Determinants in the ß and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-ß and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-ß and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, ßK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined ßK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the ß and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

The MX-Helix of Muscle nAChR Subunits Regulates Receptor Assembly and Surface Trafficking.

Nicotinic acetylcholine receptors (AChRs) are pentameric channels that mediate fast transmission at the neuromuscular junction (NMJ) and defects in receptor expression underlie neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndrome (CMS). Nicotinic receptor expression at the NMJ is tightly regulated and we previously identified novel Golgi-retention signals in the ß and δ subunit cytoplasmic loops that regulate trafficking of the receptor to the cell surface. Here, we show that the Golgi retention motifs are localized in the MX-helix, a juxta-membrane alpha-helix present in the proximal cytoplasmic loop of receptor subunits, which was defined in recent crystal structures of cys-loop receptor family members. First, mutational analysis of CD4-MX-helix chimeric proteins showed that the Golgi retention signal was dependent on both the amphipathic nature of the MX-helix and on specific lysine residues (ßK353 and δK351). Moreover, retention was associated with ubiquitination of the lysines, and ßK353R and δK351R mutations reduced ubiquitination and increased surface expression of CD4-ß and δ MX-helix chimeric proteins. Second, mutation of these lysines in intact ß and δ subunits perturbed Golgi-based glycosylation and surface trafficking of the AChR. Notably, combined ßK353R and δK351R mutations increased the amount of surface AChR with immature forms of glycosylation, consistent with decreased Golgi retention and processing. Third, we found that previously identified CMS mutations in the ε subunit MX-helix decreased receptor assembly and surface levels, as did an analogous mutation introduced into the ß subunit MX-helix. Together, these findings indicate that the subunit MX-helix contributes to receptor assembly and is required for normal expression of the AChR and function of the NMJ. In addition, specific determinants in the ß and δ subunit MX-helix contribute to quality control of AChR expression by intracellular retention and ubiquitination of unassembled subunits, and by facilitating the appropriate glycosylation of assembled surface AChR.

Acanthamoeba migration in an electric field.

PURPOSE: We investigated the in vitro response of Acanthamoeba trophozoites to electric fields (EFs). METHODS: Acanthamoeba castellanii were exposed to varying strengths of an EF. During EF exposure, cell migration was monitored using an inverted microscope equipped with a CCD camera and the SimplePCI 5.3 imaging system to capture time-lapse images. The migration of A. castellanii trophozoites was analyzed and quantified with ImageJ software. For analysis of cell migration in a three-dimensional culture system, Acanthamoeba trophozoites were cultured in agar, exposed to an EF, digitally video recorded, and analyzed at various Z focal planes. RESULTS: Acanthamoeba trophozoites move at random in the absence of an EF, but move directionally in response to an EF. Directedness in the absence of an EF is 0.08 ± 0.01, while in 1200 mV/mm EF, directedness is significantly higher at -0.65 ± 0.01 (P < 0.001). We find that the trophozoite migration response is voltage-dependent, with higher directionality with higher voltage application. Acanthamoeba move directionally in a three-dimensional (3D) agar system as well when exposed to an EF. CONCLUSIONS: Acanthamoeba trophozoites move directionally in response to an EF in a two-dimensional and 3D culture system. Acanthamoeba trophozoite migration is also voltage-dependent, with increased directionality with increasing voltage. This may provide new treatment modalities for Acanthamoeba keratitis.