Differential interactions of Na⁺ channel toxins with T-type Ca²⁺ channels

Two types of voltage-dependent Ca²⁺ channels have been identified in heart: high (I_CaL) and low (I_CaT) voltage-activated Ca²⁺ channels. In guinea pig ventricular myocytes, low voltage-activated inward current consists of I_CaT and a tetrodotoxin (TTX)-sensitive I_Ca component (I_Ca(TTX)). In this study, we reexamined the nature of low-threshold I_Ca in dog atrium, as well as whether it is affected by Na⁺ channel toxins. Ca²⁺ currents were recorded using the whole-cell patch clamp technique. In the absence of external Na⁺, a transient inward current activated near -50 mV, peaked at -30 mV, and reversed around +40 mV (HP = -90 mV). It was unaffected by 30 μM TTX or micromolar concentrations of external Na⁺, but was inhibited by 50 μM Ni²⁺ (by ∼90%) or 5 μM mibefradil (by ∼50%), consistent with the reported properties of I_CaT. Addition of 30 μM TTX in the presence of Ni²⁺ increased the current approximately fourfold (41% of control), and shifted the dose-response curve of Ni²⁺ block to the right (IC₅₀ from 7.6 to 30 μM). Saxitoxin (STX) at 1 μM abolished the current left in 50 μM Ni²⁺. In the absence of Ni²⁺, STX potently blocked I_CaT (EC₅₀ = 185 nM) and modestly reduced I_CaL (EC₅₀ = 1.6 μM). While TTX produced no direct effect on I _CaT elicited by expression of hCa_V3.1 and hCa _V3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni²⁺ (IC₅₀ increased to 550 μM Ni ²⁺ for Ca_V3.1 and 15 μM Ni²⁺ for Ca _V3.2); in contrast, 30 μM TTX directly inhibited hCa _V3.3-induced I_CaT and the addition of 750 μM Ni ²⁺ to the TTX-containing medium led to greater block of the current that was not significantly different than that produced by Ni²⁺ alone. 1 μM STX directly inhibited Ca_V3.1-, Ca_V3.2-, and Ca_V3.3-mediated I_CaT but did not enhance the ability of Ni²⁺ to block these currents. These findings provide important new implications for our understanding of structure-function relationships of I_CaT in heart, and further extend the hypothesis of a parallel evolution of Na⁺ and Ca²⁺ channels from an ancestor with common structural motifs.