Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells

J Physiol. 2004 Dec 1;561(Pt 2):415-32. doi: 10.1113/jphysiol.2004.075051. Epub 2004 Oct 7.


We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 microM)-activated TRPC6 currents (I(TRPC6)) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2+(o)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2+(o) solely inhibited TRPC7 currents (I(TRPC7)). Vigorous buffering of intracellular Ca2+ (Ca2+(i)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2+(o), disclosing fast potentiation (EC50: approximately 0.4 mM) and inhibition (IC50: approximately 4 mM) of I(TRPC6) and fast inhibition (IC50: approximately 0.4 mM) of I(TRPC7). This inhibition of I(TRPC6) and I(TRPC7) seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I(TRPC6) showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I(TRPC6) or its acceleration by Ca2+(o) probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 microM), coexpression of Ca2+-insensitive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+(i) via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I(TRPC6) was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Calcium / pharmacology
  • Calcium / physiology*
  • Calcium Channels / biosynthesis
  • Calcium Channels / genetics
  • Calcium Channels / metabolism*
  • Carbachol / pharmacology
  • Cations, Divalent
  • Cell Line
  • Humans
  • Ion Channels / biosynthesis
  • Ion Channels / genetics
  • Ion Channels / metabolism*
  • Membrane Potentials / drug effects
  • Membrane Potentials / physiology
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Structural Homology, Protein*
  • TRPC Cation Channels
  • TRPC6 Cation Channel
  • TRPM Cation Channels
  • Transfection


  • Calcium Channels
  • Cations, Divalent
  • Ion Channels
  • Membrane Proteins
  • TRPC Cation Channels
  • TRPC6 Cation Channel
  • TRPC6 protein, human
  • TRPC7 protein, human
  • TRPM Cation Channels
  • TRPM2 protein, human
  • Carbachol
  • Calcium