The up-regulation of α4β2* nicotinic acetylcholine receptors (nAChRs) by chronic nicotine is a cell-delimited process and may be necessary and sufficient for the initial events of nicotine dependence. Clinical literature documents an inverse relationship between a person’s history of tobacco use and his or her susceptibility to Parkinson’s disease; this may also result from up-regulation. This study visualizes and quantifies the subcellular mechanisms involved in nicotine-induced nAChR up-regulation by using transfected fluorescent protein (FP)-tagged α4 nAChR subunits and an FP-tagged Sec24D endoplasmic reticulum (ER) exit site marker. Total internal reflection fluorescence microscopy shows that nicotine (0.1 µM for 48 h) up-regulates α4β2 nAChRs at the plasma membrane (PM), despite increasing the fraction of α4β2 nAChRs that remain in near-PM ER. Pixel-resolved normalized Förster resonance energy transfer microscopy between α4-FP subunits shows that nicotine stabilizes the (α4)2(β2)3 stoichiometry before the nAChRs reach the trans-Golgi apparatus. Nicotine also induces the formation of additional ER exit sites (ERES). To aid in the mechanistic analysis of these phenomena, we generated a β2enhanced-ER-export mutant subunit that mimics two regions of the β4 subunit sequence: the presence of an ER export motif and the absence of an ER retention/retrieval motif. The α4β2enhanced-ER-export nAChR resembles nicotine-exposed nAChRs with regard to stoichiometry, intracellular mobility, ERES enhancement, and PM localization. Nicotine produces only small additional PM up-regulation of α4β2enhanced-ER-export receptors. The experimental data are simulated with a model incorporating two mechanisms: (1) nicotine acts as a stabilizing pharmacological chaperone for nascent α4β2 nAChRs in the ER, eventually increasing PM receptors despite a bottleneck(s) in ER export; and (2) removal of the bottleneck (e.g., by expression of the β2enhanced-ER-export subunit) is sufficient to increase PM nAChR numbers, even without nicotine. The data also suggest that pharmacological chaperoning of nAChRs by nicotine can alter the physiology of ER processes.

The mouse mutant ducky, a model for absence epilepsy, is characterized by spike-wave seizures and cerebellar ataxia. A mutation in Cacna2d2, the gene encoding the α2δ-2 voltage-dependent calcium channel accessory subunit, has been found to underlie the ducky phenotype. The α2δ-2 mRNA is strongly expressed in cerebellar Purkinje cells. We show that du/du mice have abnormalities in their Purkinje cell dendritic tree. The mutation in α2δ-2 results in the introduction of a premature stop codon and predicts the expression of a truncated protein encoded by the first three exons of Cacna2d2, followed by 8 novel amino acids. We show that both mRNA and protein corresponding to this predicted transcript are expressed indu/du cerebellum and present in Purkinje cells. Whereas the α2δ-2 subunit increased the peak current density of the Ca_V2.1/β₄ channel combination when co-expressed in vitro, co-expression with the truncated mutant α2δ-2 protein reduced current density, indicating that it may contribute to the du phenotype.

We have cloned and characterized a new member of the voltage‐dependent Ca2+ channel γ subunit family, with a novel gene structure and striking properties. Unlike the genes of other potential γ subunits identified by their homology to the stargazin gene, CACNG7 is a five‐, and not four‐exon gene whose mRNA encodes a protein we have designated γ7. Expression of human γ7 has been localized specifically to brain. N‐type current through CaV2.2 channels was almost abolished when co‐expressed transiently with γ7 in either Xenopus oocytes or COS‐7 cells. Furthermore, immunocytochemistry and western blots show that γ7 has this effect by causing a large reduction in expression of CaV2.2 rather than by interfering with trafficking or biophysical properties of the channel. No effect of transiently expressed γ7 was observed on pre‐existing endogenous N‐type calcium channels in sympathetic neurones. Low homology to the stargazin‐like γ subunits, different gene structure and the unique functional properties of γ7 imply that it represents a distinct subdivision of the family of proteins identified by their structural and sequence homology to stargazin.

Carbon dioxide (CO ₂ ) movement across cellular membranes is passive and governed by Fick's law of diffusion. Until recently, we believed that gases cross biological membranes exclusively by dissolving in and then diffusing through membrane lipid. However, the observation that some membranes are CO ₂ impermeable led to the discovery of a gas molecule moving through a channel; namely, CO ₂ diffusion through aquaporin-1 (AQP1). Later work demonstrated CO ₂ diffusion through rhesus (Rh) proteins and NH ₃ diffusion through both AQPs and Rh proteins. The tetrameric AQPs exhibit differential selectivity for CO ₂ versus NH ₃ versus H ₂ O, reflecting physico-chemical differences among the small molecules as well as among the hydrophilic monomeric pores and hydrophobic central pores of various AQPs. Preliminary work suggests that NH ₃ moves through the monomeric pores of AQP1, whereas CO ₂ moves through both monomeric and central pores. Initial work on AQP5 indicates that it is possible to create a metal-binding site on the central pore's extracellular face, thereby blocking CO ₂ movement. The trimeric Rh proteins have monomers with hydrophilic pores surrounding a hydrophobic central pore. Preliminary work on the bacterial Rh homologue AmtB suggests that gas can diffuse through the central pore and three sets of interfacial clefts between monomers. Finally, initial work indicates that CO ₂ diffuses through the electrogenic Na/HCO ₃ cotransporter NBCe1. At least in some cells, CO ₂ -permeable proteins could provide important pathways for transmembrane CO ₂ movements. Such pathways could be amenable to cellular regulation and could become valuable drug targets.

Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) is linked with high penetrance to several distinct nicotinic receptor (nAChR) mutations. We studied (α4)₃(β2)₂ versus (α4)₂(β2)₃ subunit stoichiometry for five channel-lining M2 domain mutations: S247F, S252L, 776ins3 in α4, V287L, and V287M in β2. α4 and β2 subunits were constructed with all possible combinations of mutant and wild-type (WT) M2 regions, of cyan and yellow fluorescent protein, and of fluorescent and nonfluorescent M3-M4 loops. Sixteen fluorescent subunit combinations were expressed in N2a cells. Förster resonance energy transfer (FRET) was analyzed by donor recovery after acceptor photobleaching and by pixel-by-pixel sensitized emission, with confirmation by fluorescence intensity ratios. Because FRET efficiency is much greater for adjacent than for nonadjacent subunits and the α4 and β2 subunits occupy specific positions in nAChR pentamers, observed FRET efficiencies from (α4)₃(β2)₂ carrying fluorescent α4 subunits were significantly higher than for (α4)₂(β2)₃; the converse was found for fluorescent β2 subunits. All tested ADNFLE mutants produced 10 to 20% increments in the percentage of intracellular (α4)₃(β2)₂ receptors compared with WT subunits. In contrast, 24- to 48-h nicotine (1 μM) exposure increased the proportion of (α4)₂(β2)₃ in WT receptors and also returned subunit stoichiometry to WT levels for α4S248F and β2V287L nAChRs. These observations may be relevant to the decreased seizure frequency in patients with ADNFLE who use tobacco products or nicotine patches. Fluorescence-based investigations of nAChR subunit stoichiometry may provide efficient drug discovery methods for nicotine addiction or for other disorders that result from dysregulated nAChRs.

The mouse γ-aminobutyric acid (GABA) transporter mGAT1 was expressed in neuroblastoma 2a cells. 19 mGAT1 designs incorporating fluorescent proteins were functionally characterized by [3H]GABA uptake in assays that responded to several experimental variables, including the mutations and pharmacological manipulation of the cytoskeleton. Oligomerization and subsequent trafficking of mGAT1 were studied in several subcellular regions of live cells using localized fluorescence, acceptor photobleach Förster resonance energy transfer (FRET), and pixel-by-pixel analysis of normalized FRET (NFRET) images. Nine constructs were functionally indistinguishable from wild-type mGAT1 and provided information about normal mGAT1 assembly and trafficking. The remainder had compromised [3H]GABA uptake due to observable oligomerization and/or trafficking deficits; the data help to determine regions of mGAT1 sequence involved in these processes. Acceptor photobleach FRET detected mGAT1 oligomerization, but richer information was obtained from analyzing the distribution of all-pixel NFRET amplitudes. We also analyzed such distributions restricted to cellular subregions. Distributions were fit to either two or three Gaussian components. Two of the components, present for all mGAT1 constructs that oligomerized, may represent dimers and high-order oligomers (probably tetramers), respectively. Only wild-type functioning constructs displayed three components; the additional component apparently had the highest mean NFRET amplitude. Near the cell periphery, wild-type functioning constructs displayed the highest NFRET. In this subregion, the highest NFRET component represented ∼30% of all pixels, similar to the percentage of mGAT1 from the acutely recycling pool resident in the plasma membrane in the basal state. Blocking the mGAT1 C terminus postsynaptic density 95/discs large/zona occludens 1 (PDZ)-interacting domain abolished the highest amplitude component from the NFRET distributions. Disrupting the actin cytoskeleton in cells expressing wild-type functioning transporters moved the highest amplitude component from the cell periphery to perinuclear regions. Thus, pixel-by-pixel NFRET analysis resolved three distinct forms of GAT1: dimers, high-order oligomers, and transporters associated via PDZ-mediated interactions with the actin cytoskeleton and/or with the exocyst.