SUMMARY Thyrotoxic hypokalemic periodic paralysis (TPP) is characterized by acute attacks of weakness, hypokalemia, and thyrotoxicosis of various etiologies. These transient attacks resemble those of patients with familial hypokalemic periodic paralysis (hypoKPP) and resolve with treatment of the underlying hyperthyroidism. Because of the phenotypic similarity of these conditions, we hypothesized that TPP might also be a channelopathy. While sequencing candidate genes, we identified a previously unreported gene (not present in human sequence databases) that encodes an inwardly rectifying potassium (Kir) channel, Kir2.6. This channel, nearly identical to Kir2.2, is expressed in skeletal muscle and is transcriptionally regulated by thyroid hormone. Expression of Kir2.6 in mammalian cells revealed normal Kir currents in whole-cell and single-channel recordings. Kir2.6 mutations were present in up to 33% of the unrelated TPP patients in our collection. Some of these mutations clearly alter a variety of Kir2.6 properties, all altering muscle membrane excitability leading to paralysis.

The L-type (Ca v 1.2) voltage-gated calcium channels play critical roles in membrane excitability, gene expression, and muscle contraction. The generation of splice variants by the alternative splicing of the poreforming Ca v 1.2 ␣ 1 -subunit (␣ 1 1.2) may thereby provide potent means to enrich functional diversity. To date, however, no comprehensive scan of ␣ 1 1.2 splice variation has been performed, particularly in the human context. Here we have undertaken such a screen, exploiting recently developed "transcript scanning" methods to probe the human gene. The degree of variation turns out to be surprisingly large; 19 of the 55 exons comprising the human ␣ 1 1.2 gene were subjected to alternative splicing. Two of these are previously unrecognized exons and two others were not known to be spliced. Comparisons of fetal and adult heart and brain uncovered a large IVS3-S4 variability resulting from combinatorial utilization of exons 31-33. Electrophysiological characterization of such IVS3-S4 variation revealed unmistakable shifts in the voltage dependence of activation, according to an interesting correlation between increased IVS3-S4 linker length and activation at more depolarized potentials. Steady-state inactivation profiles remained unaltered. This systematic portrait of splice variation furnishes a reference library for comprehending combinatorial arrangements of Ca v 1.2 splice exons, especially as they impact development, physiology, and disease.Rapid influx of Ca 2ϩ through the Ca v 1.2 channels initiates physiological responses like gene expression, neurotransmitter release, cardiac or smooth muscle contraction, and regulation of Ca 2ϩ -dependent ion channels (1-4). In these capacities, the functional profile of Ca v 1.2 calcium channels can be customized by combinatorial assembly of the Ca v 1.2 ␣ 1 with several different auxiliary ␤-and ␣ 2 ␦-subunits (5). Even greater flexibility in functional tuning could arise from alternative splicing of the ␣ 1 -subunit genes (6); splicing of the human ␣ 1 1.2 subunit gene is known to generate variants with tissue-specific biases and with distinct pharmacological properties (7). At present, 15 of 53 known exons (Fig. 1) of the human ␣ 1 1.2 gene have been reported to be subjected to alternative splicing (8). However, the full set of possible splice loci and variations and their distributions in heart and brain could far exceed this initial view.Recently, genome-wide analyses suggest that as high as 74% of human genes are alternatively spliced (9). Alternative splicing of pre-mRNA has been implicated in development, physiology, and pathophysiology, and the inclusion or exclusion of exons can be regulated in a tissue-specific or temporal manner (10, 11). Splice variations of human ␣ 1 1.2 subunit confer on the channel isoforms altered properties such as sensitivity to blockade by antagonists, regulation by protein kinase, current density, and activation and inactivation characteristics (12)(13)(14). However, these studies have reported generally on the impact of al...

P/Q-type (Ca v 2.1) calcium channels support a host of Ca 2ϩ -driven neuronal functions in the mammalian brain. Alternative splicing of the main ␣ 1A (␣ 1 2.1) subunit of these channels may thereby represent a rich strategy for tuning the functional profile of diverse neurobiological processes. Here, we applied a recently developed "transcript-scanning" method for systematic determination of splice variant transcripts of the human ␣ 1 2.1 gene. This screen identified seven loci of variation, which together have never been fully defined in humans. Genomic sequence analysis clarified the splicing mechanisms underlying the observed variation. Electrophysiological characterization and a novel analytical paradigm, termed strength-current analysis, revealed that one focus of variation, involving combinatorial inclusion and exclusion of exons 43 and 44, exerted a primary effect on current amplitude and a corollary effect on Ca 2ϩ -dependent channel inactivation. These findings significantly expand the anticipated scope of functional diversity produced by splice variation of P/Q-type channels.

An estimate of up to 60% of genes are subjected to alternative splicing, and 15% of human genetic diseases are associated with mutation of the splice sites [Krawczak M, Reiss J, and Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992; 90: 41-54; Cooper TA, and Mattox W. The regulation of splice-site selection, and its role in human disease. Am J Hum Genet 1997; 61: 259-66; Modrek B and Lee CJ. Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat Genet 2003; 34: 177-80; Modrek B, Resch A, Grasso C, and Lee C. Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 2001; 29: 2850-9; Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860-921] . The molecular diversity of alternatively spliced transcripts provides templates for a myriad of protein structures that are potentially crucial to sustaining the complexity of human physiology. The extensive alternative splicing of the alpha(1)1.2-subunit of the L-type Ca(v)1.2 channel, producing splice variants with distinct electrophysiological and pharmacological properties, would impact directly on the function of the cardiovascular system. Cell-selective expression of Ca(v)1.2 channels containing a specific alternatively spliced exon increases the functional variations for specific cellular activities in response to changing physiological signals. However, the regulation or control of the alpha(1)1.2-subunit alternative splicing machinery is unknown, and the role of numerous splice variants expressed in a cell is a mystery. A systematic and concerted effort is required to determine all the possible combinations of alternatively spliced exons in alpha(1)1.2-subunits in smooth and cardiac muscles. This will provide useful information to monitor changes on the usage of the entire suite of alternatively spliced exons to help relate altered Ca(v)1.2 channel function to physiology and disease.

Voltage-gated calcium channels play a major role in many important processes including muscle contraction, neurotransmission, excitation-transcription coupling, and hormone secretion. To date, 10 calcium channel ␣ 1 -subunits have been reported, of which four code for L-type calcium channels. In our previous work, we uncovered by transcript-scanning the presence of 19 alternatively spliced exons in the L-type Ca v 1.2 ␣ 1 -subunit. Here, we report the smooth muscle-selective expression of alternatively spliced exon 9* in Ca v 1.2 channels found on arterial smooth muscle. Specific polyclonal antibody against exon 9* localized the intense expression of 9*-containing Ca v 1.2 channels on the smooth muscle wall of arteries, but the expression on cardiac muscle was low. Whole-cell patch clamp recordings of the 9*-containing Ca v 1.2 channels in HEK293 cells demonstrated ؊9 and ؊11-mV hyperpolarized shift in voltage-dependent activation and current-voltage relationships, respectively. The steady-state inactivation property and sensitivity to blockade by nifedipine of the ؎exon 9* splice variants were, however, not significantly different. Such cell-selective expression of an alternatively spliced exon strongly indicates the customization and fine tuning of calcium channel functions through alternative splicing of the pore-forming ␣ 1 -subunit. The generation of proteomic variations by alternative splicing of the calcium channel Ca v 1.2 ␣ 1 -subunit can potentially provide a flexible mechanism for muscle or neuronal cells to respond to various physiological signals or to diseases.

Rab proteins are a family of small GTPases that regulate intracellular vesicle traffic. Rab8b, because of its homology with Rab8, has been suggested to function in vesicle transport to the plasma membrane. Using the yeast two-hybrid system, we identified a Rab8b interacting clone, termed TRIP8b, from a rat brain cDNA library. The gene encodes a 66-kDa protein with homology to the peroxisomal targeting signal 1 receptor. The interaction between Rab8b and TRIP8b was further verified by in vitro binding assays and co-immunoprecipitation studies. Additional experiments with Rab8b mutants demonstrated that Rab8b requires a guanine nucleotide but not prenylation for its interaction with TRIP8b. Western immunoblot analysis showed that TRIP8b was primarily expressed in brain. Subcellular fractionation of AtT20 cells revealed that TRIP8b was present in both cytosolic and membrane fractions. To investigate the function of Rab8b and TRIP8b in secretion, we examined the release of ACTH from AtT20 cells. Results from stable cell lines expressing Rab8b or TRIP8b indicated that both proteins had a stimulatory effect on cAMP-induced secretion of ACTH. In summary, these data suggest that Rab8b and TRIP8b interact with each other and are involved in the regulated secretory pathway in AtT20 cells.Rab proteins are a family of small GTP-binding proteins that are important regulators of vesicle transport (1-3). They undergo nucleotide exchange to establish the active GTP-bound form and are incorporated onto transport vesicles either during or after vesicle formation. The GTP-bound form of Rab proteins recruit effectors, either directly or indirectly, to target vesicles to the appropriate sites on acceptor membranes. These effectors include motor proteins which link the vesicles to the cytoskeleton (4, 5), docking complexes which are recruited from the cytosol or tethering factors that mediate the initial contact of membranes that are destined to fuse (6, 7).To date, about 40 distinct Rab proteins have been identified and each is believed to be specifically associated with a particular vesicle transport pathway (2,8). However, only a fraction of known Rab proteins have their functions characterized in detail. Among these, Rab8 (which we term Rab8a) has been shown to be a key regulator of constitutive polarized vesicle transport to the dendrites in the neurons or to the basolateral membrane in epithelial cells (9 -11). Using immunofluorescence and electron microscopy, Rab8a is localized to the Golgi region, cytoplasmic vesicular structures, and the plasma membrane of Madine-Darby canine kidney epithelial cells. When the wild type and dominant active mutant forms of Rab8a are overexpressed in baby hamster kidney fibroblast cells, a dramatic change in cell morphology occurs. The cells form elongated processes as a result of a reorganization of both their actin filaments and microtubules. In this case, newly synthesized vesicular stomatitis virus, a basolateral marker protein is preferentially delivered into these cell outgrowths.While th...

The corticotropin-releasing factor (CRF) peptides CRF and urocortins 1 to 3 are crucial regulators of mammalian stress and inflammatory responses, and they are also implicated in disorders such as anxiety, depression, and drug addiction. There is considerable interest in the physiological mechanisms by which CRF receptors mediate their widespread effects, and here we report that the native CRF receptor 1 (CRFR1) endogenous to the human embryonic kidney 293 cells can functionally couple to mammalian Ca V 3.2 T-type calcium channels. Activation of CRFR1 by either CRF or urocortin (UCN) 1 reversibly inhibits Ca V 3.2 currents (IC 50 of ϳ30 nM), but it does not affect Ca V 3.1 or Ca V 3.3 channels. Blockade of CRFR1 by the antagonist astressin abolished the inhibition of Ca V 3.2 channels. The CRFR1-dependent inhibition of Ca V 3.2 channels was independent of the activities of phospholipase C, tyrosine kinases, Ca 2ϩ /calmodulin-dependent protein kinase II, protein kinase C, and other kinase pathways, but it was dependent upon a cholera toxin-sensitive G protein-mediated mechanism relying upon G protein ␤␥ subunits (G␤␥). The inhibition of Ca V 3.2 channels via the activation of CRFR1 was due to a hyperpolarized shift in their steady-state inactivation, and it was reversible upon washout of the agonists. Given that UCN affect multiple aspects of cardiac and neuronal physiology and that Ca V 3.2 channels are widespread throughout the cardiovascular and nervous systems, the results point to a novel and functionally relevant CRFR1-Ca V 3.2 T-type calcium channel signaling pathway.The corticotropin-releasing factor (CRF) family, consisting of CRF, urocortin 1 (UCN), UCN2, and UCN3, are critical regulators of stress and inflammatory responses, and they have been variously associated with being cardioprotective and contributing toward alcohol and drug dependencies (Reul and Holsboer, 2002;Bale and Vale, 2004;Bruijnzeel and Gold, 2005;Gravanis and Margioris, 2005). The two major receptors for CRF and UCNs, CRF receptor (CRFR)1 and CRFR2, have been identified as G protein-coupled receptors (GPCRs) that can mediate responses via activation of the protein kinase signaling pathways (Bruijnzeel and Gold, 2005;Gravanis and Margioris, 2005). CRF has a higher affinity for CRFR1 than for CRFR2, UCN shows high affinity for both CRFR1 and CRFR2, whereas UCN2 and UCN3 are selective for CRFR2 (Bale and Vale, 2004). The CRFR1 is expressed primarily in the brain and pituitary, and activation of CRFR1 exerts numerous central and peripheral effects associated with pathological diseases (Dautzenberg and Hauger, 2002). Within the hypothalamus-pituitary axis, CRF and CRF-related peptides such as UCN activate CRFR1 receptors to regulate pituitary function in response to stress T.W

Iron is a crucial cofactor for several physiological functions in the brain including transport of oxygen, DNA synthesis, mitochondrial respiration, synthesis of myelin, and neurotransmitter metabolism. If iron concentration exceeds the capacity of cellular sequestration, excessive labile iron will be harmful by generating oxidative stress that leads to cell death. In patients suffering from Parkinson disease, the total amount of iron in the substantia nigra was reported to increase with disease severity. High concentrations of iron were also found in the amyloid plaques and neurofibrillary tangles of human Alzheimer disease brains. Besides iron, nitric oxide (NO) produced in high concentration has been associated with neurodegeneration. NO is produced as a co-product when the enzyme NO synthase converts L-arginine to citrulline, and NO has a role to support normal physiological functions. When NO is produced in a high concentration under pathological conditions such as inflammation, aberrantly S-nitrosylated proteins can initiate neurodegeneration. Interestingly, NO is closely related with iron homeostasis. Firstly, it regulates iron-related gene expression through a system involving iron regulatory protein and its cognate iron responsive element (IRP-IRE). Secondly, it modified the function of iron-related protein directly via S-nitrosylation. In this review, we examine the recent advances about the potential role of dysregulated iron homeostasis in neurodegeneration, with an emphasis on AD and PD, and we discuss iron chelation as a potential therapy. This review also highlights the changes in iron homeostasis caused by NO. An understanding of these mechanisms will help us formulate strategies to reverse or ameliorate iron-related neurodegeneration in diseases such as AD and PD.