hKv4.3 Channel Characterization and Regulation by Calcium Channel Antagonists Thierry P. G. Calmels, 1 Jean-Franc ¸ois Faivre, 1,2 Brigitte Cheval, Jean-Luc Javre ´, Sabine Rouanet, and Antoine Bril Department of Cardiovascular Pharmacology, SmithKline Beecham Laboratories Pharmaceutiques, 4 Rue du Chesnay Beauregard, 35760 Saint-Gre ´goire, France Received December 26, 2000 Relative expression pattern of short and long iso- forms of hKv4.3 channels was evaluated by RT-PCR and RPA. Electrophysiological studies were per- formed in HEK293 cells transfected with short or long hKv4.3 cDNA. The long variant L-hKv4.3 was the only form present in lung, pancreas, and small intestine. The short variant S-hKv4.3 was predominant in brain whereas expression levels of the two isoforms were similar in cardiac and skeletal muscles. Properties of the ionic channels encoded by L-hKv4.3 and S-hKv4.3 cDNAs were essentially similar. Cadmium chloride and verapamil inhibited hKv4.3 current (with EC50s of 0.110  0.004 mM and 492.9  15.1 M, respectively). Verapamil also accelerated current inactivation. An- other calcium channel antagonist nicardipine was found inactive. In conclusion, this study confirms that both isoforms underlie the transient outward potas- sium current. Moreover, calcium channel inhibitors markedly affect hKv4.3 current, an effect which must be considered when evaluating transient outward po- tassium channel properties in native tissues. © 2001 Academic Press Key Words: heart; K-channel; I to current; Kv4.3; cal- cium channel antagonist; tissue localisation; RT-PCR; Rnase protection assay. The transient outward potassium current I to plays a major role in the repolarisation process of many differ- ent cell types. In cardiac myocytes, it is involved in the early repolarization phase of the action potential and its modulation is known to affect notably the shape of cardiac action potential (1, 2). The physiological and pathophysiological relevance of this current has been well established: In normal heart, the density of the I to current is not uniformely distributed and this current is considered as an important determinant of the elec- trophysiological heterogeneity characterized in the dif- ferent cardiac cell types (3, 4). On another hand, in pathological situations, and particularly in congestive heart failure, I to current density is reduced, an effect which at least in part explains the prolongation of the cardiac action potential observed in these pathological conditions (5–7). The cardiac I to current is activated by membrane depolarization in a voltage range similar to that of the L-type calcium current (I Ca-L ) (see (8)). Because I Ca-L is present in all cardiac cells where I to is expressed, the characterization of I to in native cardiac cells requires the previous inhibition of the I Ca-L current. This strat- egy has been used indeed in most of the studies aimed at characterizing the I to current in cardiac cells. Inter- estingly, several reports indicate that calcium channel antagonists can interfere with native potassium chan- nels (9 –11) including I to channels (12–14). However, data evaluating the direct influence of calcium channel inhibitors on the I to current are scarce because of the obvious difficulty to record I to in real control conditions. The recent identification of the molecular correlate for the cardiac I to current (15) gives an interesting opportunity to address specifically this question. It is now generally accepted that Kv4.3 underly the tran- sient outward potassium current in human heart (5, 16, 17). The distribution pattern of the two known hKv4.3 isoforms differing by a 19 amino acid insert was evaluated in different tissues by RT-PCR methodology. The relative expression of both isoforms was assessed using RNAse protection assay. The properties of the channels encoded by both isoforms were found essen- tially similar. Finally, the effects of typical calcium channel inhibitors on the hKv4.3 were evaluated by investigating the effects of cadmium chloride, vera- pamil and nicardipine as representatives for inorganic blockers, phenylalkilamines and dihydropyridines, re- spectively. 1 Both authors contributed equally to this study. 2 To whom correspondence should be addressed. Fax: (33) 2 99 28 04 44. E-mail: Jean_Francois_Faivre@sbphrd.com. Biochemical and Biophysical Research Communications 281, 452– 460 (2001) doi:10.1006/bbrc.2001.4396, available online at http://www.idealibrary.com on 452 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.