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Protection of TRPC7 cation channels from calcium inhibition by closely associated SERCA pumps

National Institute of Environmental Health Sciences, NIH, Department of Health and Human Services, Research Triangle Park, North Carolina, USA

²Correspondence: NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709 USA. E-mail: putney{at}niehs.nih.gov

SPECIFIC AIMS

Among the different structures involved in the regulation of Ca²⁺ homeostasis, the transient receptor potential (TRP) channels superfamily is one of the most intensively studied. TRPs were first cloned from Drosophila melanogaster as a gene necessary for normal visual signal transduction. Numerous studies have demonstrated that members of the TRP superfamily of channels are involved in regulated Ca²⁺ entry, in some instances as store-operated channels, in others as non-store-operated channels. Most Ca²⁺-permeable channels are themselves regulated by Ca²⁺, often in complex ways. In the current study, we have investigated the regulation of TRPC7, stably expressed in human embryonic kidney (HEK293) cells by Ca²⁺, and more specifically by sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pumps. TRPC7 was chosen for this study because it is a channel known to be potentially activated by both store-operated mechanisms and non-store-operated mechanisms involving diacylglycerols.

PRINCIPAL FINDINGS

1. Activation of TRPC7 channels by the synthetic diacylglycerol, oleyl acetyl glycerol (OAG), was blocked by pretreatment with the SERCA inhibitors thapsigargin and cyclopiazonic acid (Fig. 1 )
The structurally related channel TRPC3 was similarly inhibited. This inhibition was seen when TRPC7 activity was assessed by use of Ca²⁺-sensitive fluorescent indicators in intact cells, or when channel activity was more directly evaluated by measurement of transmembrane current in the whole cells mode.

View larger version (27K): [in this window] [in a new window]   Figure 1. The OAG-induced current in the TRPC7-HEK cells is inhibited by SERCA inhibitors. A) Average time courses (mean±SE) of ITRPC7 development at –100 and +100 mV in the presence of 30 µM OAG in cells exposed (Tg, down triangle, n=5) or not (ctrl, circle, n=5) to 5 µM thapsigargin. The application of OAG is indicated by a horizontal bar; thapsigargin was added at time 0. B) I/V relationship of the currents observed in a representative cell before (ctrl) and after (OAG) an application of 30 µM OAG, and in a cell exposed to thapsigargin before (+Tg, gray trace) and after OAG (OAG+Tg). C) Average time courses (mean±SE) of ITRPC7 development in the presence of 30 µM OAG in cells exposed (CPA, up triangle, n=5) or not (ctrl, circle, n=5) to 20 µM cyclopiazonic acid. Cyclopiazonic acid was added at time 0.

2. Thapsigargin inhibited TRPC7 currents whether activated by OAG or by a receptor agonist, methacholine
This effect depended on extracellular calcium and on the driving force for Ca²⁺ entry because changing the holding potential to a value near the Nernst potential for Ca²⁺ prevented inhibition. The inhibition is not due to calcium entry via store-operated channels, because it was not blocked by 1 µM Gd³⁺, which completely blocks the endogenous store-operated channels in HEK293 cells. Rather, it seems to result from calcium entry through TRPC7 channels themselves.

3. Partial reduction of single TRPC7 channel conductance by use of submaximal concentrations of TRPC7 channel inhibitors (10 µM Gd³⁺, 30 µM 2-aminoethoxydiphenylborane (2APB)) largely prevented inhibition by thapsigargin (Fig. 2 )
The effect of thapsigargin was prevented by inhibition of calmodulin and was mimicked by pharmacological disruption of the actin cytoskeleton. When channels were first activated at a holding potential of +50 mV, whereby Ca²⁺ entry is prevented, no inhibition by thapsigargin occurred; returning the holding potential to 0 mV did not result in the development of inhibition. This latter observation suggests that the SERCA pump protection of TRPC7 channels is important during the initial activation by diacylglycerols.

View larger version (33K): [in this window] [in a new window]   Figure 2. Thapsigargin effect on ITRPC7 is prevented by TRPC7 inhibitors. A) Average time courses (mean±SE) of the OAG-induced ITRPC7 at –100 and +100 mV in the presence of 1 µM Gd3+ in cells treated (Tg, down triangle, n=4) or not (ctrl, circle, n=5) with 5 µM thapsigargin (added at time 0). B, C) Same experimental protocol as in A, but in the presence of 10 µM Gd3+ (B; ctrl: n=4; Tg: n=5) or of 30 µM 2-APB (C; ctrl: n=4; Tg: n=5).

CONCLUSIONS AND SIGNIFICANCE

These results suggest the presence of a novel mechanism involving negative regulation of TRPC7 by calcium entering through the channel itself. In the presence of thapsigargin, no global increase in Ca²⁺ occurs in response to OAG. Our results indicate that regulation of TRPC7 channels in intact cells involves Ca²⁺ entering specifically through TRPC7 channels. Surprisingly, this inhibition occurs in the presence of a 10 mM BAPTA Ca²⁺ buffer in the patch pipette solution (7 mM free BAPTA), indicating that the SERCA pumps exert powerful and likely highly localized buffering of Ca²⁺ to protect TRPC7 channels from feedback inhibition. Our results suggest the presence of a spatially organized structure, including TRPC7 channels and closely associated SERCA pumps, and depending on an organized actin cytoskeleton. In this model, Ca²⁺ entering the cell through TRPC7 channels is sequestered in the ER via SERCA pumps; this sequestration occurs at a rate sufficient to buffer Ca²⁺ near sites where it could act to inhibit TRPC7 channels. When these pumps are inhibited, an increased local elevation of the Ca²⁺ concentration leads to inhibition of the TRPC7 channels (Fig. 3 ). This hypothesis is supported by two observations. First, 7 mM of free BAPTA in the intracellular solution is unable to prevent inhibition of I_TRPC7 when the SERCA pumps are blocked, indicating that SERCA pumps provide a highly efficient buffering system close to the channels. Second, in the presence of 2APB or Gd³⁺ at concentrations that inhibit the single-channel conductance of TRPC7 by 20%, we observed restoration of I_TRPC7 in cells exposed to thapsigargin: in this case, the reduction of Ca²⁺ entry resulting from a decrease in the TRPC7 single channel conductance is sufficient to keep the intracellular Ca²⁺ concentration below a critical threshold that would otherwise lead to channel inhibition. According to our whole-cell experiments, this threshold could lie between 100 and 500 nM Ca²⁺, values close to those reported in earlier work from other laboratories. In the current study, we demonstrate that, in intact cells, modulation by Ca²⁺ is substantially tempered by closely associated SERCA pumps. Thapsigargin did not inhibit the basal, constitutive activity of TRPC7 channels, indicating that it is the signaling mechanism by which OAG activates, rather than fundamental properties of the channels that is affected. This is consistent with our previous conclusion that OAG activation of TRPC channels is unlikely to result from a direct action on the channel molecule.

View larger version (31K): [in this window] [in a new window]   Figure 3. Schematic diagram.

A new function for SERCA pumps has been revealed—regulation of surface membrane Ca²⁺-permeable channels. Mutations in a gene encoding an isoform of a SERCA pump (ATP2A2) result in a syndrome known as Darier’s disease. Symptoms include dyskeratosis of the skin, but patients also suffer neurological abnormalities, including mild mental retardation, epilepsy, schizophrenia, and bipolar disease. As TRPC channels are highly enriched in the brain, it is conceivable that abnormalities in SERCA function could influence neuronal ion channel signaling, leading to neurological deficiencies.

FOOTNOTES

¹ Present address: Neuro3d S.A., Parc d'Innovation, ESBS, Blvd. Sebastien Brandt, Illkirch 67412, France.

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4714fje;

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