INVESTIGADORES
ENNIS Irene Lucia
artículos
Título:
Novel structural determinants of mu-conotoxin (GIIIB) block in rat skeletal muscle (mu1) Na+ channels
Autor/es:
LI RA; ENNIS IL; VELEZ P; TOMASELLI GF; MARBAN E
Revista:
JOURNAL OF BIOLOGICAL CHEMISTRY
Editorial:
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Referencias:
Año: 2000 p. 27551 - 27558
ISSN:
0021-9258
Resumen:
μ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+
channel (μ1), we examined the contribution of charged residues between
the P loops and S6 in all four domains to μ-CTX block.
Conversion of the negatively charged domain II
(DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by ∼100-fold (wild-type = 22.3 ± 7.0 nm; D762C = 2558 ± 250 nm; E765C = 2020 ± 379 nm).
Restoration or reversal of charge by external modification of the
cysteine-substituted channels with methanethiosulfonate
reagents (methanethiosulfonate ethylsulfonate
(MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect
μ-CTX
block (D762C: IC50, MTSEA+ = 2165.1 ± 250 nm; IC50, MTSES− = 2753.5 ± 456.9 nm; E765C: IC50, MTSEA+ = 2200.1 ± 550.3 nm; IC50, MTSES− = 3248.1 ± 2011.9 nm) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nm) and E765D (IC50 = 22.0 ± 7.0 nm) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nm) and E765K (IC50 = 4202.7 ± 1088.0 nm)
destabilized μ-CTX block even further, suggesting a prominent
electrostatic component of the interactions between these
DII residues and μ-CTX. Kinetic analysis of μ-CTX
block reveals that the changes in toxin sensitivity are largely due to
accelerated
toxin dissociation (k
off) rates with little changes in association (k
on) rates. We conclude that the acidic
residues at positions 762 and 765 are key determinants of μ-CTX block,
primarily by virtue
of their negative charge. The inability of the
bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places
steric constraints
on the sites of toxin interaction.