Specificity for block by saxitoxin and divalent cations at a residue which determines sensitivity of sodium channel subtypes to guanidinium toxins.

Abstract

Tyrosine 401 of the skeletal muscle isoform (0`1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn ~+ and Cd 2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na § current for a series of point mutations at residue Tyr401 of the rat 0`1 Na channel expressed in Xen0-pus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na § currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with/~ values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7-and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn 2+ (/~ = 8-23 0`M) required Cys, His, or Asp, three residues commonly found to coordinate direcdy with Zn 2+ in metalloproteins. For the Cys mutant of 0.1 and also for the cardiac isoform Na channel (rhl) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na § conductance by Zn ~ § reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The ASp mutant also displayed enhanced affinity for inhibition of conductance by Ca 2+ (/~ = 0.3 mM vs ~40 mM in wild type), but block by Ca ~ § was incomplete, saturating at ~69% inhibition. In contrast, Cd 2 § completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the particular residue at

Interactions of Key Charged Residues Contributing to Selective Block of Neuronal Sodium Channels by μ-Conotoxin KIIIA

“…The mutation Na v 1.4 Y401F had little effect on the K d of KIIIA-K7A but increased the maximal block to 69%, near that for Na v 1.2. This suggests that with KIIIA-K7A bound, ion passage through the channel is limited by Tyr401, which is a primary determinant of tetrodotoxin and saxiton affinity (Favre et al, 1995).…”

L-Cysteine Prevents Oxidation-Induced Block of the Cardiac Na+ Channel Via Interaction With Heart-Specific Cysteinyl Residues in the P-Loop Region.

“…The block of Na + channels by L-cysteine was partial and reached a plateau at a concentration greater than 3,000 mol/L, which is similar to the Na + channel block by Zn 2+ via creation of a subconductance state of the Na + channels. 16 Thus L-cysteine may produce a subconductive state via a conformational change of the channel pore; the residual currents observed at 3,000 mol/L L-cysteine could form the subconductive state of the single Na + channel.…”

Heterologous expression of NaV1.9 chimeras in various cell systems

“…It was already shown that human Na V 1.9 currents are nearly unaffected by high TTX concentrations [38]. This might be due to the serine residue at position 360 in the pore loop of domain I, i.e., the residue that determines the TTX and STX sensitivity in other Na V channels (e.g., [11,13,30,32]). It is of course expected that human Na V 1.9 channels are as insensitive to TTX and STX, much like Na V 1.8.…”

“…TTX (International Clinical Service GmbH, Munich, Germany) and STX (Sigma) were stored in aqueous solutions at −20°C. The concentration of STX was estimated based on its potency of blocking Na V 1.4 channels according to published IC 50 values [11].…”