Lanthanides Potentiate TRPC5 Currents by an Action at Extracellular Sites Close to the Pore Mouth

Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1–7) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. Unlike most other TRP-related channels, which are inhibited by La3+ and Gd3+, currents through TRPC4 and TRPC5 are potentiated by La3+. 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 La3+or Gd3+, whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La3+ or Gd3+ 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 La3+, 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 Glu543and Glu595/Glu598, situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu595/Glu598 in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La3+ and Gd3+ have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La3+ 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. Mammalian members of the classical transient receptor potential channel (TRPC) subfamily (TRPC1–7) are Ca2+-permeable cation channels involved in receptor-mediated increases in intracellular Ca2+. Unlike most other TRP-related channels, which are inhibited by La3+ and Gd3+, currents through TRPC4 and TRPC5 are potentiated by La3+. 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 La3+or Gd3+, whereas millimolar concentrations were inhibitory. By comparison, TRPC6 was blocked to a similar extent by La3+ or Gd3+ 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 La3+, 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 Glu543and Glu595/Glu598, situated close to the extracellular mouth of the channel pore, resulted in a loss of potentiation, and, for Glu595/Glu598 in a modification of channel inhibition. We conclude that in the micromolar range, the lanthanide ions La3+ and Gd3+ have opposite effects on whole cell currents through TRPC5 and TRPC6 channels. The potentiation of TRPC4 and TRPC5 by micromolar La3+ 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. classical transient receptor potential channel 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid diacylglycerol enhanced green fluorescent protein human embryonic kidney N-methyl-d-glucamine the product of the number (N) of channels in the patch and the open probability 1-oleoyl-2-acetyl-sn-glycerol wild type yellow fluorescent protein Mammalian isoforms of the classical transient receptor potential channel (TRPC)1 subfamily, TRPC1–7, are likely candidates for cation channels mediating phospholipase C-dependent, receptor-operated Ca2+ influx (for reviews, see Refs. 1Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text C. C. Full Text Full Text C. Neurosci. C. The and of TRPCs have in By is on role in native cells. Schultz G. Plant T.D. that cation currents in to for of the of TRPC channels may be to the involvement of TRPC in receptor-operated cation conductances in native channels, other cation channels, have to have intracellular and and a and on amino acid the members of the TRPC subfamily be C. Neurosci. C. C. Schultz G. Full Text Full Text and is also by of the is the of channel in is that receptor-mediated of phospholipase is a in the of TRPC that currents by or be by diacylglycerol of protein C. Schultz G. 2000; Full Text Full Text Full Text Full Text is the of Trends Neurosci. Full Text Full Text By TRPC4 and TRPC5 are by and that of the phospholipase other or the channels Full Text Full Text Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text a G. Neurosci. 2000; to be a channel in C. C. Schultz G. Full Text Full Text 2000; Full Text Full Text Full Text Full Text but have to in the of channels in C. G. Full Text Full Text Schultz G. The a for G. other that channels in Schultz G. 2000; in that are also of of have a or C. Schultz G. Full Text Full Text Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Full Text Full Text 2000; Full Text Full Text Plant T.D. Schultz G. Full Text Full Text at the single channel the of single channel are the of and TRPC6 are C. Schultz G. C. C. Schultz G. G. G. G. Full Text Full Text of TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text Neurosci. 2000; the of the lanthanides and are of cation channels and other Ca2+-permeable channels. have that La3+ potentiating effects on and human TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text the of La3+ on TRPC4 and TRPC5 have characterized By other TRPCs are inhibited by micromolar concentrations of La3+ or Gd3+ Full Text Full Text Full Text Full Text G. C. 2000; Full Text Full Text C. Full Text Full Text the different effects of lanthanides are of the TRPC channels, we characterized the effect of these ions on TRPC5 in detail and on a of we the Full Text Full Text which amino acids at the and characterized Unlike to be by but by 1-oleoyl-2-acetyl-sn-glycerol Full Text Full Text whole cell patch we that TRPC5 was by potentiation at micromolar concentrations by at millimolar TRPC6 was inhibited by micromolar concentrations and showed no potentiation. the single channel the effects of La3+ on TRPC5 are the single channel the open and the of channel By of extracellular negatively charged amino we have close to the pore that are involved in potentiation of TRPC5 by the we that currents by TRPC5 and TRPC6 are by the lanthanides La3+ and whole cell currents through TRPC6 were inhibited by through TRPC5 were potentiated by concentrations but inhibited by The effect of La3+ on TRPC5 was also observed at the single channel and involved a of effect on channel and increase in channel open By of we close to the extracellular mouth of the pore, which are involved in and channel effects of different and La3+ and Gd3+, have for most Ca2+-permeable channels. by of lanthanide ions for members of the TRPC subfamily of channels, for human and Full Text Full Text Full Text Full Text G. C. 2000; Full Text Full Text for TRPC6 G. Full Text Full Text and for human C. Full Text Full Text is in the in Ca2+ in The for TRPC6 in the was in the in whole cell patch for TRPC6 and human C. 2000; Full Text Full Text increase in currents in micromolar La3+ in the are in on channel Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text and we have to Gd3+, which is of the is that lanthanide concentrations were in potentiating the and reversibly inhibited currents by concentrations of Ca2+ also potentiated currents and the effects of micromolar La3+, that may also at the for different of lanthanides on the channel was by the effects of La3+ on single channel currents in outside-out patches. La3+ a in single channel at the the channel open probability effects were observed at a of The of the increase in in outside-out the increase in whole cell the potentiation on in outside-out in whole cell Because of to currents at millimolar concentrations of La3+, is the single channel potentiation and in single channel is at for the in whole cell are of potentiating effects of La3+ on channel and to are no that effects on channel of La3+ have observed in whole cell for and human and currents Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text and for receptor-operated currents in and TRPC5 C. G. Full Text Full Text the of and TRPC5 are to be have a reduced single channel at a potential of The single channel was by the of La3+ in the native cation is of a potentiation of the native in by La3+, of single channel open were to be the of the effect of La3+. in the TRPC4 is in to TRPC4 and be that by micromolar lanthanide concentrations Full Text Full Text C. 2000; loss of the potentiating effects of La3+ and Ca2+ in of sites and to of channel are opposite other at the and of the and extracellular of amino acids are in TRPC4 at the to Glu543and in but acid amino acids are at in and in of in The the TRPC isoforms for the of the potentiating effect for TRPC4 and these sites are to in which are involved in channel potentiation and channel 2000; and to the 2000; 2000; at the and have Because the leading to of channel and the are is La3+ or Ca2+ to the extracellular sites in potentiation. By to of and to potentiation of the 2000; 2000; is to that La3+ or by the the of TRPC5 to TRPC5 also by La3+, whereas the wild type the reduction in single channel by La3+ at increase in open channel is of a at a the of whole cell currents in the which potentiation, was the showed a at the single channel and a potential of whole cell currents reduced currents. The loss of the by at the extracellular and the potential of the that in La3+ the channel the The effect of on by La3+, the increase in single channel and the increase in that close or the the in channel by La3+, is that the increase in single channel in a reduction in by a By to other channels similar the a negatively charged the extracellular pore in at negatively charged amino acids may cation the that potentiation of TRPC5 and of TRPC6 by lanthanides extracellular of La3+ is by the of the effects in intracellular and in the of concentrations of have a for C. 2000; Full Text Full Text that La3+ and human channels the of the and that different different for lanthanide ions in different cell extracellular of on TRPC6 and on we intracellular of the in different the of on TRPC channels to native channels for at to observed are for native channels in Schultz G. Plant T.D. the by Trends Neurosci. Full Text Full Text the other TRPC channels, are the and of the of different TRPC channels may be to the role of TRPC channels in native cells. TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text other TRPC channels and other cation channels in that are potentiated by La3+ in the micromolar effect is a that be to TRPCs or TRPCs and may be is the of the have a a potential close to in also in for the of currents is larger for and TRPC6 for TRPC4 and of and TRPC5 a different of a reduction in at which in a for currents C. G. Full Text Full Text The of and by in a protein is a of of channels, other TRPC channel C. Schultz G. Full Text Full Text that the be to in by Full Text Full Text be in the of channels, the effect of TRPC6 and TRPC6 but and in the the open of TRPC5 and TRPC6 single channel were to be and have in to be for human C. C. Schultz G. G. G. G. Full Text Full Text and human TRPC6 C. Schultz G. whereas open have for and TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text Neurosci. 2000; is on single channel of differential effects of by and single channel open promising to of the of the phospholipase cation currents are the potentiation of TRPC5 by micromolar Gd3+, a TRPC4 but most other cation channels, negatively charged amino acids situated close to the extracellular pore Mammalian isoforms of the classical transient receptor potential channel (TRPC)1 subfamily, TRPC1–7, are likely candidates for cation channels mediating phospholipase C-dependent, receptor-operated Ca2+ influx (for reviews, see Refs. 1Harteneck C. Plant T.D. Schultz G. Trends Neurosci. 2000; 23: 159-166Abstract Full Text Full Text C. C. Full Text Full Text C. Neurosci. C. The and of TRPCs have in By is on role in native cells. Schultz G. Plant T.D. that cation currents in to for of the of TRPC channels may be to the involvement of TRPC in receptor-operated cation conductances in native cells. channels, other cation channels, have to have intracellular and and a and on amino acid the members of the TRPC subfamily be C. Neurosci. C. C. Schultz G. Full Text Full Text and is also by of the is the of channel in is that receptor-mediated of phospholipase is a in the of TRPC that currents by or be by diacylglycerol of protein C. Schultz G. 2000; Full Text Full Text Full Text Full Text is the of Trends Neurosci. Full Text Full Text By TRPC4 and TRPC5 are by and that of the phospholipase other or the channels Full Text Full Text Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text a G. Neurosci. 2000; to be a channel in C. C. Schultz G. Full Text Full Text 2000; Full Text Full Text Full Text Full Text but have to in the of channels in C. G. Full Text Full Text Schultz G. The a for G. other that channels in Schultz G. 2000; in that are also of of have a or C. Schultz G. Full Text Full Text Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Full Text Full Text 2000; Full Text Full Text Plant T.D. Schultz G. Full Text Full Text at the single channel the of single channel are the of and TRPC6 are C. Schultz G. C. C. Schultz G. G. G. G. Full Text Full Text of TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text Neurosci. 2000; the of the lanthanides and are of cation channels and other Ca2+-permeable channels. have that La3+ potentiating effects on and human TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text the of La3+ on TRPC4 and TRPC5 have characterized By other TRPCs are inhibited by micromolar concentrations of La3+ or Gd3+ Full Text Full Text Full Text Full Text G. C. 2000; Full Text Full Text C. Full Text Full Text Because the different effects of lanthanides are of the TRPC channels, we characterized the effect of these ions on TRPC5 in detail and on a of we the Full Text Full Text which amino acids at the and characterized Unlike to be by but by 1-oleoyl-2-acetyl-sn-glycerol Full Text Full Text whole cell patch we that TRPC5 was by potentiation at micromolar concentrations by at millimolar TRPC6 was inhibited by micromolar concentrations and showed no potentiation. the single channel the effects of La3+ on TRPC5 are the single channel the open and the of channel By of extracellular negatively charged amino we have close to the pore that are involved in potentiation of TRPC5 by La3+. the we that currents by TRPC5 and TRPC6 are by the lanthanides La3+ and whole cell currents through TRPC6 were inhibited by through TRPC5 were potentiated by concentrations but inhibited by The effect of La3+ on TRPC5 was also observed at the single channel and involved a of effect on channel and increase in channel open By of we close to the extracellular mouth of the pore, which are involved in and channel effects of different and La3+ and Gd3+, have for most Ca2+-permeable channels. by of lanthanide ions for members of the TRPC subfamily of channels, for human and Full Text Full Text Full Text Full Text G. C. 2000; Full Text Full Text for TRPC6 G. Full Text Full Text and for human C. Full Text Full Text is in the in Ca2+ in The for TRPC6 in the was in the in whole cell patch for TRPC6 and human C. 2000; Full Text Full Text increase in currents in micromolar La3+ in the are in on channel Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text and we have to Gd3+, which is of the is that lanthanide concentrations were in potentiating the and reversibly inhibited currents by concentrations of Ca2+ also potentiated currents and the effects of micromolar La3+, that may also at the for different of lanthanides on the channel was by the effects of La3+ on single channel currents in outside-out patches. La3+ a in single channel at the the channel open probability effects were observed at a of The of the increase in in outside-out the increase in whole cell the potentiation on in outside-out in whole cell Because of to currents at millimolar concentrations of La3+, is the single channel potentiation and in single channel is at for the in whole cell are of potentiating effects of La3+ on channel and to are no that effects on channel of La3+ have observed in whole cell for and human and currents Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text and for receptor-operated currents in and TRPC5 C. G. Full Text Full Text the of and TRPC5 are to be have a reduced single channel at a potential of The single channel was by the of La3+ in the native cation is of a potentiation of the native in by La3+, of single channel open were to be the of the effect of La3+. in the TRPC4 is in to TRPC4 and be that by micromolar lanthanide concentrations Full Text Full Text C. 2000; loss of the potentiating effects of La3+ and Ca2+ in of sites and to of channel are opposite other at the and of the and extracellular of amino acids are in TRPC4 at the to Glu543and in but acid amino acids are at in and in of in The the TRPC isoforms for the of the potentiating effect for TRPC4 and these sites are to in which are involved in channel potentiation and channel 2000; and to the 2000; 2000; at the and have Because the leading to of channel and the are is La3+ or Ca2+ to the extracellular sites in potentiation. By to of and to potentiation of the 2000; 2000; is to that La3+ or by the the of TRPC5 to TRPC5 also by La3+, whereas the wild type the reduction in single channel by La3+ at increase in open channel is of a at a the of whole cell currents in the which potentiation, was the showed a at the single channel and a potential of whole cell currents reduced currents. The loss of the by at the extracellular and the potential of the that in La3+ the channel the The effect of on by La3+, the increase in single channel and the increase in that close or the the in channel by La3+, is that the increase in single channel in a reduction in by a By to other channels similar the a negatively charged the extracellular pore in at negatively charged amino acids may cation the that potentiation of TRPC5 and of TRPC6 by lanthanides extracellular of La3+ is by the of the effects in intracellular and in the of concentrations of have a for C. 2000; Full Text Full Text that La3+ and human channels the of the and that different different for lanthanide ions in different cell extracellular of on TRPC6 and on we intracellular of the in different the of on TRPC channels to native channels for at to observed are for native channels in Schultz G. Plant T.D. the by Trends Neurosci. Full Text Full Text the other TRPC channels, are the and of the of different TRPC channels may be to the role of TRPC channels in native cells. TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text other TRPC channels and other cation channels in that are potentiated by La3+ in the micromolar effect is a that be to TRPCs or TRPCs and may be is the of the have a a potential close to in also in for the of currents is larger for and TRPC6 for TRPC4 and of and TRPC5 a different of a reduction in at which in a for currents C. G. Full Text Full Text The of and by in a protein is a of of channels, other TRPC channel C. Schultz G. Full Text Full Text that the be to in by Full Text Full Text be in the of channels, the effect of TRPC6 and TRPC6 but and in the the open of TRPC5 and TRPC6 single channel were to be and have in to be for human C. C. Schultz G. G. G. G. Full Text Full Text and human TRPC6 C. Schultz G. whereas open have for and TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text Neurosci. 2000; is on single channel of differential effects of by and single channel open promising to of the of the phospholipase cation currents are the potentiation of TRPC5 by micromolar Gd3+, a TRPC4 but most other cation channels, negatively charged amino acids situated close to the extracellular pore the we that currents by TRPC5 and TRPC6 are by the lanthanides La3+ and whole cell currents through TRPC6 were inhibited by through TRPC5 were potentiated by concentrations but inhibited by The effect of La3+ on TRPC5 was also observed at the single channel and involved a of effect on channel and increase in channel open By of we close to the extracellular mouth of the pore, which are involved in and channel potentiation. effects of different and La3+ and Gd3+, have for most Ca2+-permeable channels. by of lanthanide ions for members of the TRPC subfamily of channels, for human and Full Text Full Text Full Text Full Text G. C. 2000; Full Text Full Text for TRPC6 G. Full Text Full Text and for human C. Full Text Full Text is in the in Ca2+ in The for TRPC6 in the was in the in whole cell patch for TRPC6 and human C. 2000; Full Text Full Text The increase in currents in micromolar La3+ in the are in on channel Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text and we have to Gd3+, which is of the is that lanthanide concentrations were in potentiating the and reversibly inhibited currents by concentrations of Ca2+ also potentiated currents and the effects of micromolar La3+, that may also at the for different of lanthanides on the channel was by the effects of La3+ on single channel currents in outside-out patches. La3+ a in single channel at the the channel open probability effects were observed at a of The of the increase in in outside-out the increase in whole cell the potentiation on in outside-out in whole cell Because of to currents at millimolar concentrations of La3+, is the single channel potentiation and in single channel is at for the in whole cell are of potentiating effects of La3+ on channel and to are no that effects on channel of La3+ have observed in whole cell for and human and currents Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text and for receptor-operated currents in and TRPC5 C. G. Full Text Full Text the of and TRPC5 are to be have a reduced single channel at a potential of The single channel was by the of La3+ in the native cation is of a potentiation of the native in by La3+, of single channel open were to be the of the effect of La3+. in the TRPC4 is in to TRPC4 and be that by micromolar lanthanide concentrations Full Text Full Text C. 2000; The loss of the potentiating effects of La3+ and Ca2+ in of sites and to of channel are opposite other at the and of the and extracellular of amino acids are in TRPC4 at the to Glu543and in but acid amino acids are at in and in of in The the TRPC isoforms for the of the potentiating effect for TRPC4 and these sites are to in which are involved in channel potentiation and channel 2000; and to the 2000; 2000; at the and have Because the leading to of channel and the are is La3+ or Ca2+ to the extracellular sites in potentiation. By to of and to potentiation of the 2000; 2000; is to that La3+ or by the the of TRPC5 to The TRPC5 also by La3+, whereas the wild type the reduction in single channel by La3+ at increase in open channel is of a at a the of whole cell currents in the which potentiation, was the showed a at the single channel and a potential of whole cell currents reduced currents. The loss of the by at the extracellular and the potential of the that in La3+ the channel the The effect of on by La3+, the increase in single channel and the increase in that close or the the in channel by La3+, is that the increase in single channel in a reduction in by a By to other channels similar the a negatively charged the extracellular pore in at negatively charged amino acids may cation the that potentiation of TRPC5 and of TRPC6 by lanthanides extracellular of La3+ is by the of the effects in intracellular and in the of concentrations of have a for C. 2000; Full Text Full Text that La3+ and human channels the of the and that different different for lanthanide ions in different cell extracellular of on TRPC6 and on we intracellular Because of the in different the of on TRPC channels to native channels for at to observed are for native channels in Schultz G. Plant T.D. the by Trends Neurosci. Full Text Full Text the other TRPC channels, are the and of the of different TRPC channels may be to the role of TRPC channels in native cells. TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text other TRPC channels and other cation channels in that are potentiated by La3+ in the micromolar effect is a that be to TRPCs or TRPCs and may be is the of the have a a potential close to in also in for the of currents is larger for and TRPC6 for TRPC4 and of and TRPC5 a different of a reduction in at which in a for currents C. G. Full Text Full Text The of and by in a protein is a of of channels, other TRPC channel C. Schultz G. Full Text Full Text that the be to in by Full Text Full Text be in the of channels, the effect of TRPC6 and TRPC6 but and in the the open of TRPC5 and TRPC6 single channel were to be and have in to be for human C. C. Schultz G. G. G. G. Full Text Full Text and human TRPC6 C. Schultz G. whereas open have for and TRPC4 and TRPC5 Plant T.D. Schultz G. 2000; Full Text Full Text C. G. Full Text Full Text Plant T.D. Schultz G. Full Text Full Text Neurosci. 2000; is on single channel of differential effects of by and single channel open promising to of the of the phospholipase cation currents are the potentiation of TRPC5 by micromolar Gd3+, a TRPC4 but most other cation channels, negatively charged amino acids situated close to the extracellular pore We for