Phoneutria nigriventer Toxin Tx3‐1 Blocks A‐Type K⁺ Currents Controlling Ca²⁺ Oscillation Frequency in GH₃ Cells

Abstract

GH 3 cells present spontaneous Ca 2ϩ action potentials and oscillations of intracellular Ca 2ϩ , which can be modified by altering the activity of K ϩ or Ca 2ϩ channels. We took advantage of this spontaneous activity to screen for effects of a purified toxin (Tx3-1) from the venom of Phoneutria nigriventer on ion channels. We report that Tx3-1 increases the frequency of Ca 2ϩ oscillations, as do two blockers of potassium channels, 4-aminopyridine and charybdotoxin. Whole-cell patch clamp experiments show that Tx3-1 reversibly inhibits the A-type K ϩ current (I A ) but does not block other K ϩ currents (delayed-rectifying, inward-rectifying, and largeconductance Ca 2ϩ -sensitive) or Ca 2ϩ channels (T and L type) in these cells. In addition, we describe the sequence of a full cDNA clone of Tx3-1, which shows that Tx3-1 has no homology to other known blockers of K ϩ channels and gives insights into the processing of this neurotoxin. We conclude that Tx3-1 is a selective inhibitor of I A , which can be used to probe the role of this channel in the control of cellular function. Based on the effect of Tx3-1, we suggest that I A is an important determinant of the frequency of Ca 2ϩ oscillations in unstimulated GH 3 cells.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

“…GH3 cells acquired from ATCC (Rockville, MD, USA) were cultured and maintained as described elsewhere (11,12). The standard pipette solution contained 100 mM CsCl, 10 mM EGTA, 2 mM MgCl 2 , and 40 mM HEPES, pH adjusted to 7.2 with CsOH.…”

Pharmacologically active spider peptide toxins

“…This result is reminiscent of the effect described for hanatoxins on Kv2.1 and Kv4.2 channels. Another potassium channel blocker (PnTx3-1) has also been isolated from the venom of the South American spider Phoneutria nigriventer [32]. PnTx3-1 reversibly inhibits the outward-rectifying A-type K + current (I A ) but does not block other K + currents (delayed rectifying, inward rectifying, and large conductance Ca 2+ sensitive) or Ca 2+ channels.…”

Tx2-6 toxin of the Phoneutria nigriventer spider potentiates rat erectile function

“…The partial proteome of this venom was recently described (Richardson et al, 2006). Most of the toxins that have been purified from this venom seem to act on ionic channels, including those for sodium (Araujo et al, 1993;De Lima et al, 2007;Martin-Moutot et al, 2006;Matavel et al, 2002), calcium (Dos Santos et al, 2002;Guatimosim et al, 1997;Leão et al, 2000;Vieira et al, 2005) and potassium (Kushmerick et al, 1999). Some of these toxins have insecticidal activity (for a review see De Lima et al, 2007), interfer with the uptake of L-glutamate on synaptosomes of rat brain (Mafra et al, 1999) and inhibit NMDA-evoked currents in rat hippocampal neurons (Figueiredo et al, 2001).…”