Nicotinic receptor assembly requires multiple regions throughout the gamma subunit.

Assembly of ionotropic neurotransmitter receptors typified by acetylcholine receptors (AChRs) is thought to be directed by an N-terminal extracellular domain of a subunit. Consistent with this hypothesis, chimeras with the delta subunit N-terminal domain fused to the rest of the gamma subunit can substitute for delta, but not gamma, subunits during AChR assembly. However, chimeras with the gamma subunit N-terminal domain fused to the rest of the delta subunit cannot substitute for gamma or delta subunits during assembly. Furthermore, expression of this chimera with the four wild-type subunits prevents the formation of alpha-bungarotoxin (Bgt) binding sites. Instead of AChR pentamers, complexes are assembled containing only the chimera and either alpha or beta subunits. Based on the results of additional gamma-delta chimeras, there are at least two different regions within the C-terminal half of the chimera required for the dominant-negative effect. Our results indicate that the N-terminal domain of the gamma subunit mediates the initial subunit associations, whereas signals in the C-terminal half of the subunit are required for subsequent subunit interactions.

Transient expression of heteromeric ion channels.

Transient transfection is an excellent method for the expression and study of cell-surface, heteromeric ion channels. The cell type, the total amount of DNA, the combination of subunits and the ratio of subunit DNA are all important parameters to consider when attempting to optimize expression. A serious drawback of this method is that the efficiency of subunit assembly is very low in comparison to the efficiency of assembly for stably expressed heteromeric ion channels. The low efficiency of assembly prevents use of transient expression methods for detailed studies of heteromeric AChR assembly, and caution should be taken in the use of these methods for the study of intracellular heteromeric ion channel subunits. After the transient expression of heteromeric AChR subunits, virtually all of the expressing cells contained all four AChR subunits. However, the subunits were heterogeneously distributed among the cells, and the low efficiency of AChR assembly appears to be due to cell-to-cell variations in the ratio of the four subunits.

Differential regulation of phenylethanolamine N-methyltransferase expression in two distinct subpopulations of bovine chromaffin cells.

Chromaffin cells were isolated from bovine adrenal glands and fractionated into two distinct subpopulations by density gradient centrifugation on Percoll. Cells in the more dense fraction stored epinephrine (E) as their predominant catecholamine (81% of total catecholamines), contained high levels of phenylethanolamine N-methyltransferase (PNMT) activity, and exhibited intense PNMT immunoreactivity. This population of chromaffin cells was termed the E-rich cell population. Cells in the less dense fraction, the norepinephrine (NE)-rich cell population, stored predominantly NE (75% of total catecholamines). Although the NE-rich cells had only 3% as much PNMT activity as did the E-rich cells, 20% of the NE-rich cells were PNMT immunoreactive. This suggested that the PNMT-positive cells in the NE-rich cell cultures contained less PNMT per cell than did E-rich cells and may not be typical adrenergic cells. The regulation of PNMT mRNA levels and PNMT activity in primary cultures of E-rich and NE-rich cells was compared. At the time the cells were isolated, PNMT mRNA levels in NE-rich cells were approximately 20% of those in E-rich cells; within 48 h in culture, PNMT mRNA in both populations declined to almost undetectable levels. Treatment with dexamethasone increased PNMT mRNA levels and PNMT activity in both populations. In E-rich cells, dexamethasone restored PNMT mRNA to the level seen in freshly isolated cells and increased PNMT activity twofold. In NE-rich cells, dexamethasone increased PNMT mRNA to levels twice those found in freshly isolated cells and increased PNMT activity sixfold. Cycloheximide blocked the effects of dexamethasone on PNMT mRNA expression in NE-rich cells but had little effect in E-rich cells. Angiotensin II, forskolin, and phorbol 12,13-dibutyrate elicited large increases in PNMT mRNA levels in E-rich cells but had no effect in NE-rich cells. Our data suggest that PNMT expression is regulated differently in the two chromaffin cell subpopulations.