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Technology Review Atomic Absorption Spectroscopy in Ion 5315_10_p569-574 11/5/04 12:39 PM Page 569 ASSAY and Drug Development Technologies Volume 2, Number 5, 2004 © Mary Ann Liebert, Inc. • Technology Review • Atomic Absorption Spectroscopy in Ion Channel Screening Larisa Stankovich, David Wicks, Sasko Despotovski, and Dong Liang Abstract: This article examines the utility of atomic absorption spectroscopy, in conjunction with cold flux assays, to ion channel screening. The multiplicity of ion channels that can be interrogated using cold flux assays and atomic absorption spectroscopy is summarized. The importance of atomic absorption spectroscopy as a screening tool is further elaborated upon by providing examples of the relevance of ion channels to various physiological processes and targeted diseases. Towards Ion Channel Drug Discovery Channelopathies and Development With ion channels playing such an important role in ECENT YEARS HAVE SEEN a significant shift of re- many physiological functions, it is not surprising that ion Rsources in the pharmaceutical industry towards the channel dysfunction has been implicated in a number area of ion channel drug discovery and development. This of diseases and disorders. Diseases that are caused by shift was compelled by the issue of drug-induced QT pro- defective ion channel proteins are termed “channelo- longation and the emergence of the S7B, E14 guidance pathies.” Indeed, ion channels represent one of today’s documents for drug safety assessment. The importance more promising and exciting classes of therapeutic tar- of the hERG ion channel to drug development created gets on account of the broad range of conditions that are significant interest in other ion channel targets. As a re- poised to benefit from agents that modulate ion channel sult, over 6 billion dollars in yearly sales and 15% of the activity. Table 2 lists some of the diseases and disorders top-selling drugs are targeted for ion channels.1–3 in which ion channel dysfunction has been implicated. Physiological Significance of Ion Channels Flux Assays in Ion Channel Screening Ion channels are integral membrane proteins that per- A number of technologies are available to screen mit the flow of ions, such as calcium, potassium, sodium, against ion channel targets. Xu et al.46 segmented the and chloride, into and out of cells. Ion channels are pres- ion channel assay market and identified the advantages ent in all human cells and are involved in functions such and disadvantages of various technologies. Available as nerve transmission, cellular homeostasis, hormonal se- technologies include manual patch-clamp, automated cretion, and the heartbeat. The wide range of physiolog- patch-clamp instruments, fluorescence-based platforms, ical processes in which ion channels are involved is sug- membrane binding assays, radioactive ionic flux assays, gested by Table 1. and non-radioactive flux assays. However, this review Aurora Biomed Inc., Vancouver, BC, Canada. ABBREVIATIONS: CFTR, cystic fibrosis transmembrane regulator; hERG, human ether-a-go-go-related gene. 569 5315_10_p569-574 11/5/04 12:39 PM Page 570 570 Stankovich et al. TABLE 1. PHYSIOLOGICAL PROCESSES INVOLVING ION CHANNELS Physiological process Ion channel or gene encoding ion channel Apoptosis MAC4 Purinergic P2X5 Bicarbonate secretion SLC266 Bone resorption CIC-77 RyR8 Cardiac repolarization KCNQ1, hERG, SCN5A, KCNE1, KCNE29 Chloride absorption CFTR10 Inflammation ASIC’s11 12 Insulin secretion KATP (Kir6.2 and SUR1 subunits) Metastasis CLCA13 Neuronal excitability KCNQ2/KCNQ314 KCNQ515 T-type Ca channels16 Neuronal migration Kv4.317 Nociception L-type Ca channels18 Regulation of basal metabolic rate Kv1.319 Smooth muscle relaxation 20 Cavernosal BKCa 21 Urinary SKCa, BKCa, and RyR Vascular KCNMB122 Sperm motility PKD223 T cell activation Kv1.1, Kv1.324 TABLE 2. DISEASES OR DISORDERS INVOLVING ION CHANNELS Disease or disorder Ion channel or gene encoding ion channel Andersen-Tawil’s syndrome KCNJ225 Bartter’s syndrome SLC12A2, KCNJ1, ROMK, CIC-Ka, CICKb26 Benign familial neonatal convulsions KCNQ2, KCN314 SCN2A27 Brody’s disease SERCA128 Brugada’s syndrome SCN5A29 Periodic paralysis SCN4A30,31 Cancer CLCA13 Cystic fibrosis CFTR10 SLC266 Epilepsy T-type Ca channels16 20 Erectile dysfunction BKCa Familial hemiplegic migraine CACNA1A32 Heart failure Cardiac P2X433 Hypertension KCNMB122 Isolated cardiac conduction disease SCN5A34 21 Incontinence SKCa, BKCa, and RyR Inflammation ASIC’s11 Lenegre’s disease SCN5A35 Long-QT syndrome KCNQ1, hERG, SCN5A, KCNE1, KCNE29 HCN436 Male sterility PKD223 Multiple sclerosis Kv1.337 Nav1.2, Nav1.6, Naϩ/Ca2ϩ exchanger38 Myotonia congenita CLCN139,40 Myotonic dystrophy SERCA128 Neuropathic pain Nav1.841 Nav1.942 Obesity Kv1.319 Osteopetrosis CIC-77 Polycystic kidney disease PKD243 Schizophrenia SK344 45 Type 1 diabetes KATP (Kir6.2 subunit) 12 Type 2 diabetes KATP (Kir6.2 and SUR1 subunits) 5315_10_p569-574 11/5/04 12:39 PM Page 571 Atomic Absorption Spectrometry 571 TABLE 3. CHANNELS THAT HAVE BEEN EXAMINED USING THE COLD FLUX ASSAY APPROACH Family Ion channel or gene encoding ion channel K hERG49,50 KCNQ251 48,52 BKCa 48 SKCa Kv1.1, Kv1.448 Kv1.353 Na SCN5A54 Cl CFTR55 Ca Cardiac L-type56 Non-selective cation Nicotinic acetylcholine48 Purinergic P2X48 Transporter Naϩ/Kϩ pump57 Kϩ-ClϪ co-transporter58 will focus on the non-radioactive (cold) flux assay tech- screening has steadily increased, and publications nology from Aurora Biomed Inc. (Vancouver, BC, describing the various channels examined are listed in Canada). Table 3. Non-radioactive flux assay technology utilizes highly Hot flux (radioactive) assays, on the other hand, have sensitive flame atomic absorption spectrometers capable been used for many years to assess ion channel function. of dealing with the low sample volumes that are typically Hot flux assays employ radiotracers to monitor ion flux used in pharmaceutical research. Cold flux assays use through channels. Some of the ion channels that have tracer ions: molecules that are similar in size and charge been examined using radiotracers are listed in Table 4. to the ion of interest. To study potassium and sodium Overall, Tables 3 and 4 represent represent some of the channels, the rubidium and lithium ions, respectively, are most recent and significant literature on their respective used. For chloride channels, the chloride ion itself is used topics. with an indirect detection procedure involving precipita- Any of the channels studied using the hot flux assay tion with silver nitrate and measurement of the concen- approach should, in theory, be amenable to the cold flux tration of free silver. assay approach. The cold flux assays that have been de- The use of flux assays in high-throughput screening of veloped for the Ion Channel Readers by the Aurora Bio- ion channels for pharmaceutical research has been well med team include the Rb flux assay against the hERG50 summarized by Gill et al.47 The cold flux assay as ap- and Kv1.356 channels as well as against the Naϩ/Kϩ plied to the study of ion channel function is a relatively pump57 and Kϩ-ClϪ co-transporter58; the Li flux assay novel approach and was first proposed by Terstappen.48 against the SCN5A channel53; and the Cl flux assay for Since then, the use of cold flux assays in ion channel the CFTR channel.54 The multiplicity of ion channels that TABLE 4. CHANNELS THAT HAVE BEEN EXAMINED USING THE HOT FLUX ASSAY APPROACH Family Ion channel or gene encoding ion channel Radiotracer K hERG59 86Rbϩ 60 86 ϩ BKCa Rb 61,62 86 ϩ IKCa Rb 63 86 ϩ SKCa Rb Kv63 86Rbϩ KCh63 86Rbϩ 64 22 ϩ Na Neuronal Nav Na Cl CFTR65 125IϪ 66 45 2ϩ Ca Neuronal Cav Ca Non-selective cation Nicotinic acetylcholine67,68 86Rbϩ Purinergic P2X69 86Rbϩ Transporter Naϩ/Kϩ pump70 86Rbϩ Kϩ-ClϪ co-transporter71,72 86Rbϩ Naϩ-KϪ-ClϪ co-transporter73,74 86Rbϩ, 125IϪ 5315_10_p569-574 11/5/04 12:39 PM Page 572 572 Stankovich et al. can be examined using the cold flux assay approach is 14. Singh NA, Westenskow P, Charlier C, Pappas C, Leslie J, indicative of the versatility of this technique for ion chan- Dillon J, Anderson VE, Sanguinetti MC, Leppert MF, nel screening. BFNC Physician Consortium: KCNQ2 and KCNQ3 potas- sium channel genes in benign familial neonatal convul- sions: expansion of the functional and mutation spectrum. Brain 2003;126:2726–2737. References 15. Schroeder BC, Hechenberger M, Weinreich F, Kubisch C, Jentsch TJ: KCNQ5, a novel potassium channel broadly 1. Augustin HG: Antiangiogenic tumour therapy: will it expressed in brain, mediates M-type currents. J Biol Chem work? Trends Pharmacol Sci 1998;19:216–222. 2000;275:24089–24095. 2. Baxter DF, Kirk M, Garcia AF, Raimondi A, Holmqvist 16. Lacinova L: Pharmacology of recombinant low-voltage ac- MH, Flint KK, Bojanic D, Distefano PS, Curtis R, Xie Y: tivated calcium channels. Curr Drug Targets 2004;3:105– A novel membrane potential-sensitive fluorescent dye im- 111. proves cell-based assays for ion channels. J Biomol Screen 17. Hsu YH, Huang HY, Tsaur ML: Contrasting expression of 2001;7:79–85. Kv4.3, an A-type Kϩ channel, in migrating Purkinje cells 3. Berman FW, Murray TF: Brevetoxin-induced autocrine ex- and other post-migratory cerebellar neurons. Eur J Neu- citotoxicity is associated with manifold routes of Ca2ϩ in- rosci 2003:18;601–612. flux. J Neurochem 2000;74:1443–1451. 18. Todorovic SM, Pathirathna S, Meyenburg A, Jevtovic- 4. Guo L, Pietkiewicz D, Pavlov EV, Grigoriev SM, Todorovic V: Mechanical and thermal anti-nociception in Kasianowicz JJ, Dejean LM, Korsmeyer SJ, Antonsson B, rats after systemic administration of verapamil. Neurosci Kinnaly KW: Effects of cytochrome c on the mitochondr- Lett 2004;360:57–60. ial apoptosis-induced channel MAC. Am J Physiol Cell 19. Xu J, Koni PA, Wang P, Li G, Kaczmarek L, Wu Y, Li Y, Physiol 2003;286:C1109–C1117. Flavell RA, Desir GV: The voltage-gated potassium chan- 5. Sellick GS, Rudd M, Eve P, Allinson R, Matutes E, nel Kv1.3 regulates energy homeostasis and body weight.
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