Localization of the pannexin1 protein at postsynaptic sites in the cerebral cortex and hippocampus.

Pannexins (Panx) constitute a new family of gap junction type proteins. Functional expression in paired Xenopus oocytes indicated that pannexins are capable of forming communicating junctions but also proved to be active in forming of unopposed hemichannels. In the vertebrate brain pannexins have been found in neurons. However, the subcellular cerebral localization of pannexin proteins which could gain first clues on their putative function is essentially unknown. Here we demonstrate by light and electron microscopical immunohistochemistry that Panx1 reveals postsynaptic localization in rodent hippocampal and cortical principal neurons accumulating at postsynaptic densities. The postsynaptic localization was corroborated by co-localization of Panx1 with postsynaptic density protein 95 (PSD-95), a prominent postsynaptic scaffolding protein, in hippocampal neurons expressing tagged versions of these proteins. The asymmetric synaptic distribution of Panx1 suggests that it may function in neurons as non-junctional channels (pannexons) at postsynaptic sites and comprises a novel component of the postsynaptic protein complex.

Evidence for a role of the N-terminal domain in subcellular localization of the neuronal connexin36 (Cx36).

The expression and functional properties of connexin36 (Cx36) have been investigated in two neuroblastoma cell lines (Neuro2A, RT4-AC) and primary hippocampal neurons transfected with a Cx36-enhanced green fluorescent protein (EGFP) expression vector. Transfected cells express Cx36-EGFP mRNA, and Cx36-EGFP protein is localized in the perinuclear area and cell membrane. Upon differentiation of cell lines, Cx36-EGFP protein was detectable in processes with both axonal and dendritic characteristics. Small gap junction plaques were found between adjacent cells, and electrophysiological recordings demonstrated that the electrical properties of these gap junctions were virtually indistinguishable from those reported for native Cx36. Mutagenesis of Cx36 led to the identification of a structural element that interferes with normal protein localization. In contrast, site directed mutagenesis of putative protein phosphorylation motifs did not alter subcellular localization. This excludes phosphorylation/dephosphorylation as a major regulatory step in Cx36 protein transport.

Haptenylation of antibodies during affinity purification: a novel and convenient procedure to obtain labeled antibodies for quantification and double labeling.

Haptenylation of primary antibodies is a useful technique for multiple purposes. It is a technically straightforward procedure, as many haptens are available as N-hydroxysuccinimide esters or isothiocyanates. Unfortunately, the hapten group may become covalently attached to or close to the combining site of antibodies, lectins, or other ligand-binding proteins during the process of haptenylation. Thus, the interaction of the corresponding protein with its ligand may become severely hampered. To overcome this restriction, we developed a novel procedure for the haptenylation of polyclonal antibodies that combines purification and haptenylation. Haptenylation during adsorption to the affinity matrix combines two advantages: the antigen binding site is protected and the labeling procedure becomes most convenient, as overlabeled proteins and unreacted haptens are easily removed by simple washing. Haptenylation during adsorption to the affinity matrix is a two-phase reaction, which requires different conditions to the conventional procedure. To obtain such optimal conditions, stabilities and reactivities of N-hydroxysuccinimide esters and isothiocyanate groups were investigated with a newly developed assay. Based on this information, antibodies against two recently described calcium-binding proteins, NCS-1 and NVP-3, were biotinylated or digoxigenylated. The haptenylated antibodies were successfully applied for biochemical determination and simultaneous immunoenzymatic double labeling of the two proteins.