Assignment of voltage-gated potassium channel blocking activity to kappa-KTx1.3, a non-toxic homologue of kappa-hefutoxin-1, from Heterometrus spinifer venom.
A new family of weak K(+) channel toxins (designated kappa-KTx) with a novel "bi-helical" scaffold has recently been characterized from Heterometrus fulvipes (Scorpionidae) venom. Based on the presence of the minimum functional dyad (Y5 and K19), kappa-hefutoxin-1 (kappa-KTx1.1) was investigated and found to block Kv 1.2 (IC(50) approximately 40 microM) and Kv 1.3 (IC(50) approximately 150 microM) channels. In the present study, kappa-KTx1.3, that shares approximately 60% identity with kappa-hefutoxin 1, has been isolated from Heterometrus spinifer venom. Interestingly, despite the presence of the functional dyad (Y5 and K19), kappa-KTx1.3 failed to reproduce the K(+) channel blocking activity of kappa-hefutoxin-1. Since the dyad lysine in kappa-KTx1.3 was flanked by another lysine (K20), it was hypothesized that this additional positive charge could hinder the critical electrostatic interactions known to occur between the dyad lysine and the Kv 1 channel selectivity filter. Hence, mutants of kappa-KTx1.3, substituting K20 with a neutral (K20A) or a negatively (K20E) or another positively (K20R) charged amino acid were synthesized. kappa-KTx1.3 K20E, in congruence with kappa-hefutoxin 1 with respect to subtype selectivity and affinity, produced blockade of Kv 1.2 (IC(50) = 36.8+/-4.9 microM) and Kv 1.3 (IC(50)=53.7+/-6.7 microM) but not Kv 1.1 channels. kappa-KTx1.3 K20A produced blockade of both Kv 1.2 (IC(50) = 36.9+/-4.9 microM) and Kv 1.3 (IC(50)=115.7+/-7.3 microM) and in addition, acquired affinity for Kv 1.1 channels (IC(50) =1 10.7+/-7.7 microM). kappa-KTx1.3 K20R failed to produce any blockade on the channel subtypes tested. These data suggest that the presence of an additional charged residue in a position adjacent to the dyad lysine impedes the functional block of Kv 1 channels produced by kappa-KTx1.3.
Nirthanan
Selvanayagam
S
Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Pil
Joost
J
Abdel-Mottaleb
Yousra
Y
Sugahara
Yuko
Y
Gopalakrishnakone
Ponnampalam
P
Joseph
Jeremiah S
JS
Sato
Kazuki
K
Tytgat
Jan
J
D000595:Amino Acid Sequence; D004220:Disulfides / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D024642:Potassium Channels, Voltage-Gated / Q000037:antagonists & inhibitors* / Q000737:chemistry; D012604:Scorpion Venoms / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014118:Toxins, Biological / Q000494:pharmacology*
D004220:Disulfides; D026902:Potassium Channel Blockers; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms; D014118:Toxins, Biological