Efficient expression of rat brain type IIA Na+ channel alpha subunits in a somatic cell line.

Type IIA rat brain Na+ channel alpha subunits were expressed in CHO cells by nuclear microinjection or by transfection using a vector containing both metallothionein and bacteriophage SP6 promoters. Stable cell lines expressing Na+ channels were isolated, and whole-cell Na+ currents of 0.9-14 nA were recorded. The mean level of whole-cell Na+ current (4.5 nA) corresponds to a cell surface density of approximately 2 channels active at the peak of the Na+ current per microns 2, a density comparable to that observed in the cell bodies of central neurons. The expressed Na+ channels had the voltage dependence, rapid activation and inactivation, and rapid recovery from inactivation characteristic of Na+ channels in brain neurons, bound toxins at neurotoxin receptor sites 1 and 3 with normal properties, and were posttranslationally processed to a normal mature size of 260 kd. Expression of Na+ channel cDNA in CHO cells driven by the metallothionein promoter accurately and efficiently reproduces native Na+ channel properties and provides a method for combined biochemical and physiological analysis of Na+ channel structure and function.

Synthesis of potent cyclic hexapeptide NK-1 antagonists. Use of a minilibrary in transforming a peptidal somatostatin receptor ligand into an NK-1 receptor ligand via a polyvalent peptidomimetic.

The endogenous peptides somatostatin (SRIF) and substance P comprise very different structures. Although both bind G-protein-coupled receptors, the SRIF receptors (SSTR 1-5) recognize SRIF and related peptides which retain its beta-turn such as the potent cyclic hexapeptide SRIF agonist L-363,301 (6a), but not substance P. Conversely the NK-1 receptor binds substance P but not the above ligands. In contrast, the beta-D-glucosides 1 and 2, designed to mimic the beta-turn of 6a, bind both receptors. This observation led us to attempt the conversion of 6a into the first potent, selective cyclic hexapeptide ligand for the NK-1 receptor. To this end, we combined design with a minilibrary approach. The goal was accomplished with surprising ease, leading to the NK-1 receptor antagonist 9 (IC50 2.0 +/- 0.4 nM). This demonstrates that peptidomimetics, incorporating in this case the promiscuous beta-D-glucose scaffold, can provide valuable clues about receptor similarities not revealed by their endogenous ligands. In addition, this work suggests that the use of libraries and rational design need not be mutually exclusive approaches to lead discovery.