Abstract
Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1-7) are Ca(2+)-permeable cation channels involved in receptor-mediated increases in intracellular Ca(2+). Unlike most other TRP-related channels, which are inhibited by La(3+) and Gd(3+), currents through TRPC4 and TRPC5 are potentiated by La(3+). Because these differential effects of lanthanides on TRPC subtypes may be useful for clarifying the role of different TRPCs in native tissues, we characterized the potentiating effect in detail and localized the molecular determinants of potentiation by mutagenesis. Whole cell currents through TRPC5 were reversibly potentiated by micromolar concentrations of La(3+) or Gd(3+), whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La(3+) or Gd(3+) at micromolar concentrations and showed no potentiation. Dual effects of lanthanides on TRPC5 were also observed in outside-out patches. Even at micromolar concentrations, the single channel conductance was reduced by La(3+), but reduction in conductance was accompanied by a dramatic increase in channel open probability, leading to larger integral currents. Neutralization of the negatively charged amino acids Glu(543) and Glu(595)/Glu(598), situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu(595)/Glu(598) in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La(3+) and Gd(3+) have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La(3+) at extracellular sites close to the pore mouth is a promising tool for identifying the involvement of these isoforms in receptor-operated cation conductances of native cells.
Highlights
Mammalian isoforms of the classical transient receptor potential channel (TRPC)1 subfamily, TRPC1–7, are likely candidates for cation channels mediating phospholipase C-depend
Receptor-activated Currents through TRPC5 and TRPC6 — We first performed whole cell voltage clamp recordings to compare currents mediated by TRPC5 with those mediated by TRPC6
We show that currents mediated by TRPC5 and TRPC6 are affected differently by the lanthanides La3ϩ and Gd3ϩ
Summary
Molecular Biology and Stable Transfection—The isolation of TRPC5 from mouse brain total RNA has been described previously [15]. For cloning of TRPC6, total RNA was prepared from rat brain or A7r5 smooth muscle cells using a TriZol reagent (Invitrogen) according to the standard protocol. For the generation of a stably transfected TRPC6 cell line, the KpnI/ BamHI fragment of the cloned cDNA was ligated into the tetracyclineinducible eukaryotic expression vector pcDNA4/TO and transfected into T-REx-293 cells (both from Invitrogen) using the transfection reagent FuGENE 6 (Roche Molecular Biochemicals). The standard intracellular solution contained 110 mM cesium methanesulfonate, 25 mM CsCl, 2 mM MgCl2, 3.62 mM CaCl2, 10 mM EGTA, and 30 mM HEPES (pH 7.2 with CsOH) with a calculated [Ca2ϩ] of 100 nM. A pipette solution with stronger Ca2ϩ buffering (30 mM BAPTA) with a calculated [Ca2ϩ] of 100 nM was used It contained 50 mM cesium methanesulfonate, 25 mM CsCl, 2 mM MgCl2, Lanthanide Potentiation of TRPC5. Differences were regarded as statistically significant for p Ͻ 0.05 and as highly statistically significant for p Ͻ 0.01
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