TRPC5 channels undergo changes in gating properties during the activation-deactivation cycle.
TRPC5 are non-specific cation channels activated through phospholipase C-dependent pathways, although the precise gating mechanism is unknown. TRPC5 current-voltage relationships (I-Vs) change systematically during the activation-deactivation cycle, shifting between outwardly rectifying and doubly rectifying shapes. Since several TRP family members exhibit voltage-dependent properties, we investigated whether the various I-V relationships were due to changes in gating. Using patch-clamp recordings of rat TRPC5 transfected HEK293 cells, we found that TRPC5 currents had distinct biophysical characteristics correlated with individual I-V shapes, a phenomenon we call 'phases.' At rest, channels were closed at most potentials, although strong depolarizations (>+80 mV) stimulated small outward currents (Phase 0). For 10-15 sec after activation, voltage steps evoked small inward and large outward currents with time- and voltage-dependent kinetics (Phase 1, outwardly-rectifying I-Vs). At maximal inward amplitude, currents were voltage-independent at all potentials (Phase 2, doubly-rectifying I-Vs owing to Mg2+ block). During desensitization (Phase 3), currents reverted to a Phase 1-like voltage-dependence. La3+ ions potentiated inward TRPC5 currents by promoting a reversible transition from Phase 3 to Phase 2. Single channel recordings revealed asymmetric conductance properties with values of approximately 40 pS at negative potentials and approximately 130 pS at >+60 mV. Mutation of D633, a cytoplasmic residue that mediates Mg2+ block, decreased channel activity at negative potentials during Phase 2. We conclude that TRPC5 gating properties can switch reversibly between voltage-dependent and voltage-independent states. The modulation of phase transitions by external agents such as La3+ and EBP50, a scaffolding protein, may constitute a novel mechanism for regulation of channel activity.
Obukhov
Alexander G
AG
Department Pharmacology & Physiology, UMDNJ, New Jersey Medical School, Newark, New Jersey 07103, USA.
Nowycky
Martha C
MC
D000818:Animals; D002460:Cell Line; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006632:Histamine / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D007811:Lanthanum / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D050052:TRPC Cation Channels / Q000235:genetics / Q000378:metabolism*
D050052:TRPC Cation Channels; C503500:Trpc5 protein, rat; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D007811:Lanthanum; D006632:Histamine