© 1996 American Heart Association, Inc.
Articles |
Antiarrhythmic Drug Binding Sites in Cardiac K+ Channels
From the Division of Cardiovascular and Metabolic Diseases, Wyeth-Ayerst Research, Princeton, NJ.
Correspondence to Thomas J. Colatsky, PhD, Division of Cardiovascular and Metabolic Diseases, Wyeth-Ayerst Research, CN 8000, Princeton, NJ 08543. E-mail colatst@war.wyeth.com.
Key Words: Editorials • quinidine • K+ channels • drug binding sites • drug-channel interactions • drug specificity
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Introduction |
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The picture that most of us carry in our minds of how drugs block ion channels is taken from the classic studies of Hille1 involving local anesthetics and Armstrong2 involving tetraethylammonium (TEA) derivatives. This picture is elegant in its simplicity yet robust enough to have provided a conceptual framework for explaining a variety of experimental results and for developing a rigorous model of the drug-channel interaction.3 4 We see cationic drugs entering the channel from the cytoplasmic side, only after the channel has opened, and binding to a site 20% to 50% of the way into the membrane field. Neutral drugs are not restricted to the open pore but can also access and leave the blocking site via a separate hydrophobic pathway. The movement of drugs into and out of the channel is ultimately governed by membrane potential and channel gating, producing use-dependent changes in membrane current and electrophysiological activity. Larger and more hydrophilic drugs move slowly and have slower use-dependent kinetics. Smaller and more hydrophobic drugs show rapid use dependence.
Molecular biology techniques have greatly refined this picture over the past several years by providing new and important structural detail. For K+ channels, TEA binding has been localized to a critical threonine residue in the P region of the channel that is highly conserved among different K+ channel subtypes.5 Specific amino acid residues in the predicted transmembrane S6 segment of domain IV (IVS6) were found to be critical determinants of local anesthetic action in Na+ channels6 7 and to govern the block of Ca2+ channels by phenylalkylamines.8 The article by Yeola et al9 in this issue of Circulation Research complements and extends these earlier findings by analyzing the hydrophobic components of quinidine binding in the cloned human Kv1.5 delayed rectifier K+ channel (hKv1.5). Using site-directed mutagenesis to modify nonconserved amino acid residues from the S6 region, these investigators have been able to demonstrate that quinidine binding is stabilized by hydrophobic interactions at the inner mouth of the channel and that the S6 domain is an important contributor to open-channel block. These findings have important implications for understanding the basic mechanisms by which drugs block cardiac K+ channels and also lend support to the possibility of defining a unique "receptor" site within the channel pore that can be exploited in the rational design of new K+ channel–specific antiarrhythmic drugs.
One is struck by several findings in the article by Yeola et al.9 First, the so-called TEA binding site within the P loop (T477) of hKv1.5 appears to be less important for antiarrhythmic drug binding than one might have supposed on the basis of previous models. Mutations that reduce TEA binding in Shaker and Kv2.1 K+ channels >10-fold (ie, T477S and L508M) produce only modest changes in quinidine block of hKv1.5, suggesting that other (eg, hydrophobic) components are perhaps of greater (or at least equal) importance in determining quinidine's affinity to the "receptor." This suggests a more complex and specific relationship between the drug and the channel and helps to explain observed differences in potency and blocking properties that can occur despite homologous pore structures and the presence of a ubiquitous tertiary amine group believed to be the primary site of attachment of the drug to the channel. Second, increasing the hydrophobicity of amino acid residues at key sites within the S6 segment markedly enhances quinidine binding, although not to the same extent as do the same amino substitutions in the homologous regions of Shaker and Kv2.1. The quantitatively different responses of Shaker, Kv2.1, and hKv1.5 channels to similar mutations suggest a strong possibility that channel-specific agents can be designed. Third, the mutations producing the largest changes in quinidine affinity are those associated with a reduction in dissociation rate constant, consistent with a drug sticking better to the channel rather than having improved access. Overall, these results suggest that marked differences in binding affinity can be associated with what appear to be relatively small and specific changes in channel structure.
A comparison between local anesthetic blocking mechanisms in Na+ and K+ channels is valuable in identifying common elements of the drug-channel interaction. Previous work by Ragsdale et al6 has established that portions of the IVS6 segment are involved in regulating local anesthetic binding and use-dependent block in heart and brain Na+ channels. Mutating F1764 near the middle of the IVS6 segment of the rat brain Na+ channel to alanine dramatically reduces the affinity of both open and inactivated channels to lidocaine and etidocaine, whereas mutations at N1769 increase the degree of resting block.6 In the cardiac Na+ channel (rH1), the quaternary lidocaine derivative QX-314 was found to bind to a single receptor site (F1762) whether applied from the external or internal surface; the threonine at location 1755 in rH1 controlled the ability of QX-314 to access this site from either side of the membrane, whereas the wild-type brain Na+ channel could only be blocked by internally applied drug.7 As stated by Yeola et al,9 the Na+ channel residues 1764 and 1771 correspond to hKv1.5 residues 502 and 508, which places the local anesthetic binding site in Na+ channels somewhat deeper into the membrane field, consistent with electrophysiological findings. Nevertheless, the parallels between these results remain striking, particularly given that some local anesthetic molecules like quinidine and procainamide can simultaneously block both Na+ and K+ channels. Further comparisons are likely to provide a number of new insights that may be useful in drug design. For example, it has long been appreciated that changing the substituent on the aromatic moiety of the local anesthetic pharmacophore from an electron donating group (eg, NH2) to an electron withdrawing (eg, NO2) or electron neutral group (eg, N-acetyl) can eliminate the ability of a drug to block Na+ channels while retaining its blocking activity at K+ channels.10 The results presented in this editorial, together with recent results obtained on brain and heart Na+ channels, begin to shed some light on the molecular basis for these structure-activity relations. Any points of correspondence between the critical sites in these channels that determine blocking characteristics need to be clearly and rigorously defined. Existing observations, like the relative insensitivity of the K+ channel blockers to changes in the electronic nature of the aromatic ring, also need to be fitted into the emerging picture of the complete receptor site. In fact, a pharmacological study in which the physicochemical properties of the local anesthetic molecule are systematically varied would nicely complement the careful mutational analysis of channel structure presented by Yeola et al.9
In closing, the results reported by Yeola et al9 have moved us closer toward a molecular understanding of antiarrhythmic drug action in cardiac K+ channels. Although the studies were conducted with hKv1.5, which has been tentatively assigned to a rapid delayed rectifier current in atrial muscle,11 and with quinidine, which is a fairly promiscuous blocker of a variety of K+ channels, the results should have broader relevance given the overall similarities between the blocking mechanisms observed in other K+ channel subtypes and with other, more specific classes of drug. Some critical pieces of the puzzle remain missing from the study, because the mutations needed to confirm a particular finding have resulted in nonfunctional channels or expression levels too low to resolve currents of sufficient magnitude. However, a closer examination of how the mechanisms of Na+ and K+ channel block differ and a prediction of the three-dimensional topology of the putative receptor binding site will add new texture to the picture of the drug-channel interaction and may lead us in the direction of newer and better designed therapeutic agents.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionReferences |
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2.
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