Extracellular hemin is a reverse use-dependent gating modifier of cardiac voltage-gated Na⁺ channels.

Heme (Fe²⁺-protoporphyrin IX) is a well-known protein prosthetic group; however, heme and hemin (Fe³⁺-protoporphyrin IX) are also increasingly viewed as signaling molecules. Among the signaling targets are numerous ion channels, with intracellular-facing heme-binding sites modulated by heme and hemin in the sub-µM range. Much less is known about extracellular hemin, which is expected to be more abundant, in particular after hemolytic insults. Here we show that the human cardiac voltage-gated sodium channel hNa_V1.5 is potently inhibited by extracellular hemin (<i>IC</i> ₅₀ ≈ 80 nM), while heme, dimethylhemin, and protoporphyrin IX are ineffective. Hemin is selective for hNa_V1.5 channels: hNa_V1.2, hNa_V1.4, hNa_V1.7, and hNa_V1.8 are insensitive to 1 µM hemin. Using domain chimeras of hNa_V1.5 and rat rNa_V1.2, domain II was identified as the critical determinant. Mutation N803G in the domain II S3/S4 linker largely diminished the impact of hemin on the cardiac channel. This profile is reminiscent of the interaction of some peptide voltage-sensor toxins with Na_V channels. In line with a mechanism of select gating modifiers, the impact of hemin on Na_V1.5 channels is reversely use dependent, compatible with an interaction of hemin and the voltage sensor of domain II. Extracellular hemin thus has potential to modulate the cardiac function.