Identification of a peptide toxin from Grammostola spatulata spider venom that blocks cation-selective stretch-activated channels.
We have identified a 35 amino acid peptide toxin of the inhibitor cysteine knot family that blocks cationic stretch-activated ion channels. The toxin, denoted GsMTx-4, was isolated from the venom of the spider Grammostola spatulata and has <50% homology to other neuroactive peptides. It was isolated by fractionating whole venom using reverse phase HPLC, and then assaying fractions on stretch-activated channels (SACs) in outside-out patches from adult rat astrocytes. Although the channel gating kinetics were different between cell-attached and outside-out patches, the properties associated with the channel pore, such as selectivity for alkali cations, conductance ( approximately 45 pS at -100 mV) and a mild rectification were unaffected by outside-out formation. GsMTx-4 produced a complete block of SACs in outside-out patches and appeared specific since it had no effect on whole-cell voltage-sensitive currents. The equilibrium dissociation constant of approximately 630 nM was calculated from the ratio of association and dissociation rate constants. In hypotonically swollen astrocytes, GsMTx-4 produces approximately 40% reduction in swelling-activated whole-cell current. Similarly, in isolated ventricular cells from a rabbit dilated cardiomyopathy model, GsMTx-4 produced a near complete block of the volume-sensitive cation-selective current, but did not affect the anion current. In the myopathic heart cells, where the swell-induced current is tonically active, GsMTx-4 also reduced the cell size. This is the first report of a peptide toxin that specifically blocks stretch-activated currents. The toxin affect on swelling-activated whole-cell currents implicates SACs in volume regulation.
Suchyna
T M
TM
Department of Physiology and Biophysics, State University of New York at Buffalo, Buffalo, New York 14214, USA. suchyna@acsu.buffalo.edu
Johnson
J H
JH
Hamer
K
K
Leykam
J F
JF
Gage
D A
DA
Clemo
H F
HF
Baumgarten
C M
CM
Sachs
F
F
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000502:physiology*; D002412:Cations / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006352:Heart Ventricles / Q000166:cytology; D015640:Ion Channel Gating / Q000187:drug effects; D007473:Ion Channels / Q000502:physiology; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D018485:Muscle Fibers, Skeletal / Q000502:physiology; D009206:Myocardium / Q000166:cytology; D018408:Patch-Clamp Techniques; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013111:Spider Venoms / Q000737:chemistry* / Q000302:isolation & purification* / Q000494:pharmacology; D013112:Spiders; D013314:Stress, Mechanical
D002412:Cations; D007473:Ion Channels; D013111:Spider Venoms; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid