^−/− cerebella and striata. A–C, Immunostaining of WT and Kv4.2^−/− sagittal cerebellar sections for Kv4.2 (A), KChIP1 (B), and KChIP3 (C) in red and Slo1 (A, B) and Kv1.2 (C) in green. Anterior, Left; posterior, right. Scale bar, 200 μm. D, Staining of KChIP2 in WT and Kv4.2^−/− striatum. Scale bar, 100 μm. E, Fluorescence intensity measurements from 50 × 50 μm square fields from the granule cell layer in anterior or posterior cerebellar cortex from five WT (white bars) and five Kv4.2^−/− (black bars) mice. The highest mean value for a given subunit (either WT or Kv4.2^−/−) was set at 100%, and the remaining three mean values were subsequently normalized. Values are mean ± SE. Asterisks are values from Kv4.2^−/− sections that are significantly different (p < 0.05; n = 5) from WT. ant, Anterior; post, posterior. F, Fluorescence intensity measurements from 400 × 400 μm square striatal fields from five WT (white bars) and four Kv4.2^−/− (black bars) mice, as described in D.