Characterization of a Dihydropyridine‐insensitive Cav1.3 Channel

Abstract

The L‐type voltage‐gated calcium channels (L‐VGCCs), Cav1.2 and Cav1.3, play an important role in different physiological processes such as action potential firing, exocytosis and muscle contraction. Due to a lack of selectivity for the dihydropyridine (DHP) class of L‐VGCC blockers, a Cav1.3 mutant that is insensitive to DHPs (Cav1.3/DHPi) has been used to dissect the role of Cav1.3 in glucose‐stimulated insulin secretion and other cellular activities. However, the Cav1.3/DHPi mutant channel has not been biophysically characterized. Our lab created the Cav1.3/DHPi by mutating Thr1033 and Gln1037 that are located in segment 5 of domain III (IIIS5) of the rat neuronal Cav1.3 clone (accession no. AF30010.1) to Tyr and Met, respectively. Ba2+ current (IBa) elicited by different voltage steps from tsA cells expressing Cav channels with and without the presence of drugs was measured. The voltage‐dependent activation and inactivation of Cav1.3/DHPi (V1/2 inact.= ‐26.6±1.2mV, V1/2 act.= ‐22.3±1.1mV) resembles that of wild type Cav1.3 (Cav1.3/WT) (V1/2 inact.= ‐36.0±1.3mV, V1/2 act.= ‐29.8±1.5mV). Cav1.3/DHPi is over 100 fold more insensitive to nifedipine compared to Cav1.3/WT (IC50 for Cav1.3/WT= ~0.55μM; IC50 for Cav1.3/DHPi=~70μM). However, the non‐DHP blockers, diltiazem (500μM) and the recently debated Cav1.3 selective blocker, compound 8 (50μM) achieved comparable inhibition of Cav1.3/WT and Cav1.3/DHPi‐mediated current. Our findings showed that Thr1033 and Gln1037 are not only critical, but also specific for DHP block of Cav1.3. The Cav1.3/DHPi mutant is a useful tool to screen for Cav1.3 specific blockers by removing the DHP binding site which is common to both Cav1.2 and Cav1.3.