A cDNA with 403 nucleotides encoding the precursor of the toxin PnTx2-6 was cloned and sequenced. Subsequent analysis revealed that the precursor begins with a signal peptide and a glutamate-rich propeptide. The succeeding peptide con¢rmed the reported sequence of PnTx2-6. The puri¢ed toxin exerted complex e¡ects on Na + current of frog skeletal muscle. There was a marked decrease of the inactivation kinetics, and a shift to hyperpolarizing potentials of both the Na + conductance and the steady-state inactivation voltage dependences, along with a reduction of the current amplitude. The concentration dependence of the modi¢ed current suggests a K D of 0.8 W WM for the toxin^channel complex. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

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.

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