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¹ Excigen, Inc., Baltimore, MD 21224
² Department of Medicine, Université de Montréal, and Research Centre, Montréal Heart Institute, Montréal, Québec, Canada
³ School of Kinesiology, Faculty of Applied Sciences, Simon Fraser University, Vancouver, British Columbia, Canada V6B 5K3
⁴ Department of Physiology and Biophysics, Hotchkiss Brain Institute, University of Calgary, Calgary, Alberta, Canada T2N 1N4
⁵ Department of Pharmacology, Center of Biomedical Research Excellence (COBRE), University of Nevada School of Medicine, Reno, NV 89557

Correspondence to Normand Leblanc: nleblanc{at}medicine.nevada.edu

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 Ni²⁺ 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 Ni²⁺ 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.

Abbreviations used in this paper: HP, holding potential; LVA, low voltage–activated inward Ca²⁺ current; SF, selectivity filter; STX, saxitoxin; TTX, tetrodotoxin.

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