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.