Differential Interactions of Na(+) Channel Toxins with T-type Ca(2+) Channels

Two types of voltage-dependent Ca(2+) channels have been identified in heart: high (I(CaL)) and low (I(CaT)) voltage-activated Ca(2+) 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. Ca(2+) 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 Ni(2+) (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 Ni(2+) increased the current approximately fourfold (41% of control), and shifted the dose–response curve of Ni(2+) block to the right (IC(50) from 7.6 to 30 μM). Saxitoxin (STX) at 1 μM abolished the current left in 50 μM Ni(2+). In the absence of Ni(2+), STX potently blocked I(CaT) (EC(50) = 185 nM) and modestly reduced I(CaL) (EC(50) = 1.6 μM). While TTX produced no direct effect on I(CaT) elicited by expression of hCa(V)3.1 and hCa(V)3.2 in HEK-293 cells, it significantly attenuated the block of this current by Ni(2+) (IC(50) increased to 550 μM Ni(2+) for Ca(V)3.1 and 15 μM Ni(2+) for Ca(V)3.2); in contrast, 30 μM TTX directly inhibited hCa(V)3.3-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 Ni(2+) alone. 1 μM STX directly inhibited Ca(V)3.1-, Ca(V)3.2-, and Ca(V)3.3-mediated I(CaT) but did not enhance the ability of Ni(2+) 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 Ca(2+) channels from an ancestor with common structural motifs.