Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin-resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction.

Abstract

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxinsensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na + in heart and Cd 2+ in brain yielded similar Kn values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca 2+, Mg 2+, Mn 2+, Co z+, Ni 2+, Cd 2+, and Zn z÷) the sequence for [aH]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd 2+ and Zn 2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd 2+ and Zn ~+ in heart and Zn z÷ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of K~ for the higher affinity sites were similar between the channel types, but the lower affinity K(s were larger for heart. On the other hand, the slopes of competition curves for Cd 2+ and Zn 2+ were close to -1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site. On the other hand n-ethylmaleimide (NEM) preferentially reduced the affinity of [3H]STX binding to cardiac channels, and this was prevented by coincubation with STX. The NEM treatment also reduced the competition of Cd 2+ for STX binding to cardiac channels. Diethylpyrocarbonate and phenylglyoxyl abolished binding to both channel types. We conclude that divalent ions act at two sites on the Na channel that affect STX binding. Although both channel types contain one or more carboxyl groups, the charged regions are not identical. The STX binding region in cardiac Na channels contai...

No Apparent Requirement for Neuronal Sodium Channels in Excitation-Contraction Coupling in Rat Ventricular Myocytes

“…4 However, it is known that TTX affinity depends on [Na] o . 22,23 Our K D is similar to previous reports using low [Na] o in cardiac ventricular myocytes 5,13 and after correction for the effect of [Na] o 22 is in agreement with the micromolar range reported previously. The dose-response curve at Ϫ10 mV is well fitted with a double Hill curve (gray line, Figure 1B): if it is assumed that the K D of TTX-resistant current is 628 nmol/L (and the Hill coefficients of both TTX-resistant and TTX-sensitive currents are 1), this gives a K D for the TTX-sensitive current of 8.8 nmol/L (ESE, 6.4; CI, 27) and the fraction of TTX-resistant and TTX-sensitive currents as 0.86 and 0.14 (ESE, 0.03; CI, 0.11), respectively.…”

“…The K D of the TTX-sensitive current is consistent with published data (1 to 25 nmol/L), 4 even after correction for the effect of [Na] o (which has a similar effect on cardiac and neuronal Na channels). 22 The fraction of TTX-sensitive I Na (14Ϯ3%) is in the range of other cardiac preparations (5% to 30%). 5,6,10,13 …”

“…The K D of the TTX-sensitive current is consistent with published data (1 to 25 nmol/L), 4 even after correction for the effect of [Na] o (which has a similar effect on cardiac and neuronal Na channels). 22 The fraction of TTX-sensitive I Na (14Ϯ3%) is in the range of other cardiac preparations (5% to 30%). 5,6,10,13 Characterization of TTX-Resistant and TTX-Sensitive I Na in Rat Ventricular Myocytes I Na subtypes were characterized by their differential TTX sensitivity: 100 nmol/L TTX is expected to block Ϸ92% of TTX-sensitive I Na , with little effect on TTX-resistant I Na (Ϸ14% block, assuming the K D given above).…”

“…At a concentration of 200 nmol/L, TTX is expected to block Ϸ92% of TTX-sensitive I Na , with little effect on TTX-resistant I Na (Ϸ14% block), after accounting for the use of physiological [Na] o , which modifies TTX affinity. 22,23 …”

Developmental Biology of Neoplastic Growth

“…One of these (trimethyloxonium ion) revealed that acidic amino acids were important in the binding of TTX and STX. Pretreatment with this reagent abolished the channel's ability to bind the toxin, whereas pre-exposure of the channel to the toxin protected it from protein modification (Barchi and Weigele 1979;Doyle et al 1993;Spalding 1980;Worley et al 1986). Later, after Na channel genes were sequenced and successfully expressed in recombinant systems, mutagenesis studies pinpointed a series of carboxylate sidechain-containing amino acids in homologous positions within each of the four domains of the channel involved in binding the toxin (Terlau et al 1991) and were hypothesized to form two predominantly negatively charged rings near the ion pore.…”