Novel Blockers of T-Type Calcium Channels Modify Gating

Abstract

Physiological and biophysical studies of T-type calcium channels, a subclass of voltage gated ion channels with a characteristically low voltage threshold for activation, have been hampered by a lack of selective antagonists, and no specific blockers of T-type calcium channels are currently clinically available. Several compounds have recently been identified that block calcium entry into cells expressing T-type calcium channels (Uebele et al, Cell Biochem Biophys 55:81). These compounds include an amide (TTA-A2) and a quinazolinone (TTA-Q4). We used whole cell patch clamp to examine block by these two experimental drugs in HEK 293 cells heterogeneously expressing CaV3.1. When holding potential was −110 mV, the TTAs blocked resting T-type channels with sub-micromolar affinity (ED50=890nM for TTA-A2 and 560nM for TTA-Q4). Block was voltage dependent, and the affinity rose by approximately 5-fold when tested at −20 mV (ED50=200nM for TTA-A2 and 130nM for TTA-Q4). Although the extent of block was voltage dependent, the kinetics were not. Neither the rate of development of block (τ=100ms for TTA-A2 and 178ms for TTA-Q4) nor the recovery from block (τ=1.2 sec for TTA-A2 and 4 sec for TTA-Q4) changed with voltage, consistent with a guarded receptor model. An examination of gating current revealed that both TTA-A2 and TTA-Q4 inhibited the movement of gating charge at depolarized potentials, without shifting the half-point of activation. These findings suggest that TTA-A2 and TTA-Q4 inhibit T-type current by restricting the movement of one or more voltage sensors, which may prevent the opening of the channel.Support: T32HL007381 (PB), RO1HL065680.