Abstract
Voltage-gated Na+ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Nav and Cav, but not Kv, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Nav1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly Kv channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Nav1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na v channels with potential to shed new light on this process.
| Original language | English |
|---|---|
| Pages (from-to) | 12687-12697 |
| Number of pages | 11 |
| Journal | Journal of Biological Chemistry |
| Volume | 282 |
| Issue number | 17 |
| DOIs | |
| State | Published - Apr 27 2007 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Molecular interactions of the gating modifier toxin ProTx-II with Na v1.5 : Implied existence of a novel toxin binding site coupled to activation. / Smith, Jaime J.; Cummins, Theodore; Alphy, Sujith; Blumenthal, Kenneth M.
In: Journal of Biological Chemistry, Vol. 282, No. 17, 27.04.2007, p. 12687-12697.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Molecular interactions of the gating modifier toxin ProTx-II with Na v1.5
T2 - Implied existence of a novel toxin binding site coupled to activation
AU - Smith, Jaime J.
AU - Cummins, Theodore
AU - Alphy, Sujith
AU - Blumenthal, Kenneth M.
PY - 2007/4/27
Y1 - 2007/4/27
N2 - Voltage-gated Na+ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Nav and Cav, but not Kv, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Nav1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly Kv channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Nav1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na v channels with potential to shed new light on this process.
AB - Voltage-gated Na+ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies activation kinetics of Nav and Cav, but not Kv, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Nav1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly Kv channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Nav1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na v channels with potential to shed new light on this process.
UR - http://www.scopus.com/inward/record.url?scp=34250312096&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=34250312096&partnerID=8YFLogxK
U2 - 10.1074/jbc.M610462200
DO - 10.1074/jbc.M610462200
M3 - Article
C2 - 17339321
AN - SCOPUS:34250312096
VL - 282
SP - 12687
EP - 12697
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 17
ER -