A transcriptional regulatory element critical for CHRNB4 promoter activity in vivo.

Genome-wide association studies have underscored the importance of the clustered neuronal nicotinic acetylcholine receptor (nAChR) subunit genes with respect to nicotine dependence as well as lung cancer susceptibility. CHRNB4, which encodes the nAChR ß4 subunit, plays a major role in the molecular mechanisms that govern nicotine withdrawal. Thus, elucidating how expression of the ß4 gene is regulated is critical for understanding the pathophysiology of nicotine addiction. We previously identified a CA box regulatory element, (5'-CCACCCCT-3') critical for ß4 promoter activity in vitro. We further demonstrated that a 2.3-kb fragment of the ß4 promoter region containing the 5'-CCACCCCT-3' regulatory element in the ß4 gene promoter (CA box) is capable of directing cell-type specific expression of a reporter gene to a myriad of brain regions that endogenously express the ß4 gene. To test the hypothesis that the CA box is critical for ß4 promoter activity in vivo, transgenic animals expressing a mutant form of the ß4 promoter were generated. Reporter gene expression was not detected in any tissue or cell type at embryonic day 18.5 (ED 18.5). Similarly, we observed drastically reduced reporter gene expression at postnatal day 30 (PD30) when compared to wild type (WT) transgenic animals. Finally, we demonstrated that CA box mutation results in decreased interaction of the transcription factor Sp1 with the mutant ß4 promoter. Taken together these results demonstrate that the CA box is critical for ß4 promoter activity in vivo.

From smoking to lung cancer: the CHRNA5/A3/B4 connection.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that modulate key physiological processes ranging from neurotransmission to cancer signaling. These receptors are activated by the neurotransmitter, acetylcholine, and the tobacco alkaloid, nicotine. Recently, the gene cluster encoding the alpha3, alpha5 and beta4 nAChR subunits received heightened interest after a succession of linkage analyses and association studies identified multiple single-nucleotide polymorphisms in these genes that are associated with an increased risk for nicotine dependence and lung cancer. It is not clear whether the risk for lung cancer is direct or an effect of nicotine dependence, as evidence for both scenarios exist. In this study, we summarize the body of work implicating nAChRs in the pathogenesis of lung cancer, with special focus on the clustered nAChR subunits and their emerging role in this disease state.

Location and orientation of minK within the I(Ks) potassium channel complex.

The slowly activating cardiac potassium current (I(Ks)) is generated by a heteromultimeric potassium channel complex consisting of pore-forming (KvLQT1) and accessory (minK) subunits belonging to the KCNQ and KCNE gene families, respectively. Evidence indicating that minK residues line the I(Ks) pore originates from the observation that two minK cysteine mutants (G55C and F54C) render I(Ks) Cd2+-sensitive. We have identified a single cysteine residue in the KvLQT1 S6 segment (Cys-331) that contributes to Cd2+ coordination in conjunction with cysteine residues engineered into the minK transmembrane domain. This observation indicates that minK resides in close proximity to S6 in the I(Ks) channel complex. On the basis of homology modeling that compares the KvLQT1 S6 segment with the structure of the bacterial potassium channel KcsA, we predict that the sulfhydryl side chain of Cys-331 projects away from the central axis of the KvLQT1 pore and suggest that minK resides outside of the permeation pathway. A preliminary model illustrating the orientation of minK with S6 was validated by successful prediction of a novel Cd2+ binding site created within the I(Ks) channel complex by engineering additional cysteine residues into both subunits. Our results indicate the location and orientation of minK within the I(Ks) channel complex and further suggest that Cd2+ exerts its effect on I(Ks) through an allosteric mechanism rather than direct pore blockade.

MinK subdomains that mediate modulation of and association with KvLQT1.

KvLQT1 is a voltage-gated potassium channel expressed in cardiac cells that is critical for myocardial repolarization. When expressed alone in heterologous expression systems, KvLQT1 channels exhibit a rapidly activating potassium current that slowly deactivates. MinK, a 129 amino acid protein containing one transmembrane-spanning domain modulates KvLQT1, greatly slowing activation, increasing current amplitude, and removing inactivation. Using deletion and chimeric analysis, we have examined the structural determinants of MinK effects on gating modulation and subunit association. Coexpression of KvLQT1 with a MinK COOH-terminus deletion mutant (MinK DeltaCterm) in Xenopus oocytes resulted in a rapidly activated potassium current closely resembling currents recorded from oocytes expressing KvLQT1 alone, indicating that this region is necessary for modulation. To determine whether MinK DeltaCterm was associated with KvLQT1, a functional tag (G55C) that confers susceptibility to partial block by external cadmium was engineered into the transmembrane domain of MinK DeltaCterm. Currents derived from coexpression of KvLQT1 with MinK DeltaCterm were cadmium sensitive, suggesting that MinK DeltaCterm does associate with KvLQT1, but does not modulate gating. To determine which MinK regions are sufficient for KvLQT1 association and modulation, chimeras were generated between MinK and the Na(+) channel beta1 subunit. Chimeras between MinK and beta1 could only modulate KvLQT1 if they contained both the MinK transmembrane domain and COOH terminus, suggesting that the MinK COOH terminus alone is not sufficient for KvLQT1 modulation, and requires an additional, possibly associative interaction between the MinK transmembrane domain and KvLQT1. To identify the MinK subdomains necessary for gating modulation, deletion mutants were designed and coexpressed with KvLQT1. A MinK construct with amino acid residues 94-129 deleted retained the ability to modulate KvLQT1 gating, identifying the COOH-terminal region critical for gating modulation. Finally, MinK/MiRP1 (MinK related protein-1) chimeras were generated to investigate the difference between these two closely related subunits in their ability to modulate KvLQT1. The results from this analysis indicate that MiRP1 cannot modulate KvLQT1 due to differences within the transmembrane domain. Our results allow us to identify the MinK subdomains that mediate KvLQT1 association and modulation.

Formation of isoprostane-like compounds (neuroprostanes) in vivo from docosahexaenoic acid.

F2-isoprostanes are prostaglandin F2-like compounds that are formed nonenzymatically by free radical-induced oxidation of arachidonic acid. We explored whether oxidation of docosahexaenoic acid (C22:6omega3), which is highly enriched in the brain, led to the formation of F2-isoprostane-like compounds, which we term F4-neuroprostanes. Oxidation of docosahexaenoic acid in vitro yielded a series of compounds that were structurally established to be F4-neuroprostanes using a number of mass spectrometric approaches. The amounts formed exceeded levels of F2-isoprostanes generated from arachidonic acid by 3.4-fold. F4-neuroprostanes were detected esterified in normal whole rat brain and newborn pig cortex at a level of 7.0 +/- 1.4 ng/g and 13.1 +/- 8 ng/g, respectively. Furthermore, F4-neuroprostanes could be detected in normal human cerebrospinal fluid and levels in patients with Alzheimer's disease (110 +/- 12 pg/ml) were significantly higher than age-matched controls (64 +/- 8 pg/ml) (p < 0.05). F4-neuroprostanes may provide a unique marker of oxidative injury to the brain and could potentially exert biological activity. Furthermore, the formation of F4-neuroprostane-containing aminophospholipids might adversely effect neuronal function as a result of alterations they induce in the biophysical properties of neuronal membranes.