Calcium Block of Single Sodium Channels: Role of a Pore-Lining Aromatic Residue

Extracellular Ca2+ ions cause a rapid block of voltage-gated sodium channels, manifest as an apparent reduction of the amplitude of single-channel currents. We examined the influence of residue Tyr-401 in the isoform rNaV1.4 on both single-channel conductance and Ca2+ block. An aromatic residue at this position in the outer mouth of the pore plays a critical role in high-affinity block by the guanidinium toxin tetrodotoxin, primarily due to an electrostatic attraction between the cationic blocker and the system of π electrons on the aromatic face. We tested whether a similar attraction between small metal cations (Na+ and Ca2+) and this residue would enhance single-channel conductance or pore block, using a series of fluorinated derivatives of phenylalanine at this position. Our results show a monotonic decrease in Ca2+ block as the aromatic ring is increasingly fluorinated, a result in accord with a cation-π interaction between Ca2+ and the aromatic ring. This occurred without a change of single-channel conductance, consistent with a greater electrostatic effect of the π system on divalent than on monovalent cations. High-level quantum mechanical calculations show that Ca2+ ions likely do not bind directly to the aromatic ring because of the substantial energetic penalty of dehydrating a Ca2+ ion. However, the complex of a Ca2+ ion with its inner hydration shell, Ca2+(H2O)6, interacts electrostatically with the aromatic ring in a way that affects the local concentration of Ca2+ ions in the extracellular vestibule.