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

Vincent P. Santarelli ^*, Amy L. Eastwood , Dennis A. Dougherty , Christopher A. Ahern ^* and Richard Horn ^*

^* Department of Molecular Physiology and Biophysics, Institute of Hyperexcitability, Jefferson Medical College, Philadelphia, Pennsylvania; and Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California

Correspondence: Address reprint requests to Dr. Richard Horn, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125. Tel.: 215-503-6725; Fax: 215-503-2073; E-mail: Richard.Horn{at}jefferson.edu.

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 rNa_V1.4 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 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- interaction between Ca²⁺ and the aromatic ring. This occurred without a change of single-channel conductance, consistent with a greater electrostatic effect of the 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.

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