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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/23984
Title: Molecular determinants of stereoselective bupivacaine block of hKv1.5 channels
Authors: Franqueza, Laura; Longobardo, Mónica; Vicente, Javier; Delpón, Eva; Tamkun, Michael M.; Tamargo, Juan; Snyders, Dirk J.; Valenzuela, Carmen
Keywords: Local anesthetic
K+ channel
Drug binding site
Issue Date: Nov-1997
Publisher: American Heart Association
Citation: Circulation Research 81(6): 1053-1064 (1997)
Abstract: Enantiomers of local anesthetics are useful probes of ion channel structure that can reveal three-dimensional relations for drug binding in the channel pore and may have important clinical consequences. Bupivacaine block of open hKv1.5 channels is stereoselective, with the R(+)-enantiomer being 7-fold more potent than the S(-)-enantiomer (Kd = 4.1 mumol/L versus 27.3 mumol/L). Using whole-cell voltage clamp of hKv1.5 channels and site-directed mutants stably expressed in Ltk- cells, we have identified a set of amino acids that determine the stereoselectivity of bupivacaine block. Replacement of threonine 505 by hydrophobic amino acids (isoleucine, valine, or alanine) abolished stereoselective block, whereas a serine substitution preserved it [Kd = 60 mumol/L and 7.4 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. A similar substitution at the internal tetraethylammonium binding site (T477S) reduced the affinity for both enantiomers similarly, thus preserving the stereoselectivity [Kd = 45.5 mumol/L and 7.8 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. Replacement of L508 or V512 by a methionine (L508M and V512M) abolished stereoselective block, whereas substitution of V512 by an alanine (V512A) preserved it. Block of Kv2.1 channels, which carry valine, leucine, and isoleucine residues at T505, L508, and V512 equivalent sites, respectively, was not stereoselective [Kd = 8.3 mumol/L and 13 mumol/L for S(-)- and R(+)-bupivacaine, respectively]. These results suggest that (1) the bupivacaine binding site is located in the inner mouth of the pore, (2) stereoselective block displays subfamily selectivity, and (3) a polar interaction with T505 combined with hydrophobic interactions with L508 and V512 are required for stereoselective block.
Description: 18 pages, 7 figures, 3 tables.
Publisher version (URL): http://dx.doi.org/10.1161/01.RES.81.6.1053
URI: http://hdl.handle.net/10261/23984
DOI: 10.1161/01.RES.81.6.1053
ISSN: 0009-7330
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