Structural and Functional Characterization of Human Potassium Channel Subunit β1 (KCNA1B)

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Abstract

Voltage-activated Shaker-related potassium channels (Kv1) consist of α and β subunits. We have analysed the structure of the human KCNA1B (hKvβ1) gene. KCNA1B is >250 kb in size and encodes at least three Kvβ1 splice variants. The Kvβ1 open reading frame is divided into 14 exons. In contrast, genes coding for family members of KCNA (Kv1α) subunits are markedly smaller and have intronless open reading frames. The expression of Kv1α and Kvβ mRNA was compared in Northern blots of poly(A+) RNA isolated from various human brain tissues. The results suggest an intricate and cell-specific regulation of Kv1α and Kvβ mRNA synthesis such that distinct combinations of α and β subunits would occur in different nuclei of the brain. The splice variants hKvβ1.1 and hKvβ1.2 were functionally characterized in coexpression studies with hKv1.5α subunits in 293 cells. It is shown that they confer rapid inactivation on hKv1.5 channels with different potencies. This may be due to differences in their amino terminal sequences and/or inactivating domains. It is also shown that the amino terminal Kvβ1.1 and Kv1.4α inactivating domains compete with each other, probably for the binding to the same receptor site(s) on Kv1α-subunits. Copyright @ 1996 Elsevier Science Ltd

Section snippets

Isolation and analysis of cDNA clones

A human brain cDNA library in λgt10 (Clontech.) was hybridized in 6 × SET (15 mM Tris-HCl (pH 7.4), 75 mM NaCl, 0.5 mM EDTA), 1% SDS, 30% formamide, 5 × Denhardt's solution and 0.1 mg/ml denatured herring testis DNA at 42°C with a rat Kvβ1.1 cDNA probe (Rettig et al., 1994) labelled by random priming (Feinberg and Vogelstein, 1983) with (α- 32P)dCTP. Filters were washed in 1 × SET, 0.1% SDS at 50°C. Exposure to X-ray films (Kodak XAR5) was overnight with an amplifying screen (Cronex Hi+,

Characterization of the Kvβ1 gene

The characteristics of the derived Kvβ1 protein sequences Kvβ1.1, Kvβ1.2, Kvβ1.3 (Rettig et al., 1994; England et al. (1995a), England et al. (1995b); McCormack et al., 1995; Majumder et al., 1995; Morales et al., 1995) suggested that they are splice variants produced from a common Kvβ1 gene. This was confirmed by genomic DNA sequence analysis and by genomic mapping. The Kvβ1 (KCNA1B) gene has been mapped by hybridizing Kvβ1.2 probes to somatic cell hybrid panels to chromosome 3 (England et

DISCUSSION

Kv channels purified from rat (Rehm and Lazdunski, 1988) and bovine brain (Parcej and Dolly, 1989) are heterooligomeric complexes consisting of two different types of subunits: membrane-bound pore-forming α subunits and the smaller, auxiliary β subunits. They appear to be tightly associated with cytoplasmic, amino terminal domains of the Kv1α subunits (Sewing et al., 1996; Yu et al., 1996). Thus, Kv channels may form α4β4 heteromultimers in vivo (Parcej et al., 1992). It may be hypothesized

Acknowledgements

The work in this paper was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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