title: The relationship between functional inhibition and binding for K(Ca)2 channel blockers.
abstract: Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are blocked with high affinity by both peptide toxins (e.g. apamin) and small molecule blockers (e.g. UCL 1848). In electrophysiological experiments, apamin shows subtype selectivity with IC50s of ∼100 pM and ∼1 nM for block KCa2.2 and KCa2.3 respectively. In binding studies, however, apamin appears not to discriminate between KCa2.2 and 2.3 and is reported to have a significantly higher (∼20-200-fold) affinity (∼5 pM). This discrepancy between binding and block has been suggested to reflect an unusual mode of action of apamin. However, these binding and electrophysiological block experiments have not been conducted in the same ionic conditions, so it is also possible that the discrepancy arises simply because of differences in experimental conditions. We have now examined this latter possibility. Thus, we measured (125)I-apamin binding to intact HEK 293 cells expressing KCa2 channels under the same ionic conditions (i.e. normal physiological conditions) that we also used for current block measurements. We find that binding and block experiments agree well if the same ionic conditions are used. Further, the binding of apamin and other blockers showed subtype selectivity when measured in normal physiological solutions (e.g.(125)I-apamin bound to KCa2.2 with K L 91±40 pM and to KCa2.3 with K L 711±126 pM, while inhibiting KCa2.2 current at IC50 103±2 pM). We also examined KCa2 channel block in Ca(2+) and Mg(2+) free solutions that mimic conditions reported in the literature for binding experiments. Under these (non-physiological) conditions the IC50 for apamin block of KCa2.2 was reduced to 20±3 pM. Our results therefore suggest that the apparent discrepancy between blocking and binding reported in the literature can be largely accounted for by the use of non-physiological ionic conditions in binding experiments.
authors.affiliation: Department of Neuroscience, Physiology and Pharmacology, University College London, London, United Kingdom.
authors.forename: David Charles Hammond
authors.initials: DC
authors.lastname: Benton
authors.forename: Monique
authors.initials: M
authors.lastname: Garbarg
authors.forename: Guy William John
authors.initials: GW
authors.lastname: Moss
mesh_terms: D001030:Apamin / Q000378:metabolism / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D026902:Potassium Channel Blockers / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism*
chemical_list: D026902:Potassium Channel Blockers; D051640:Small-Conductance Calcium-Activated Potassium Channels; D001030:Apamin
references.citation: J Biol Chem. 2007 Feb 9;282(6):3478-86
references.pmid: 17142458
references.citation: J Neurosci. 2005 Apr 6;25(14):3489-98
references.pmid: 15814779
references.citation: Chembiochem. 2011 Aug 16;12(12):1808-12
references.pmid: 21726033
references.citation: Br J Pharmacol. 2000 Feb;129(4):627-30
references.pmid: 10683185
references.citation: Curr Med Chem. 2007;14(13):1437-57
references.pmid: 17584055
references.citation: Science. 1996 Sep 20;273(5282):1709-14
references.pmid: 8781233
references.citation: J Biol Chem. 1984 Feb 10;259(3):1491-5
references.pmid: 6319399
references.citation: J Neurosci Methods. 1998 Jun 1;81(1-2):73-83
references.pmid: 9696312
references.citation: J Biol Chem. 2010 Aug 27;285(35):27067-27077
references.pmid: 20562108
references.citation: Annu Rev Physiol. 2012;74:245-69
references.pmid: 21942705
references.citation: FEBS Lett. 2000 Mar 10;469(2-3):196-202
references.pmid: 10713270
references.citation: J Physiol. 2001 Sep 1;535(Pt 2):323-34
references.pmid: 11533126
references.citation: J Physiol. 1985 Jan;358:373-94
references.pmid: 2580085
references.citation: J Biol Chem. 1997 Sep 12;272(37):23195-200
references.pmid: 9287325
references.citation: Br J Pharmacol. 2000 Mar;129(5):991-9
references.pmid: 10696100
references.citation: J Med Chem. 2000 Sep 21;43(19):3478-81
references.pmid: 11000001
references.citation: Nature. 1981 Oct 8;293(5832):471-4
references.pmid: 6273730
references.citation: J Biol Chem. 1994 Jul 8;269(27):18053-61
references.pmid: 8027065
references.citation: Naunyn Schmiedebergs Arch Pharmacol. 2002 Nov;366(5):470-7
references.pmid: 12382077
references.citation: J Med Chem. 2008 Dec 11;51(23):7625-34
references.pmid: 18998663
references.citation: J Physiol. 2003 Nov 15;553(Pt 1):13-9
references.pmid: 14555714
references.citation: Neuropharmacology. 2001 Sep;41(3):341-50
references.pmid: 11522325
references.citation: Nature. 1998 Oct 1;395(6701):503-7
references.pmid: 9774106
references.citation: Proc Natl Acad Sci U S A. 2011 Nov 8;108(45):18494-9
references.pmid: 22025703
references.citation: J Med Chem. 1998 Jan 1;41(1):2-5
references.pmid: 9438015
references.citation: Nature. 1979 Nov 22;282(5737):415-7
references.pmid: 228203
references.citation: FEBS Lett. 1983 Feb 21;152(2):265-9
references.pmid: 6298001
references.citation: J Physiol. 1996 Nov 1;496 ( Pt 3):627-37
references.pmid: 8930831
references.citation: Proc Natl Acad Sci U S A. 1982 Feb;79(4):1308-12
references.pmid: 6122211
references.citation: J Physiol. 2000 Sep 1;527 Pt 2:283-90
references.pmid: 10970429
references.citation: Nat Rev Neurosci. 2004 Oct;5(10):758-70
references.pmid: 15378036
references.citation: Curr Med Chem. 2003 Apr;10(8):625-47
references.pmid: 12678783