Implementing Algorithm-Guided Warfarin Dosing in an Ethnically Diverse Patient Population Using Electronic Health Records and Preemptive CYP2C9 and VKORC1 Genetic Testing.

Implementation of pharmacogenetic-guided warfarin dosing has been hindered by inconsistent results from reported clinical trials and a lack of available algorithms that include alleles prevalent in non-white populations. However, current evidence indicates that algorithm-guided dosing is more accurate than empirical dosing. To facilitate multiethnic algorithm-guided warfarin dosing using preemptive genetic testing, we developed a strategy that accounts for the complexity of race and leverages electronic health records for algorithm variables and deploying point-of-care dose recommendations.

Structural elements in domain IV that influence biophysical and pharmacological properties of human alpha1A-containing high-voltage-activated calcium channels.

We have cloned two splice variants of the human homolog of the alpha1A subunit of voltage-gated Ca2+ channels. The sequences of human alpha1A-1 and alpha1A-2 code for proteins of 2510 and 2662 amino acids, respectively. Human alpha1A-2alpha2bdeltabeta1b Ca2+ channels expressed in HEK293 cells activate rapidly (tau+10mV = 2.2 ms), deactivate rapidly (tau-90mV = 148 micros), inactivate slowly (tau+10mV = 690 ms), and have peak currents at a potential of +10 mV with 15 mM Ba2+ as charge carrier. In HEK293 cells transient expression of Ca2+ channels containing alpha1A/B(f), an alpha1A subunit containing a 112 amino acid segment of alpha1B-1 sequence in the IVS3-IVSS1 region, resulted in Ba2+ currents that were 30-fold larger compared to wild-type (wt) alpha1A-2-containing Ca2+ channels, and had inactivation kinetics similar to those of alpha1B-1-containing Ca2+ channels. Cells transiently transfected with alpha1A/B(f)alpha2bdeltabeta1b expressed higher levels of the alpha1, alpha2bdelta, and beta1b subunit polypeptides as detected by immunoblot analysis. By mutation analysis we identified two locations in domain IV within the extracellular loops S3-S4 (N1655P1656) and S5-SS1 (E1740) that influence the biophysical properties of alpha1A. alpha1AE1740R resulted in a threefold increase in current magnitude, a -10 mV shift in steady-state inactivation, and an altered Ba2+ current inactivation, but did not affect ion selectivity. The deletion mutant alpha1ADeltaNP shifted steady-state inactivation by -20 mV and increased the fast component of current inactivation twofold. The potency and rate of block by omega-Aga IVA was increased with alpha1ADeltaNP. These results demonstrate that the IVS3-S4 and IVS5-SS1 linkers play an essential role in determining multiple biophysical and pharmacological properties of alpha1A-containing Ca2+ channels.

The expression of voltage-dependent calcium channel beta subunits in human hippocampus.

The beta subunits of voltage-dependent calcium channels (VDCC) modulate the electrophysiology and cell surface expression of pore-forming alpha1 subunits. In the present study we have investigated the distribution of beta1,beta2,beta3 and beta4 in the human hippocampus using in situ hybridization (ISH) and immunohistochemistry. ISH studies showed a similar distribution of expression of beta1,beta2 and beta3 subunit mRNAs, including labelling of the dentate granule cell layer, all CA pyramidal regions, and the subiculum. Relatively low levels of expression of beta1 and beta2 subunit mRNAs correlated with low protein expression in the immunocytochemical (ICC) studies. There was a relative lack of beta4 expression by both ISH and ICC in the CA1 region, compared with high levels of expression in the subiculum. Immunostaining for beta1 and beta2 subunits was weak and relatively homogeneous throughout the hippocampus. The beta3 and beta4 subunits appeared to be more discretely localized. In general, beta3-immunoreactivity was moderate both in cell bodies, and as diffuse staining in the surrounding neuropil. Strongest staining was observed in mossy fibres and their terminal region in the CA3 stratum lucidum. In contrast, beta4-immunoreactivity in the neuropil showed intense dendritic localisation. Unlike the other subunits, beta4-immunoreactivity was absent from CA1 pyramidal neurones but was present in a small population of interneurone-like cells. The localisation of beta3 and beta4 may represent presynaptic and postsynaptic compartments in some populations of hippocampal neurones. Comparison of beta subunit distribution with previously published data on alpha1 subunits indicates certain neuronal groups and subcellular compartments in which the subunit composition of native pre- and postsynaptic VDCC can be predicted.

The expression of voltage-dependent calcium channel beta subunits in human cerebellum.

The beta subunits of voltage-dependent calcium channels, exert marked regulatory effects on the biophysical and pharmacological properties of this diverse group of ion channels. However, little is known about the comparative neuronal expression of the four classes of beta genes in the CNS. In the current investigation we have closely mapped the distribution of beta1, beta2, beta3 and beta4 subunits in the human cerebellum by both in situ messenger RNA hybridization and protein immunohistochemistry. To our knowledge, these studies represent the first experiments in any species in which the detailed localization of each beta protein has been comparatively mapped in a neuroanatomically-based investigation. The data indicate that all four classes of beta subunits are found in the cerebellum and suggest that in certain neuronal populations they may each be expressed within the same cell. Novel immunohistochemical results further exemplify that the beta voltage-dependent calcium channel subunits are regionally distributed in a highly specific manner and studies of Purkinje cells indicate that this may occur at the subcellular level. Preliminary indication of the subunit composition of certain native voltage-dependent calcium channels is suggested by the observation that the distribution of the beta3 subunit in the cerebellar cortex is identical to that of alpha(1E). Our cumulative data are consistent with the emerging view that different native alpha1/beta subunit associations occur in the CNS.

Preparation and purification of antibodies specific to human neuronal voltage-dependent calcium channel subunits.

Neuronal voltage-dependent calcium channels (VDCCs) each comprising of alpha 1, alpha 2 delta, and beta subunits, are one mechanism by which excitable cells regulate the flux of calcium ions across the cell membrane following depolarisation Studies have shown the expression of several alpha 1 and beta subtypes within neuronal tissue. The comparative distribution of these in normal human brain is largely unknown. The aim of this work is to prepare antibodies directed specifically to selected subunits of human neuronal VDCCs for use in biochemical and mapping studies of calcium channel subtypes in the brain. Previous studies have defined DNA sequences specific for each subunit Comparison of these sequences allows the selection of unique amino acid sequences for use as immunogens which are prepared as glutathione-S-transferase (GST) fusion proteins in E. coli. Polyclonal antibodies raised against these fusion proteins are purified by Protein A chromatography, followed by immunoaffinity chromatography and extensive adsorptions using the appropriate fusion protein-GST Sepharose 4B columns. The resultant antibodies are analysed for specificity against the fusion proteins by ELISA, and by immunofluorescence and Western immunoblot analysis of recombinant HEK293 cells stably transfected with cDNAs encoding alpha 1, alpha 2 delta and beta subunits.

Comparative structure of human neuronal alpha 2-alpha 7 and beta 2-beta 4 nicotinic acetylcholine receptor subunits and functional expression of the alpha 2, alpha 3, alpha 4, alpha 7, beta 2, and beta 4 subunits.

cDNA clones encoding human neuronal nicotinic acetylcholine receptor alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha 7, beta 2, beta 3, and beta 4 subunits were isolated from brainstem, hippocampus, prefrontal cortex, substantia nigra, thalamus, and IMR32 libraries. Human alpha 2 and alpha 6 and full-length beta 3 and beta 4 clones have not been previously reported. Deduced amino acid sequences of the alpha 2, alpha 6, beta 3, and beta 4 predicted mature peptides are 503 residues (56.9 kDa), 464 residues (53.7 kDa), 440 residues (50.8 kDa), and 477 residues (54.1 kDa), respectively. These sequences show 84 (alpha 2), 87 (alpha 6), 89 (beta 3), and 84% (beta 4) identity to the corresponding rat sequences. The amino termini of the human alpha 2 and beta 3 mature peptides contain 23 and six additional residues, respectively, compared to those of rat alpha 2 and beta 3. Recombinant receptors were expressed in Xenopus laevis oocytes injected with in vitro transcripts encoding either alpha 7 alone or alpha 2, alpha 3, or alpha 4 in pairwise combination with beta 2 or beta 4. Inward currents were elicited by the application of acetylcholine (1-100 microM) and other agonists; these responses were blocked 65-97% by application of 10 microM d-tubocurare, confirming functional expression of human nicotinic receptors.

Cloning and functional characterization of human heteromeric N-methyl-D-aspartate receptors.

Human cDNAs encoding N-methyl-D-aspartate receptor type (NMDAR)1A, NMDAR2A and NMDAR2B subunits were cloned and receptors encoded by these cDNAs were functionally expressed by injection of the respective mRNAs in Xenopus oocytes. The pharmacological properties of recombinant human N-methyl-D-aspartate (NMDA) receptors were characterized by profiling two agonists and four antagonists at both the NMDA and glycine sites in voltage-clamped oocytes. NMDA, glycine and D-serine were significantly more potent at human NMDAR (hNMDAR)1A/2B receptors than at nNMDAR1A/2A, whereas there was no detectable subtype-dependent difference in the potency of glutamate. Of the NMDA-site antagonists tested, CGP 43487 and 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonate exhibited 5.8- and 3.9-fold greater potency, respectively, at hNMDAR1A/2A receptors than at hNMDAR1A/2B. Of the four glycine-site competitive antagonists tested, L-689,560 displayed 5-fold greater potency at hNMDAR1A/2A, whereas 5,7-dichlorokynurenic acid, HA-966 and CGP 58411 did not discriminate between hNMDAR1A/2A and hNMDAR1A/2B. Receptors resulting from injection of hNMDAR1A, hNMDAR2A and hNMDAR2B transcripts in a 1:1:1 ratio were indistinguishable from hNMDAR1A/2B receptors in terms of their sensitivity to NMDA, glycine, D-serine, CGS 19755 and CGP 40116. Ifenprodil was approximately 350-fold more potent at hNMDAR1A/2B than at hNMDAR1A/2A receptors. Ifenprodil sensitivities of receptors formed in oocytes injected with a constant amount of hNMDAR1A mRNA but varying ratios of hNMDAR2A or hNMDAR2B mRNAs were compared. The receptors expressed at a 10:1 ratio of 2A:2B transcripts displayed an ifenprodil sensitivity that would be predicted for a population in which 51% was represented by hNMDAR(1A)2(2A)3 complexes. Our results underscore the need for subtype-selective compounds acting at novel sites to sufficiently probe the pharmacological differences between NMDA receptor subtypes formed by different subunit combinations.

Distribution of alpha 1A, alpha 1B and alpha 1E voltage-dependent calcium channel subunits in the human hippocampus and parahippocampal gyrus.

The distribution of voltage-dependent calcium channel subunits in the central nervous system may provide information about the function of these channels. The present study examined the distribution of three alpha-1 subunits, alpha 1A, alpha 1B and alpha 1E, in the normal human hippocampal formation and parahippocampal gyrus using the techniques of in situ hybridization and immunocytochemistry. All three subunit mRNAs appeared to be similarly localized, with high levels of expression in the dentate granule and CA pyramidal layer. At the protein level, alpha 1A, alpha 1B and alpha 1E subunits were differentially localized. In general, alpha 1A-immunoreactivity was most intense in cell bodies and dendritic processes, including dentate granule cells, CA3 pyramidal cells and entorhinal cortex pre-alpha and pri-alpha cells. The alpha 1B antibody exhibited relatively weak staining of cell bodies but stronger staining of neuropil, especially in certain regions of high synaptic density such as the polymorphic layer of the dentate gyrus and the stratum lucidum and radiatum of the CA regions. The alpha 1E staining pattern shared features in common with both alpha 1A and alpha 1B, with strong immunoreactivity in dentate granule, CA3 pyramidal and entorhinal cortex pri-alpha cells, as well as staining of the CA3 stratum lucidum. These findings suggest regions in which particular subunits may be involved in synaptic communication. For example, comparison of alpha 1B and alpha 1E staining in the CA3 stratum lucidum with calbindin-immuno-reactivity suggested that these two calcium channels subunits may be localized presynaptically in mossy fibre terminals and therefore may be involved in neurotransmitter release from these terminals.

Chromosomal localization of the human genes for alpha 1A, alpha 1B, and alpha 1E voltage-dependent Ca2+ channel subunits.

The alpha 1 subunit genes encoding voltage-dependent Ca2+ channels are members of a gene family. We have used human brain cDNA probes to localize the neuronal isoform genes CACNL1A4 (alpha 1A), CACNL1A5 (alpha 1B), and CACNL1A6 (alpha 1E) to 19p13, 9q34, and 1q25-q31, respectively, using fluorescence in situ hybridization on human chromosomes. These genes are particularly interesting gene candidates in the pathogenesis of neuronal disorders. Although genetic disorders have been linked to loci 9q34 and 19p13, no genetic disease related to Ca2+ signaling defects has yet been linked to these loci.

The expression of neuronal voltage-dependent calcium channels in human cerebellum.

Little is known about the comparative distribution of voltage-dependent calcium channel subtypes in normal human brain. Previous studies in experimental animals have predominantly focused on the regional expression of single alpha 1 genes. We describe the preparation of riboprobes and antisera specific for human alpha 1A, alpha 1B and alpha 1E subunits and their application in comprehensive mapping studies of the human cerebellum. Within the cerebellar cortex, these pore forming proteins were found to have differential localisations when examined in adjacent sections. The alpha 1A and alpha 1B subunits broadly colocalised and were both present, though at apparently different levels, in the molecular, Purkinje and granule cell layers whilst alpha 1E was predominantly expressed in Purkinje cells. In the dentate nucleus, an area which has received little attention in previous studies, alpha 1A was highly expressed in regions in which Purkinje cell nerve terminals form synapses with deep cerebellar neurones.

Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5.

We have isolated and characterized overlapping cDNAs that encode two isoforms of the human metabotropic glutamate receptor subtype 5 (hmGluR5). The deduced amino acid sequences of human and rat mGluR5a are 94.5% identical. However, a region in the putative cytoplasmic domain (SER926-ALA1121) displays significant sequence divergence. Genomic analysis of this region showed that the sequence divergence results from species-specific differences in the genomic sequences, not from alternative splicing. The distribution of mGluR5 mRNA in human brain was most strongly detected throughout the hippocampus, with moderate levels in the caudate-putamen, cerebral cortex, thalamus, and deep cerebellar nuclei, and at low levels in the cerebellar cortex. Activation of both hmGluR5a and hmGluR5b transiently expressed in Xenopus oocytes and HEK293 cells was coupled to inositol phosphate (InsP) formation and elevation of the intracellular free calcium ([Ca2+]i). The agonist rank order of potency for activating recombinant hmGluR5a receptors in either system was quisqualate > L-glutamate > 1S,3R-ACPD. Both the quisqualate stimulated InsP and [Ca2+]i were inhibited by (+)-MCPG. Recombinant human mGluR5a was also stably expressed in mouse fibroblast Ltk- cells, in which the efficacy and potency of quisqualate were unchanged for more than 30 cell passages.

Characteristics of a human N-type calcium channel expressed in HEK293 cells.

The human alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel was stably expressed in HEK293 cells producing a human brain N-type voltage-dependent calcium channel (VDCC). Whole cell voltage-clamp electrophysiology and fura-2 based microfluorimetry have been used to study its characteristics. Calcium currents (ICa) recorded in transfected HEK293 cells were activated at potentials more depolarized than -20 mV with peak currents occurring at approx + 10 mV in 5 mM extracellular CaCl2. ICa and associated rises in intracellular free calcium concentrations ([Ca2+]i) were sensitive to changes in both the [Ca2+]o and holding potential. Steady-state inactivation was half maximal at a holding potential of -60 mV. Ba2+ was a more effective charge carrier than Ca2+ through the alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel and combinations of both Ba2+ and Ca2+ as charge carriers resulted in the anomalous mole fraction effect. Ca2+ influx into transfected HEK293 cells was irreversibly inhibited by omega-conotoxin-GVIA (omega-CgTx-GVIA; 10 nM-1 microM) and omega-conotoxin-MVIIA; 100 nM-1 microM) whereas 1 microM) whereas no reductions were seen with agents which block P or L-type Ca2+ channels. The inorganic ions, gadolinium (Gd3+), cadmium (Cd2+) and nickel (Ni2+) reduced the ICa under voltage-clamp conditions in a concentration-dependent manner. The order of potency of the three ions was Gd3+ > Cd2+ > Ni2+. These experiments suggest that the cloned and expressed alpha 1B-1 alpha 2b beta 1-2 Ca2+ channel subunits form channels in HEK293 cells that exhibit properties consistent with the activity of the native-N-type VDCC previously described in neurons.

Structure and functional characterization of neuronal alpha 1E calcium channel subtypes.

We have cloned overlapping cDNAs encoding alpha 1E Ca2+ channel subunits from mouse and human brain. We observed that these alpha 1E transcripts were widely distributed in the central nervous system. We also demonstrated the existence of two variants of the human alpha 1E subunit. Comparison of the sequence of these alpha 1E subunits to those from other species suggests that at least four alternatively spliced variants of alpha 1E exist. Expression of human alpha 1E in HEK293 cells and Xenopus oocytes produced high voltage-activated Ca2+ currents that inactivated rapidly (tau approximately 20 ms at 0 mV). The size of the currents obtained were enhanced approximately 40-fold by co-expression with human neuronal alpha 2 and beta Ca2+ channel subunits. alpha 1E currents were insensitive to the drugs and toxins previously used to define other classes of voltage-activated Ca2+ channels. Thus, alpha 1E-mediated Ca2+ channels appear to be a pharmacologically distinct class of voltage-activated Ca2+ channels.

Human neuronal voltage-dependent calcium channels: studies on subunit structure and role in channel assembly.

Voltage-dependent calcium (Ca2+) channels, expressed in the CNS, appear to be multimeric complexes comprised of at least alpha 1, alpha 2 and beta subunits. Previously, we cloned and expressed human neuronal alpha 1, alpha 2 and beta subunits to study recombinant channel complexes that display properties of those expressed in vivo. The alpha 1B-mediated channel subtype binds omega-conotoxin (CgTx) GVIA with high affinity and exhibits properties of N-type voltage-dependent Ca2+ channels. Here we describe several alpha 2 and beta splice variants and report results on the expression of omega-CgTx GVIA binding sites, assembly of the subunit complex and biophysical function of alpha 1B-mediated channel complexes containing some of these splice variants. We optimized recombinant expression in human embryonic kidney (HEK) 293 cells of alpha 1B alpha 2b beta 1 subunit complexes by controlling the expression levels of subunit mRNAs and monitored cell surface expression by binding of omega-CgTx GVIA to the alpha 1B subunit. Co-expression of either alpha 2b or beta 1 subunits with an alpha 1B subunit increased expression of binding sites while the most efficient expression was achieved when both alpha 2b and beta 1 subunits were co-expressed with an alpha 1B subunit. The presence of alpha 2b affects the affinity of omega-CgTx GVIA binding and barium (Ba2+) current magnitudes, although it does not appear to alter kinetic properties of the Ba2+ current. This is the first evidence of an alpha 2 subunit modulating the binding affinity of a cell-surface Ca2+ channel ligand. Our results demonstrate that alpha 1, alpha 2 and beta subunits together contribute to the efficient assembly and functional expression of voltage-dependent Ca2+ channel complexes.

Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel.

N-type calcium channels are omega-conotoxin (omega-CgTx)-sensitive, voltage-dependent ion channels involved in the control of neurotransmitter release from neurons. Multiple subtypes of voltage-dependent calcium channel complexes exist, and it is the alpha 1 subunit of the complex that forms the pore through which calcium enters the cell. The primary structures of human neuronal calcium channel alpha 1B subunits were deduced by the characterization of overlapping complementary DNAs. Two forms (alpha 1B-1 and alpha 1B-2) were identified in human neuroblastoma (IMR32) cells and in the central nervous system, but not in skeletal muscle or aorta tissues. The alpha 1B-1 subunit directs the recombinant expression of N-type calcium channel activity when it is transiently co-expressed with human neuronal beta 2 and alpha 2b subunits in mammalian HEK293 cells. The recombinant channel was irreversibly blocked by omega-CgTx but was insensitive to dihydropyridines. The alpha 1B-1 alpha 2b beta 2-transfected cells displayed a single class of saturable, high-affinity (dissociation constant = 55 pM) omega-CgTx binding sites. Co-expression of the beta 2 subunit was necessary for N-type channel activity, whereas the alpha 2b subunit appeared to modulate the expression of the channel. The heterogeneity of alpha 1B subunits, along with the heterogeneity of alpha 2 and beta subunits, is consistent with multiple, biophysically distinct N-type calcium channels.

Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype.

The primary structures of human neuronal alpha 1, alpha 2, and beta subunits of a voltage-dependent Ca2+ channel were deduced by characterizing cDNAs. The alpha 1 subunit (alpha 1D) directs the recombinant expression of a dihydropyridine-sensitive L-type Ca2+ channel when coexpressed with the beta (beta 2) and the alpha 2 (alpha 2b) subunits in Xenopus oocytes. The recombinant channel is also reversibly blocked by 10-15 microM omega-conotoxin. Expression of the alpha 1D subunit alone, or coexpression with the alpha 2b subunit, did not elicit functional Ca2+ channel activity. Thus, the beta 2 subunit appears to serve an obligatory function, whereas the alpha 2b subunit appears to play an accessory role that potentiates expression of the channel. The primary transcripts encoding the alpha 1D, alpha 2, and beta subunits are differentially processed. At least two forms of neuronal alpha 1D were identified. Different forms of alpha 2 and beta transcripts were also identified in CNS, skeletal muscle, and aorta tissues.

Primary structure of the gamma subunit of the DHP-sensitive calcium channel from skeletal muscle.

Affinity-purified, polyclonal antibodies to the gamma subunit of the dihydropyridine (DHP)-sensitive, voltage-dependent calcium channel have been used to isolate complementary DNAs to the rabbit skeletal muscle protein from an expression library. The deduced primary structure indicates that the gamma subunit is a 25,058-dalton protein that contains four transmembrane domains and two N-linked glycosylation sites, consistent with biochemical analyses showing that the gamma subunit is a glycosylated hydrophobic protein. Nucleic acid hybridization studies indicate that there is a 1200-nucleotide transcript in skeletal muscle but not in brain or heart. The gamma subunit may play a role in assembly, modulation, or the structure of the skeletal muscle calcium channel.

Sequence and expression of mRNAs encoding the alpha 1 and alpha 2 subunits of a DHP-sensitive calcium channel.

Complementary DNAs were isolated and used to deduce the primary structures of the alpha 1 and alpha 2 subunits of the dihydropyridine-sensitive, voltage-dependent calcium channel from rabbit skeletal muscle. The alpha 1 subunit, which contains putative binding sites for calcium antagonists, is a hydrophobic protein with a sequence that is consistent with multiple transmembrane domains and shows structural and sequence homology with other voltage-dependent ion channels. In contrast, the alpha 2 subunit is a hydrophilic protein without homology to other known protein sequences. Nucleic acid hybridization studies suggest that the alpha 1 and alpha 2 subunit mRNAs are expressed differentially in a tissue-specific manner and that there is a family of genes encoding additional calcium channel subtypes.

Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris.

The oxidation of methanol follows a well-defined pathway and is similar for several methylotrophic yeasts. The use of methanol as the sole carbon source for the growth of Pichia pastoris stimulates the expression of a family of genes. Three methanol-responsive genes have been isolated; cDNA copies have been made from mRNAs of these genes, and the protein products from in vitro translations have been examined. The identification of alcohol oxidase as one of the cloned, methanol-regulated genes has been made by enzymatic, immunological, and sequence analyses. Methanol-regulated expression of each of these three isolated genes can be demonstrated to occur at the level of transcription. Finally, DNA subfragments of two of the methanol-responsive genomic clones from P. pastoris have been isolated and tentatively identified as containing the control regions involved in methanol regulation.