The purification and N-terminal sequencing of a polypeptide that prolongs action potentials by altering Na channel inactivation from the venom of Buthus martensii Karsch.
A polypeptide that extensively prolongs action potentials (APs) in frog nerve has been isolated and purified from the venom of the scorpion Buthus martensii Karsch (BMK). The polypeptide was purified using gel filtration, ion exchange, FPLC, and HPLC chromatography. APs recorded in the presence of nanomolar concentrations of the polypeptide were extensively prolonged without much attenuation in their heights. The N-terminal sequence of BMK 11(2) was found to be: VRDGYIADDKD-AYF-GRDAYYDDDEKKKD. Sequence similarity comparisons to other alpha-scorpion toxins suggest that the two blanks in the sequences are cysteines. The molecular weight (M.W.) of BMK 11(2) was determined by LC/MS/MS to be 7216 Da. Voltage-clamp experiments conducted on plasmid-transfected human kidney cells expressing the alpha and beta subunits of the rat sodium channel showed that BMK 11(2) acted to prolong Na channel inactivation. Also, in the presence of 100-200 nM BMK 11(2), a persistent non-activating Na current was induced when the membrane was depolarized from a -120 mV holding potential. BMK 11(2) caused Na channel fast inactivation to be further slowed when the holding potential was increased, suggesting that BMK 11(2) effects are voltage dependent. Na channel slow inactivation and return from slow inactivation were unaffected by the presence of BMK 11(2). Since the polypeptide prolongs APs when both K+ and Ca+ channels were blocked and shows sequence similarity to other alpha-neurotoxins, it appears likely that BMK 11(2) acts to selectively alter Na channel inactivation to produce its effect.
Chen
Ziyi
Z
Biological Sciences, Northern Illinois University, DeKalb 60115, USA.
Reddy
Giridher
G
Kondratiev
Andrei
A
Hahin
Richard
R
D000200:Action Potentials / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007668:Kidney; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009498:Neurotoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D011898:Ranidae; D051381:Rats; D012584:Sciatic Nerve / Q000187:drug effects / Q000502:physiology; D012604:Scorpion Venoms / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D026941:Sodium Channel Blockers / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D015222:Sodium Channels / Q000502:physiology; D014162:Transfection
C460341:BMK 11(2) toxin; D009498:Neurotoxins; D010455:Peptides; D012604:Scorpion Venoms; D026941:Sodium Channel Blockers; D015222:Sodium Channels