Forward genetic analysis reveals multiple gating mechanisms of TRPV4.
TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a limited number of such growth-blocking mutations were isolated. Expressed in oocytes, wild-type channels can be strongly activated by either hypotonicity or exposure to the potent agonist 4alphaPDD, although the GOF channels behaved as if they were fully prestimulated as well as lacking a previously uncharacterized voltage-dependent inactivation. Five of six mutations occurred at or near the inner ends of the predicted core helices, giving further direct evidence that this region indeed forms the main intracellular gate in TRP channels. Surprisingly, both wild-type channels as well as these GOF channels maintain strong steady-state outward rectification that is not due to a Ca(2+) block, as has been proposed elsewhere. We conclude that TRPV4 contains an additional voltage-dependent gating mechanism in series with the main intracellular gate.
Loukin
Stephen
S
Laboratory of Molecular Biology, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA. shloukin@wisc.edu
Su
Zhenwei
Z
Zhou
Xinliang
X
Kung
Ching
C
D000483:Alleles; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D005260:Female; D005690:Galactose / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D007038:Hypotonic Solutions / Q000494:pharmacology; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D009865:Oocytes / Q000502:physiology; D018408:Patch-Clamp Techniques; D010703:Phorbol Esters / Q000494:pharmacology; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D050916:TRPV Cation Channels / Q000235:genetics / Q000502:physiology*; D014981:Xenopus
D007038:Hypotonic Solutions; D010703:Phorbol Esters; D050916:TRPV Cation Channels; C486577:Trpv4 protein, rat; C008876:phorbol-12,13-didecanoate; D005947:Glucose; D002118:Calcium; D005690:Galactose