Transfer of L-type Calcium Channel IVS6 Segment Increases Phenylalkylamine Sensitivity of α1A

Abstract

Conditioned ("use-dependent") inhibition by phenylalkylamines (PAAs) is a characteristic property of Ltype calcium (Ca 2؉ ) channels. To determine the structural elements of the PAA binding domain we transferred sequence stretches of the pore-forming regions of repeat III and/or IV from the skeletal muscle ␣ 1 subunit (␣ 1S ) to the class A ␣ 1 subunit (␣ 1A ) and expressed these chimeras together with ␤ 1a and ␣ 2 /␦ subunits in Xenopus oocytes. The corresponding barium currents (I Ba ) were tested for PAA sensitivity during trains of depolarizing test pulses (conditioned block). I Ba of oocytes expressing the ␣ 1A subunit were only weakly inhibited by PAAs (less than 10% conditioned block of I Ba during a 100-ms pulse train of 0.1 Hz). Transfer of the transmembrane segment IVS6 from ␣ 1S to ␣ 1A produced an enhancement of PAA sensitivity of the resulting ␣ 1A /␣ 1S chimera comparable to L-type ␣ 1 subunits (about 35% conditioned block of I Ba during a 100-ms pulse train of 0.1 Hz). Our results demonstrate that substitution of 11 amino acids within the segment IVS6 of ␣ 1A with the corresponding residues of ␣ 1S is sufficient to transfer L-type PAA sensitivity into the low sensitive class A Ca 2؉ channel.Voltage-gated Ca 2ϩ channels mediate the depolarization-induced influx of Ca 2ϩ into excitable cells, thereby regulating cellular processes such as muscle contraction, propagation of action potentials, secretion, and gene expression. They are heterooligomeric complexes formed by at least an ␣ 1 , ␤, and ␣ 2 /␦ subunit (1). The ␣ 1 subunit is the pore-forming membrane protein consisting of four homologous repeats (I-IV), each of them composed of six transmembrane segments (S1-S6) (2). Based on different pharmacological and biophysical properties, various types of voltage-dependent Ca 2ϩ channels (T, L, N, P, Q, and R) can be distinguished (3-5). Their sensitivity to Ca (PAAs) (6, 7). In contrast, classes A, B, and E are considered to be DHP-insensitive.To localize Ca 2ϩ antagonist interaction domains within Ltype ␣ 1 subunits we have recently shown (8) that sensitivity for DHP Ca 2ϩ channel blockers and activators can be transferred to class A ␣ 1 subunits (␣ 1A ) by substituting regions close to the channel pore in repeats III and IV (segments IIIS5, IIIS6, IVS6, and the respective S5-S6 linkers) with the corresponding L-type ␣ 1 sequences (from ␣ 1S or ␣ 1C ). The DHP sensitivity was lost after replacement of short sequence stretches within these regions by the ␣ 1A sequence. For example, when segment IIIS5 was replaced by ␣ 1A sequence, DHP sensitivity disappeared. The same effect was observed after replacing the IVS5-IVS6 linker. These results suggest that the DHP molecules interact with multiple amino acid residues located within distant regions of the primary structure.Hockerman et al. (9) recently identified three amino acid residues within segment IVS6 of a L-type Ca 2ϩ channel ␣ 1C subunit (Tyr-1463, Ala-1467, and Ile-1470, numbering according to ␣ 1C-c ) (10) as critical determinants for high affinity ...

Amino Acids in Segment IVS6 and β-Subunit Interaction Support Distinct Conformational Changes during Cav2.1 Inactivation

“…1A). Simultaneous replacement of both ␣ 1 2.1 residues by their ␣ 1 1.1 counterparts almost perfectly reproduced the phenotype of chimera AL23 (27), a chimera in which the entire IVS6 segment is of ␣ 1 1.1 sequence. The similarity between ML/II and AL23 is emphasized by the fact that these two constructs exhibit not only similar rates of I Ba inactivation ( Fig.…”

“…This study demonstrated a key role of segment IS6 and adjacent intra-and extracellular stretches in the rate of channel inactivation. 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).…”

“…Here we evaluate previously observed IVS6-mediated changes in Ca v 2.1 inactivation (27) at the level of single amino acids. The results identify a number of residues within IVS6 that represent critical determinants of voltage-dependent inactivation of Ca V 2.1.…”

Molecular Determinants of Frequency Dependence and Ca2+ Potentiation of Verapamil Block in the Pore Region of Cav1.2

“…Quaternary amine derivatives of verapamil and methoxyverapamil block Ca v 1.2 channels in smooth muscle cells only when applied to the intracellular side of the membrane, whereas a quaternary amine derivative of desmethoxyverapmil blocks Ca v 1.2 from either side of the membrane (Berjukov et al, 1996). Block of closed channels by desmethoxyverapamil involves specific amino acid residues in transmembrane segments IIIS6 and IVS6 that are unique to L-type channels (Hockerman et al, 1995(Hockerman et al, , 1997aDoring et al, 1996). PAAs preferentially block Ca v 1.2 channels undergoing high-frequency depolarizations, a property called frequency-dependent block (Lee and Tsien, 1983), which is the result of a higher drug affinity for the inactivated state of the channel (Johnson et al, 1996;Nawrath and Wegener, 1997).…”

A Region in IVS5 of the Human Cardiac L-type Calcium Channel Is Required for the Use-dependent Block by Phenylalkylamines and Benzothiazepines

“…In more recent studies, the molecular identification of the binding sites on the ␣ 1 subunit for PAA (5)(6)(7)(8), BZT (9,10), and DHPs (7,(11)(12)(13)(14) were revealed. The region YMAI in IVS6 is a common binding site for both BZT (9) and PAA (7,15).…”