, 1992), enhanced contraction of vascular smooth muscle (Antunes and Málaque, ICG-001 clinical trial 2003), effects on blood pressure (Costa et al., 1996), activation of the tissue kallikrein-kinin system (Marangoni et al., 1993) and increased nitric oxide (NO) release in cavernosum tissue (Nunes et al., 2008). In severe spider envenomations, cardiovascular alterations such as hypertension, tachycardia and arrhythmias have been described (Antunes and Málaque, 2003). Phoneutria nigriventer (Ctenidae, Labidognatha), popularly known as the “armed” spider, is an aggressive venomous spider found in South America ( Lucas, 1988), responsible for approximately 40% of the spider

bites in humans in Brazil ( Bucaretchi et al., 2000). Its venom contains a cocktail of toxins that affect ionic channels (see review

Gomez et al., 2002) including voltage gated sodium (Na+), calcium (Ca2+) and potassium (K+) channels. We have previously shown that one component of the venom, a neurotoxic peptide originally named Tx3-1 (Cordeiro et al., 1993) blocks voltage activated A-type potassium currents in the GH3 neuroendocrinal cell line. In the interest of large scale testing of this peptide, we subsequently produced recombinant Tx3-1 which maintained its channel blocking activity (Carneiro et al., 2003). In light of its potassium channel blocking activity, this toxin was recently renamed PhKv. In the present study, we describe large scale production of recombinant PhKv and investigated the effects of native and recombinant PhKv on cardiac DNA-PK inhibitor function using an isolated heart preparation and isolated ventricular cardiac myocytes. PhKv toxin was purified from the PhTx3 fraction of the P. nigriventer venom, according to Cordeiro et al. (1993). PhKv,

previously named Tx3-1, contains 40 amino acids and a molecular weight of 4584 Da. All other chemical reagents were of analytical grade. The toxin was dissolved in deionized water and MG-132 in vivo work solutions were prepared by dilution of frozen 1 mM stock solutions immediately before use. The coding region for the toxin was produced by PCR using the Tx3-1-ISEF clone (Carneiro et al., 2003) as template. Serial PCR reactions were used in order to change some of the spider cDNA codons to Escherichia coli preferential codons. The oligonucleotides Tx31F (5′GCA GAA TGC GCA GCT GTT TAT GAA CGT TGC GGT AAA G 3′) and Tx31R (5′TTT GCA CGG ACG TTC TTC ACA G 3′) were used to amplify a fragment that codes for the 5′ region of the spider cDNA and the oligonucleotides Tx31F2 (5′TGA AGA ACG TCC GTG CAA ATG C 3′) and Tx31R2 (5′ AAT TCT GCA GTC ATT CGC TGA TAA ATT TTT TGC 3′) were used to amplify a fragment that codes for the 3′ region of the spider Pskov cDNA.