Sabine Sewing

Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain.

A large variety of potassium channels is involved in regulating integration and transmission of electrical signals in the nervous system. Different types of neurons, therefore, require specific patterns of potassium channel subunit expression and specific regulation of subunit coassembly into heteromultimeric channels, as well as subunit‐specific sorting and segregation. This was investigated by studying in detail the expression of six different α‐subunits of voltage‐gated potassium channels in the rat hippocampus, cerebellum, olfactory bulb and spinal cord, combining in situ hybridization and immunocytochemistry. Specific polyclonal antibodies were prepared for five α‐subunits (KV1.1, KV1.2, KV1.3, KV1.4, KV1.6) of the Shaker‐related subfamily of rat Kv channels, which encode delayed‐rectifier type and rapidly inactivating A‐type potassium channels. Their distribution was compared to that of an A‐type potassium channel (KV3.4), belonging to the Shaw‐related subfamily of rat Kv channels. Our results show that these Kv channel α‐subunits are differentially expressed in rat brain neurons. We did not observe in various neurons a stereotypical distribution of Kv channel α‐subunits to dendritic and axonal compartments, but a complex differential subcellular subunit distribution. The different Kv channel subunits are targeted either to presynaptic or to postsynaptic domains, depending on neuronal cell type. Thus, distinct combinations of Kv1 α‐subunits are co‐localized in different neurons. The implications of these findings are that both differential expression and assembly as well as subcellular targeting of Kv channel α‐subunits may contribute to Kv channel diversity and thereby to presynaptic and postsynaptic membrane excitability.

Kvβ1 Subunit Binding Specific for Shaker-Related Potassium Channel α Subunits

Abstract Voltage-activated potassium (Kv) channels from mammalian brain are hetero-oligomers containing α and β subunits. Coexpression of Kv1α and Kvβ1 subunits confers rapid A-type inactivation on noninactivating potassium channels (delayed rectifiers) in expression systems in vitro. We have delineated a Kv1.5 amino-terminal region of up to 90 amino acids (residues 112–201) that is sufficient for interactions of Kv1.5α and Kvβ1 subunits. Within this region of the Kv1.5 amino terminus (residues 193–201), a Kvβ1 interaction site necessary for Kvβ1-mediated rapid inactivation of Kv1.5 currents was detected. This interaction site motif (FYE/QLGE/DEAM/L) is found exclusively in the Shaker -related subfamily (Kv1). The results show that hetero-oligomerization between α and Kvβ1 subunits is restricted to Shaker -related potassium channel α subunits.

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

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].

Ultrastructural localization of Shaker-related potassium channel subunits and synapse-associated protein 90 to septate-like junctions in rat cerebellar Pinceaux☆

The Pinceau is a paintbrush-like network of cerebellar basket cell axon branchlets embracing the initial segment of the Purkinje cell axon. Its electrical activity contributes to the control of the cerebellar cortical output through the Purkinje cell axon by generating an inhibitory field effect. In addition to the structural features of the Pinceau, its repertoire of voltage-gated ion channels is likely to be an important aspect of this function. Therefore, we investigated the fine structural distribution of voltage-activated potassium (Kv1.1, Kv1.2, Kv3.4) and sodium channel proteins in the Pinceau. The ultrastructural localization of potassium channel subunits was compared to the distribution of synapse-associated protein 90 (SAP90), a protein capable to induce in vitro clustering of Kv1 proteins. With an improved preembedding technique including ultrasmall gold particles, silver enhancement and gold toning, we could show that antibodies recognizing Kv1.1, Kv1.2 and SAP90 are predominantly localized to septate-like junctions, which connect the basket cell axonal branchlets. Kv3.4 immunoreactivity is not concentrated in junctional regions but uniformly distributed over the Pinceau and the pericellular basket surrounding the Purkinje cell soma. In contrast, voltage-activated sodium channels were not detected in the Pinceau, but localized to the Purkinje cell axon initial segment. The results suggest that Kv1.1 and Kv1.2 form heterooligomeric delayed rectifier type Kv channels, being colocalized to septate-like junctions by interaction with SAP90.

NIP domain prevents N-type inactivation in voltage-gated potassium channels

Shaker-related voltage-gated K+ (Kv) channels, are assembled from ion-conducting Kvα subunits, which are integral membrane proteins, and auxiliary Kvβ subunits. This leads to the formation of highly diverse heteromultimeric Kv channels that mediate outward currents with a wide range of time courses for inactivation. Two principal inactivation mechanisms have been recognized: C-type inactivation correlated with carboxy-terminal Kvα-subunit structures, and N-type inactivation conferred by ‘ball’ domains in the amino termini of certain Kvα, and Kvβ subunits. Assembly of heteromultimers with one or more Kvα,- and/or Kvβ ball domains appears to be an essential principle of the generation of A-type Kv channel diversity. Here we show that, unexpectedly, the presence of Kvα- or Kvβ-ball domains does not dominate the gating phenotype in heteromultimers containing Kv1.6α subunits. These heteromultimers mediate non-inactivating currents because of the dominant-negative activity of a new type of N-type inactivation-prevention (NIP) domain present in the Kv1.6 amino terminus. Mutations in the NIP domain lead to loss of function, and its transfer to another Kvα subunit leads to gain of function. Our discovery of the NIP domain, which neutralizes the activity of Kvα- and Kvβ-inactivation gates, establishes a new determinant for the gating behaviour of heteromultimeric Kv channels.

The xenopus oocyte as an ectopic expression system for the selection of protein isoform-specific antibodies

A panel of Xenopus oocytes, each injected with cRNA coding for one specific isoform of the rat brain RCK family of voltage gated potassium channel proteins, was employed to screen for isoform-specific monoclonal antibodies. Several days after injection, cryosections of embedded oocytes were produced and were employed in immunohistochemical analysis of antibody binding. Of the advantageous properties of the assay, it employs the native antigen, it can be applied to homooligomeric and heterooligomeric proteins, and cryosections of the same batch can be stored frozen for later tests. The method may be advantageous also for the selection of isoform-specific antibodies of other protein families.

Oligomeric and Subunit Structures of Voltage-Gated Potassium Channels

The activity of potassium (K) channels controls the electrical potential across the cell membrane and thereby may regulate cellular processes such as cell excitability, secretion, and signal transduction [1]. K channels are ubiquitous membrane proteins, which occur in most, if not all excitable and nonexcitable cells. Accordingly, K channels represent a cation channel family, which is very diverse with respect to activation, inactivation, and conductance properties. A large number of cDNAs has been cloned and characterized which encode voltage-gated K (Kv) channels [2-4]. With the availability of efficientin vitroexpression systems, remarkable progress has been made recently in deciphering the structural properties of Kvchannels. Members of the Kvchannel family are known to be oligomeric, integral membrane proteins, apparently created from different combinations of isoforms of α and β subunits. These combinations give rise to Kvchannels with distinct electrophysiological and pharmacological properties. Experimental evidence for such a fundamental principle is outlined herein, especially the relationship between subunit composition and rapid, A-type inactivation behavior of Kvchannels [5-7].