Genetic Analysis of the Mammalian K+ Channel β Subunit Kvβ2 (Kcnab2)

Ken Mccormack,Jolien X Connor,Lei Zhou,Ling Ling Ho,Barry Ganetzky,Shing-Yan Chiu,Albee Messing

doi:10.1074/jbc.m111465200

Ken Mccormack, Jolien X Connor + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m111465200

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Abstract

Kvbeta2 binds to K(+) channel alpha subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily. Proposed functions for this protein in vivo include a chaperone-like role in Kv1 alpha subunit biogenesis and catalytic activity as an AKR oxidoreductase. To investigate the in vivo function of Kvbeta2, Kvbeta2-null and point mutant (Y90F) mice were generated through gene targeting in embryonic stem cells. In Kvbeta2-null mice, Kv1.1 and Kv1.2 localize normally in cerebellar basket cell terminals and the juxtaparanodal region of myelinated nerves. Moreover, normal glycosylation patterns are observed for Kv1.1 and Kv1.2 in whole brain lysates. Thus, loss of the chaperone-like activity does not appear to account for the phenotype of Kvbeta2-null mice, which include reduced life spans, occasional seizures, and cold swim-induced tremors similar to that observed in Kv1.1-null mice. Mice expressing Kvbeta2, mutated at a site (Y90F) that abolishes AKR-like catalytic activity in other family members, have no overt phenotype. We conclude that Kvbeta2 contributes to regulation of excitability in vivo, although not directly through either chaperone-like or typical AKR catalytic activity. Rather, Kvbeta2 relies upon as yet unidentified mechanisms in the regulation of K(+) channel and/or oxidoreductive functions.

Highlights

Kv␤2 binds to K؉ channel ␣ subunits from at least two different families (Kv1 and Kv4) and is a member of the aldo-ketoreductase (AKR) superfamily
Kv␤2 binds NADPH with an affinity comparable with that found for other AKR enzymes but with only 10-fold lower affinity for NADH; NADPH and NADH bind more tightly than NADPϩ and NADϩ, respectively, indicating that Kv␤2 would be more likely to function as a reductase than an oxidase [18]; cofactor binding is not a prerequisite for multimer formation
We describe the generation of Kv␤2-null and Kv␤2-Y90F mutant mice to examine the in vivo function of Kv␤2, a major auxiliary subunit of Kv1␣ channels in the mammalian nervous system

Summary

EXPERIMENTAL PROCEDURES

The final targeting vector consisted of a contiguous 5.7-kb BamHI/BglII genomic fragment interrupted at the XhoI site with the floxed PGK-neo, with a 1.3-kb 5Ј arm of homology containing the point mutation and the 4.4-kb 3Ј arm of homology (see Fig. 1B). For the ES cells transfected with the Kv␤2-Y90F targeting vector, DNA was digested with BamHI (for the 3Ј probe) or AccI and AccI/SacII (for the neo probe). Excision of the PGKneo insert was induced through transient transfection of CMV-Cre recombinase (pBS185; Invitrogen), and recombinant clones were identified through Southern blot analysis utilizing the same 0.8-kb AvrII/SalI 3Ј probe by comparison of BamHI versus BamHI/SacII digestion as well as by PCR using primers corresponding to genomic DNA sequences 5Ј of exon 5 and 3Ј of the XhoI site in intron 5. The Kv␤2-null and Y90F point mutant mice described in this report have been given the strain designations Kcnab2tm1Mes and Kcnab2tm2Mes, respectively

Survival Analysis

Myoclonus Score Following Cold Water Swim

RESULTS

TABLE I Myoclonus scores

DISCUSSION

Chiu and Albee Messing