Characterization of a Dihydropyridine‐insensitive Ca v 1.3 Channel

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