Transfer of High Sensitivity for Benzothiazepines from L-type to Class A (BI) Calcium Channels

Abstract

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (␣ 1S or ␣ 1C ) to a weakly sensitive, nonstereoselective class A (␣ 1A ) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type ␣ 1 into the ␣ 1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (؊)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (؊)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.L-type calcium (Ca 2ϩ ) channels (classes C (formed by ␣ 1C subunits), D (␣ 1D ), and S (␣ 1S )) possess high affinity stereoselective drug receptors for Ca 2ϩ antagonists such as 1,4-dihydropyridines (DHPs), 1 phenylalkylamines (PAAs), and benzothiazepines (BTZs) (reviewed in Refs. 1-5) located on their pore-forming ␣ 1 channel subunit (6). Classes A (␣ 1A ), B (␣ 1B ), and E (␣ 1E ) Ca 2ϩ channels are insensitive for DHPs (2, 5, 7-9) and only weakly sensitive for . Essential parts of the high affinity binding sites for DHPs and PAAs on L-type Ca 2ϩ channels have been identified by replacing sequence stretches in ␣ 1C or ␣ 1S subunits by corresponding non-L-type sequences (8, 13) or by mutating single amino acids in these subunits (10, 13). Alternatively, molecular determinants of the high affinity DHP and PAA receptor sites could be localized in pore-lining regions of repeats III and/or IV by transferring L-type ␣ 1 sequences into the ␣ 1A subunit (9, 12). Transfer of segment IVS6 from ␣ 1S to ␣ 1A enhanced PAA sensitivity of the resulting ␣ 1A /␣ 1S chimera to the level of L-type ␣ 1 subunits (12).The efficacy of the BTZ diltiazem as an antiarrhythmic and antihypertensive drug is due to its voltage-and use-dependent block of L-type Ca 2ϩ channels (14). Studies on cloned ␣ 1 subunits of different Ca 2ϩ channel classes (C, B, A, and E) have enabled a more precise characterization of their pharmacological features (15). To identify the molecular determinants of the high affinity BTZ interaction domain of L-type Ca 2ϩ channels, we introduced corresponding L-type sequence stretches into ␣ 1A . The diltiazem sensitivity of the resulting ␣ 1 chimeras was measured as use-dependent barium current (I Ba ) block after coexpression with ␤ 1a (16) and ␣ 2 /␦ (17) in Xenopus oocytes. EXPERIMENTAL PROCEDURESMolecular Biology-The construction of L-type chimera Lh (repeats I-IV from ␣ 1C-a (18)) with the N terminus replaced with ␣ 1S (19), as well as construction of chimeras AL12h and AL22, were described previously (9, 12). Chimera AL20 was generated by replacing the ClaI (nucleotide position, 4925)-XbaI (3Ј-polylinker) fragment of AL9 (9) by ...

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“…An important role of segment IVS6 was subsequently demonstrated by Döring et al (27). Mutations within IVS6 of Ca v 2.1 produce profound effects on the pharmacological properties of Ca v 2.1 as well as on inactivation (28). However, the precise relationship (e.g.…”