Antibodies specific for distinct Kv subunits unveil a heterooligomeric basis for subtypes of .alpha.-dendrotoxin-sensitive potassium channels in bovine brain

Abstract

The authentic subunit compositions of neuronal K+ channels purified from bovine brain were analyzed using a monoclonal antibody (mAb 5), reactive exclusively with the Kv1.2 subunit of the latter and polyclonal antibodies specific for fusion proteins containing C-terminal regions of four mammalian Kv proteins. Western blotting of the K+ channels isolated from several brain regions, employing the selective blocker alpha-dendrotoxin (alpha-DTX), revealed the presence in each of four different Kvs. Variable amounts of Kv1.1 and 1.4 subunits were observed in the K+ channels purified from cerebellum, corpus striatum, hippocampus, cerebral cortex, and brain stem; on the other hand, contents of Kv1.6 and 1.2 subunits appeared uniform throughout. Each Kv-specific antibody precipitated a different proportion (anti-Kv1.2 > 1.1 >> 1.6 > 1.4) of the channels detectable with radioiodinated alpha-DTX in every brain region, consistent with a widespread distribution of these oligomeric subtypes. Such heterooligomeric combinations were further documented by the lack of additivity upon their precipitation with a mixture of antibodies to Kv1.1 and Kv1.2; moreover, cross-blotting of the multimers precipitated by mAb 5 showed that they contain all four Kv proteins. Collectively, these findings demonstrate that subtypes of alpha-DTX-susceptible K+ channels are prevalent throughout mammalian brain which are composed of different Kv proteins assembled in complexes, shown previously to also contain auxiliary beta-subunits [Parcej, D. N., Scott, V. E. S., & Dolly, J.O. (1992) Biochemistry 31, 11084-11088].

Do voltage‐gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

“…This was checked by immunoblotting 150 gg of soluble protein extracts with affinity-purified polyclonal anti-RCK1 antibodies [18], 48 h after injection of the oocytes. A representative Western blot containing soluble protein extracts of defolliculated native (non-injected), RCKl-injected and tandem-injected oocytes is shown in Fig.…”

“…Translational efficacy of heterologous RCK1 and tandem proteins in Xenopus laevis oocytes was checked by immunoblotting 150 lag of soluble protein extract with affinity-purified polyclonal anti-RCK1 antibodies [18]. 48 h after injection, defolliculated native and injected oocytes were lysed in a hypotonic buffer (see Section 2.5).…”

Diverse Expression and Distribution ofShakerPotassium Channels during the Development of theDrosophilaNervous System

“…Abbreviations are as in Figure 2. eration of Kch diversity (Isacoff et al, 1990;Ruppersberg et al, 1990). However, there is compelling evidence in the literature to support both the colocalization and the segregation of different Kch subunits in mammalian brain (Sheng et al, 1992(Sheng et al, , 1994Wang et al, 1993Wang et al, , 1994Scott et al, 1994;Mi et al, 1995;Weiser et al, 1995). PCR , in situ hybridization (TsengCrank et al, 1991), and reporter gene engineering (Mottes and Iverson, 1995) experiments indicate that Sh splice variants can be expressed differentially in some areas of the nervous tissue, retina, and muscle of Drosophila.…”

Alternative Splicing in the Pore-Forming Region ofshakerPotassium Channels

“…Shak er channels are subject to post-translational modifications, including phosphorylation (Rehm et al, 1989;Moran et al, 1991;Drain et al, 1992;Isacoff et al, 1992;Levitan, 1994;Scott et al, 1994;Holmes et al, 1996) and glycosylation (Rehm et al, 1989;Scott et al, 1990Scott et al, , 1994. As indicated in Figure 2, Panulirus shaker contains two putative N-linked glycosylation sites (Hubbard and Ivatt, 1981;Kornfeld and Kornfeld, 1985), four putative protein kinase C phosphorylation sites (K ishimoto et al, 1985;Woodgett et al, 1986), 10 putative casein kinase II phosphorylation sites (Kuenzel et al, 1987;Aitken, 1990), and a single putative cAMPdependent protein kinase site (Krebs and Beavo, 1979;Aitken, 1990).…”