点击显示 收起
Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin
Abstract
In this issue of Molecular Pharmacology, Hahn et al. (p. 457) present a study of previously uncharacterized single amino acid variants of the human norepinephrine transporter. Intracellular trafficking, surface expression, transport properties, interaction with antagonists, and regulation by a protein kinase C-linked regulatory pathway were studied by heterologous expression in COS-7 cells. In recent years, there has been increasing focus on the natural variations and roles of nonsynonymous single-nucleotide polymorphisms in human membrane transporter genes (and their protein products) in human disease. As this information is assimilated and understood at the molecular and genetic level, the relationship between transporter pharmacogenomics and therapeutics in the age of individualized medicine will be greatly impacted.
Over the past 30 years, the time between basic science discoveries and the application of new information to the treatment of human disease has become increasingly compressed. For example, consider the translational relationship between the purification and characterization of the nicotinic neuromuscular junction acetylcholine receptor (Patrick and Lindstrom, 1973; Berman and Patrick, 1980; Lindstrom, 2000, 2002) and the application of this knowledge to therapeutic treatment regimens for myasthenia gravis. Likewise, consider the discoveries and exhaustive characterization over a few short years related to drug metabolism and exemplified in the P450 field. The dramatic effects that this information has provided to our understanding, not only of drug pharmacodynamics in humans but also of the ethnic genetic variations in world populations, are truly profound (Cascorbi, 2003; Schwarz, 2003). Knowledge of the effects of P450 polymorphisms in drug metabolism is influencing the approaches by which physicians treat human diseases (Dahl, 2002; Roots et al., 2004; Tempfer et al., 2004; Kirchheiner and Brockmoller, 2005). New drugs are now synthesized and screened in the pharmaceutical industry to meet strict requirements dictated by known drug metabolic pathways (Rodrigues and Rushmore, 2002).
Many researchers have been engaged in exciting and highly significant experiments identifying and understanding the consequences of single-nucleotide polymorphisms (SNPs) and other genetically transmitted alterations in neurotransmitter receptors (Michel and Insel, 2003; Small et al., 2003; Kirstein and Insel, 2004; Liggett, 2004), ion channels (Furutani et al., 1999; Zhou et al., 1999; Anson et al., 2004), and enzymes important in cellular and organismal homeostasis.
Membrane transporters play a critical role in maintaining physiological balance in the biological kingdom. Both plasma membrane and intracellular membrane transporters compartmentalize metabolic processes, including the synthesis and sequestration of neurotransmitters and the elimination of toxic waste products and xenobiotics. Many natural variations resulting in SNPs in transporter genes that are drug targets have been identified (Leabman et al., 2003). These transporters include the ATP binding cassette superfamily (e.g., ATP binding cassette transporters, MDR1), which removes xenobiotics from cells, and solute carrier superfamily transporters (e.g., OCT1, DAT, NET, VMAT1), which sequester neurotransmitters and small molecules into and within cells.
The monoamine transporters for norepinephrine (NE), dopamine (DA) and serotonin belong to the SLC6A family of Na+/Cl--dependent transporters. Several SNPs for the human norepinephrine transporter (NET), the human dopamine transporter, and the human serotonin transporter have been reported and many have been characterized at the cellular level (Hahn and Blakely, 2002a). Some transporter variants that relate to human phenotypes have been successfully linked within families (Hahn and Blakely, 2002a).
In this issue of Molecular Pharmacology, the article titled "Single Nucleotide Polymorphisms in the Human Norepinephrine Transporter Gene Affect Expression, Trafficking, Antidepressant Interaction, and Protein Kinase C Regulation" characterizes several properties of the norepinephrine transporter (NET) with regard to amino acid variants that have not been previously characterized in human populations that include cardiovascular phenotypes. Hahn et al. (2005) have significantly enhanced our knowledge of the characterization of hNET SNPs that influence transporter function at the cellular level. These investigators previously identified an SNP of hNET (A457P) that is directly linked to an autonomic disorder, orthostatic intolerance (Shannon et al., 2000; Hahn et al., 2003). This condition is characterized by elevated heart rate and increased plasma norepinephrine levels in family members. Because NET resequesters most of the NE released in the heart, this reuptake system in the sympathetic neurons serving the heart assumes a singular role, compared with the brain, in which regulation between several neurotransmitter systems (including NE) is shared through interactive regulatory pathways (Hahn and Blakely, 2002a,b).
Most of the hNET variants reported by Hahn et al. (2005) have also been identified in populations having cardiovascular phenotypes that include extremes of blood pressure or long QT syndrome (Halushka et al., 1999; Iwasa et al., 2001) but have not been previously characterized at the cellular level. Figure 1 shows the nonsynonymous SNPs that were characterized in this study () in the background of additional nonsynonymouns SNPs previously identified (). Expression levels of each SNP were assessed by Western blots after transient transfection in COS-7 cells. Surface expression of the hNET variants was identified by sensitivity to surface biotinylation. Transporter function was measured by NE and DA uptake.
The hNET variants V244I (TM4), V356L (TM7), N375S (ECL4), and K463R (TM9/ECL5) had little effect on hNET expression or transport of NE and DA. These data indicate that the relatively conservative amino acid substitutions at these positions are stably tolerated, as further evidenced by comparison of monoamine transporter sequences from several species in which various amino acid substitutions at these analogous positions are allowed.
On the other hand, several SNPs were demonstrated to result in unfavorable substitutions. The most dramatic substitution involved A369P (TM7/ECL4), which resulted in the total loss of surface expression of the mature 110-kDa transporter and loss of NE and DA transport of the surface-expressed 54-kDa molecules. In addition, this hNET variant, as previously reported for the A457P allele as well (Hahn et al., 2003), decreased total and surface levels of hNET, demonstrating an important dominant-negative suppression of wild-type transporter expression and function. The potential synergism of diminished wild-type neurotransmitter transporter activity through dominant-negative oligomerization in individuals carrying variant alleles is particularly significant and is likely to further exacerbate cardiovascular deficiencies.
Three additional striking ideas that were generated in the current study could have profound consequences: 1) The hNET F528C (TM2) allele showed a selective change in transport for NE over DA. This analogous region of the serotonin transporter has been identified as extremely important for determining the potencies of substrates and antidepressants and similarly for functional activity of the dopamine transporter. The notion that variant alleles in the TM2 region (including hNET) could impact drug responses in these individuals is especially significant. 2) In addition, as the authors correctly point out, variants in the Phe528 region of hNET could well alter the balance of monoamine neurotransmission in areas of the brain (e.g., the prefrontal cortex) where free DA is cleared by NET (Gresch et al., 1995), with resultant elevation of both NE and DA and significant global consequences after use of selective NET antagonists. 3) Finally, altered functional responses of hNET variants (expressed separately or in concert with wild-type hNET) to protein kinase C regulation raises additional significant concerns related to hNET activity when variant alleles are present. Further experimental work is needed to fully understand the consequence of SNPs on PKC-mediated regulation of hNET. Certainly it is reasonable to conclude that PKC regulation of variant transporters will be affected during both the basal and regulated "state" (compare F528C with R121Q), but the complete elucidation of functional interactions and PKC regulation must await further experimental investigation.
Taken together, these data add significantly to our knowledge regarding the properties of SNPs in hNET. The authors do an excellent job of correlating in a broader sense the significance of these mutations with regard to function and regulation and the correlative changes that have been observed in other monoamine transporters such as the serotonin transporter (Lesch et al., 1996; Hahn and Blakely, 2002a; Kilic et al., 2003). Eventually, a thorough database will be needed to fully understand human conditions that are affected by SNPs in transporters and other drug targets. This process is already under development by the National Institutes of General Medical Sciences (NIGMS) through the Pharmacogenomics Network (http://www.PharmGKB.org) as a repository of pharmacologically relevant genes (Hewett et al., 2002; Klein and Altman, 2004). Several entries are currently present from the solute carrier transporter superfamily, including the hNET SNP A457P, which is linked to orthostatic intolerance. As additional relevant linkages of genotypes and phenotypes for transporters are discovered, this pharmacogenomic knowledge base will be invaluable.
Although there is a long and exciting journey ahead to further define the role of genetic polymorphisms in many drug targets, the transporter families are already well seeded and nourished. The substantial significance of the work represented in this article (Hahn et al., 2005) and analogous experiments performed in many other laboratories are worthy of our notice and support. Individualized medicine, with a molecular mechanism basis, long a dream of many, is one step closer to reality.
Please see the related article on page 457.
References
Anson BD, Ackerman MJ, Tester DJ, Will ML, Delisle BP, Anderson CL, and January CT (2004) Molecular and functional characterization of common polymorphisms in HERG (KCNH2) potassium channels. Am J Physiol 286: H2434-H2441.
Berman PW and Patrick J (1980) Linkage between the frequency of muscular weakness and loci that regulate immune responsiveness in murine experimental myasthenia gravis. J Exp Med 152: 507-520.
Cascorbi I (2003) Pharmacogenetics of cytochrome P4502D6: genetic background and clinical implication. Eur J Clin Investig 33 (Suppl 2): 17-22.
Dahl ML (2002) Cytochrome p450 phenotyping/genotyping in patients receiving antipsychotics: useful aid to prescribing Clin Pharmacokinet 41: 453-470.
Furutani M, Trudeau MC, Hagiwara N, Seki A, Gong Q, Zhou Z, Imamura S, Nagashima H, Kasanuki H, Takao A, et al. (1999) Novel mechanism associated with an inherited cardiac arrhythmia: defective protein trafficking by the mutant HERG (G601S) potassium channel. Circulation 99: 2290-2294.
Gresch PJ, Sved AF, Zigmond MJ, and Finlay JM (1995) Local influence of endogenous norepinephrine on extracellular dopamine in rat medial prefrontal cortex. J Neurochem 65: 111-116.
Hahn MK and Blakely RD (2002a) Gene organization and polymorphisms of monoamine transporters: relationship to psychiatric and other complex diseases, in Neurotransmitter Transporters: Structure, Function and Regulation (Reith MEA ed), pp 111-169, Contempory Neuroscience series, Humana Press Inc, Totowa, NJ.
Hahn MK and Blakely RD (2002b) Monoamine transporter gene structure and polymorphisms in relation to psychiatric and other complex disorders. Pharmacogenomics J 2: 217-235.
Hahn MK, Mazei-Robison MS, and Blakely RD (2005) Single nucleotide polymorphisms in the human norepinephrine transporter gene affect expression, trafficking, antidepressant interaction, and protein kinase C regulation. Mol Pharmacol 68: 457-466.
Hahn MK, Robertson D, and Blakely RD (2003) A mutation in the human norepinephrine transporter gene (SLC6A2) associated with orthostatic intolerance disrupts surface expression of mutant and wild-type transporters. J Neurosci 23: 4470-4478.
Halushka MK, Fan JB, Bentley K, Hsie L, Shen N, Weder A, Cooper R, Lipshutz R, and Chakravarti A (1999) Patterns of single nucleotide polymorphisms in candidate genes for blood-pressure homeostasis. Nat Genet 22: 239-247.
Hewett M, Oliver DE, Rubin DL, Easton KL, Stuart JM, Altman RB, and Klein TE (2002) PharmGKB: the pharmacogenetics knowledge base. Nucleic Acids Res 30: 163-165.
Iwasa H, Kurabayashi M, Nagai R, Nakamura Y, and Tanaka T (2001) Genetic variations in five genes involved in the excitement of cardiomyocytes. J Hum Genet 46: 549-552.
Kilic F, Murphy DL, and Rudnick G (2003) A human serotonin transporter mutation causes constitutive activation of transport activity. Mol Pharmacol 64: 440-446.
Kirchheiner J and Brockmoller J (2005) Clinical consequences of cytochrome P450 2C9 polymorphisms. Clin Pharmacol Ther 77: 1-16.
Kirstein SL and Insel PA (2004) Autonomic nervous system pharmacogenomics: a progress report. Pharmacol Rev 56: 31-52.
Klein TE and Altman RB (2004) PharmGKB: the pharmacogenetics and pharmacogenomics knowledge base. Pharmacogenomics J 4(1): 1.
Leabman MK, Huang CC, DeYoung J, Carlson EJ, Taylor TR, de la Cruz M, Johns SJ, Stryke D, Kawamoto M, Urban TJ, et al. (2003) Natural variation in human membrane transporter genes reveals evolutionary and functional constraints. Proc Natl Acad Sci USA 100: 5896-5901.
Lesch KP, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S et al. (1996) Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science (Wash DC) 274: 1527-1531.
Liggett SB (2004) Polymorphisms of beta-adrenergic receptors in heart failure. Am J Med 117: 525-527.
Lindstrom J (2002) Autoimmune diseases involving nicotinic receptors. J Neurobiol 53: 656-665.
Lindstrom JM (2000) Acetylcholine receptors and myasthenia. Muscle Nerve 23: 453-477.
Michel MC and Insel PA (2003) Receptor gene polymorphisms: lessons on functional relevance from the beta 1-adrenoceptor. Br J Pharmacol 138: 279-282.
Patrick J and Lindstrom J (1973) Autoimmune response to acetylcholine receptor. Science (Wash DC) 180: 871-872.
Rodrigues AD and Rushmore TH (2002) Cytochrome P450 pharmacogenetics in drug development: in vitro studies and clinical consequences. Curr Drug Metab 3: 289-309.
Roots I, Gerloff T, Meisel C, Kirchheiner J, Goldammer M, Kaiser R, Laschinski G, Brockmoller J, Cascorbi I, Kleeberg U, et al. (2004) Pharmacogenetics-based new therapeutic concepts. Drug Metab Rev 36: 617-638.
Schwarz UI (2003) Clinical relevance of genetic polymorphisms in the human CYP2C9 gene. Eur J Clin Investig 33 (Suppl 2): 23-30.
Shannon JR, Flattem NL, Jordan J, Jacob G, Black BK, Biaggioni I, Blakely RD, and Robertson D (2000) Orthostatic intolerance and tachycardia associated with norepinephrine-transporter deficiency. N Engl J Med 342: 541-549.
Small KM, McGraw DW, and Liggett SB (2003) Pharmacology and physiology of human adrenergic receptor polymorphisms. Annu Rev Pharmacol Toxicol 43: 381-411.
Tempfer CB, Schneeberger C, and Huber JC (2004) Applications of polymorphisms and pharmacogenomics in obstetrics and gynecology. Pharmacogenomics 5: 57-65.
Zhou Z, Gong Q, and January CT (1999) Correction of defective protein trafficking of a mutant HERG potassium channel in human long QT syndrome. Pharmacological and temperature effects. J Biol Chem 274: 31123-31126.