An Analysis of the Variations in Potency of Grayanotoxin Analogs in Modifying Frog Sodium Channels of Differing Subtypes

Abstract

Responses of tetrodotoxin-sensitive (TTX-s) and insensitive (TTX-i) Na(+) channels, in frog dorsal root ganglion (DRG) cells and frog heart Na(+) channels, to two grayanotoxin (GTX) analogs, GTX-I and alpha-dihydro-GTX-II, were examined using the patch clamp method. GTX-evoked modification occurred only when repetitive depolarizing pulses preceded a single test depolarization; modification, during the test pulse, was manifested by a decrease in peak Na(+) current accompanied by a sustained Na(+) current. GTX-evoked modification of whole-cell Na(+) currents was quantified by normalizing the conductance for sustained currents through GTX-modified Na(+) channels to that for the peak current through unmodified Na(+) channels. The dose-response relation for GTX-modified Na(+) channels was constructed by plotting the normalized slope conductance against GTX concentration. With respect to DRG TTX-i Na(+) channels, the EC(50) and maximal normalized slope conductance were estimated to be 31 microM and 0.23, respectively, for GTX-I, and 54 microM and 0.37, respectively, for alpha-dihydro-GTX-II. By contrast, TTX-s Na(+) channels in DRG cells and Na(+) channels in ventricular myocytes were found to have a much lower sensitivity to both GTX analogs. In single-channel recording on DRG cells and ventricular myocytes, Na(+) channels modified by the two GTX analogs (both at 100 microM), had similar relative conductances (range, 0.25-0.42) and open channel probabilities (range, 0.5-0.71). From these observations, we conclude that the differences in responsiveness of DRG TTX-i, and ventricular whole cell Na(+) currents to the GTX analogs studied are related to the number of Na(+) channels modified.

Structural determinants for the action of grayanotoxin in D1 S4–S5 and D4 S4–S5 intracellular linkers of sodium channel α-subunits

“…These investigators reported that mutation of I287N in the D1S4-S5 linker confers a modest level of resistance to pyrethroids; moreover, insects having mutations in S6 segments plus the S4-S5 linkers in D1, D2 and D3 responded to the pyrethroid by a factor of one-hundred times less than wild type. In this connection, it is worthwhile to note certain similarities in the pharmacological effects of pyrethroids and GTX on Na + channels: 1) both pyrethroids and GTX are known to prolong open time of Na + channels and to shift the sodium conductance-voltage curve in the hyperpolarizing direction (12,14); 2) treatment with these toxins induced a characteristic, long-lasting opening of single Na + channels when rectangular pulses were applied (12,14). However, some differences also exist: 1) GTX modifies Na + channels strictly in their open state (4), whereas pyrethroids can modify Na + channels in all states (14), including the channel's open configuration, which still has higher susceptibility to GTX than the resting or closed states; 2) single channel conductance of Na + channels modified by pyrethroids remains the same as that in control (14), whereas GTX modification reduces channel conductance to one-third of the control value (7,12).…”

“…Because GTX exclusively binds to Na + 2) the open channel probabilities are in the range from 0.78 to 0.8; and consequently 3) any difference in GTX action among Na + channel isoforms should be due to a change in the number of channels modified (7,12). Therefore, it is reasonable to use the ratio of chord conductances of GTX-modified to unmodified channels as a measure of channel sensitivity to GTX modification, which is referred to as the relative chord conductance.…”

Sodium Channel Inactivation: Molecular Determinants and Modulation

“…In contrast to toxins of other groups, they also affect ion permeation, impressively demonstrated with VT by a sudden reduction of single-channel conductance on binding (27,413,477) which explains the much reduced macroscopic I Na in the presence of VT (453). GTX-modified channels also have a reduced conductance (508). Likewise sodium channels from eel electroplax inserted in planar bilayers have reduced conductances in the sequence unmodified Ͼ BTX Ͼ GTX Ͼ VT (115,364).…”

Three New Highly Acylated 3,4-seco-Grayanane Diterpenoids from the Fruits of Pieris formosa

“…[20][21][22][23][24][25][26][27] Our previous research on the flowers of P. formosa led to the discovery of a new grayanane diterpenoid, grayanotoxin XXII.…”

“…[20][21][22][23][24][25][26][27] Our previous research on the flowers of P. formosa led to the discovery of a new grayanane diterpenoid, grayanotoxin XXII. 28) Since the secondary metabolites of the different parts of the medicinal plants often differ even growing in the same ecological environments, we further explored the fruits of this plant, in order to look for structural unique and bioactive diterpenoids.…”