Transfer of 1,4-Dihydropyridine Sensitivity from L-Type to Class A (BI) Calcium Channels

Abstract

L-type Ca2+ channels are characterized by their unique sensitivity to organic Ca2+ channel modulators like the 1,4-dihydropyridines (DHPs). To identify molecular motifs mediating DHP sensitivity, we transferred this sensitivity from L-type Ca2+ channels to the DHP-insensitive class A brain Ca2+ channel, BI-2. Expression of chimeras revealed minimum sequence stretches conferring DHP sensitivity including segments IIIS5, IIIS6, and the connecting linker, as well as the IVS5-IVS6 linker plus segment IVS6. DHP agonist and antagonist effects are determined by different regions within the repeat IV motif. Sequence regions responsible for DHP sensitivity comprise only 9.4% of the overall primary structure of a DHP-sensitive alpha 1A/alpha 1S construct. This chimera fully exhibits the DHP sensitivity of channels formed by L-type alpha 1 subunits. In addition, it displays the electrophysiological properties of alpha 1A, as well as its sensitivity toward the peptide toxins omega-agatoxin IVA and omega-conotoxin MVIIC.

Voltage‐dependent binding and calcium channel current inhibition by an anti‐α1D subunit antibody in rat dorsal root ganglion neurones and guinea‐pig myocytes

“…Results presented here show that an áÔD antibody raised against a VDCC region postulated to be involved in DHP agonist sensitivity (Grabner et al 1996) inhibited IBa in cultured rat DRG neurones (Fig. 4).…”

“…Our evidence suggests that incubation of cultured DRGs with áÔD antibody for 2 h is not a sufficient period of time to induce calcium channel internalization. In addition to being located close to a region which contributes to the áÔD VDCC pore, the region to which the antibody binds is in a domain that was found to confer DHP agonist sensitivity to DHP-insensitive calcium channels (Grabner et al 1996). Consequently, it was unsurprising to find that, in addition to attenuating whole-cell VDCC current, the anti-áÔD antibody reduced the ability of the DHP agonist S-(−)-Bay K 8644 to potentiate IBa in DRG neurones.…”

“…The linker region between IVS5 and IVS6 appeared to be a major site for DHP action. Further studies (Grabner, Wang, Hering, Streissnig & Glossmann, 1996) demonstrated that the minimum áÔC sequence stretches that conferred DHP sensitivity to an áÔA subunit consisted of regions of IIIS5, IIIS6 and their connecting linker, and in addition that the IVS5-IVS6 linker and a segment of IVS6 were required. Differences between agonist and antagonist action appeared to centre on binding differences within repeat IV.…”

“…In this study, the presence of a DHP-sensitive áÔD subunit in both cultured rat DRG neurones and guinea-pig cardiac myocytes has been demonstrated using the reverse transcriptase-polymerase chain reaction (RT-PCR). An áÔD subunit-specific antibody targeted to amino acids 1417-1434 of the rat brain áÔD clone, a sequence corresponding to part of a region reported to confer DHP agonist sensitivity (Grabner et al 1996), was raised. The effect of this antibody on calcium channel currents in DRGs and cardiac myocytes and its effect on DHP sensitivity were also investigated.…”

Dissection of Functional Domains of the Voltage-Dependent Ca2+Channel α2δ Subunit

“…These changes in ␣ 1C conformation then would be responsible for an increase not only in the accessibility of the drug to its site but also in the opening probability of the channel, as it has been observed in Xenopus oocytes (Shistik et al, 1995). Because the binding site for DHPs has been localized to the IIIS5-S6 and IVS5-S6 regions (Grabner et al, 1996;Peterson et al, 1996), these sites also may have been involved in the interaction with the ␣ 2 ␦ complex.…”