Overexpression of human transient receptor potential M5 upregulates endogenous human transient receptor potential A1 in a stable HEK cell line.
Transient receptor potential M5 (TRPM5), a monovalent cation channel, is primarily activated by increases in intracellular calcium. However, we found unexpectedly that allyl isothiocyanate (AITC) and structural analogs triggered a membrane potential and calcium dye responses in TRPM5-HEK cells (AITC EC₅₀ = 9.0 ± 2.4 μM, n = 5). Although AITC and its analogs were more potent on transient receptor potential A1 (TRPA1)-HEK cells (AITC EC₅₀ = 0.23 ± 0.03 μM, n = 4), the rank order potency of these compounds were similar for TRPM5- and TRPA1-HEK cells. No response to these compounds was seen in parental HEK cells, TRPM5-CHO cells, and TRPM4b-, TRPM8-, or TRPV1-transfected HEK cells. An AITC-evoked current in TRPM5-HEK cells was confirmed in whole-cell voltage clamp recording. AITC elicited an intracellular calcium increase that was not dependent on phorpholipase C(β)₂ (PLC(β)₂) activation but was dependent on extracellular calcium concentration. TRPA1 mRNA was upregulated fourfold in TRPM5-HEK cells compared with parental cells. In contrast, TRPA1 was not upregulated in HEK cells transfected in a similar manner with TRPV1 or TRPM8 genes. The AITC response was blocked by a TRPA1 inhibitor and reduced by a TRPM5 inhibitor and by targeted TRPA1 siRNA. These results suggest that TRPM5 may play a role in upregulating endogenous expression of TRPA1, that TRPA1 activation may be an additional trigger for co-expressed calcium-dependent ion channels such as TRPM5, and that TRPM5 may amplify responses to TRPA1 ligands.
Redpoint Bio Corporation, Ewing, New Jersey, USA.
M Tulu
MT
Buber
Rok
R
Cerne
Rosa Y
RY
Cortés
Robert W
RW
Bryant
Seunghun Paul
SP
Lee
D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D057809:HEK293 Cells; D006801:Humans; D017879:Isothiocyanates / Q000494:pharmacology; D008564:Membrane Potentials; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D018408:Patch-Clamp Techniques; D000074025:TRPA1 Cation Channel; D050053:TRPM Cation Channels / Q000235:genetics / Q000502:physiology*; D050916:TRPV Cation Channels / Q000235:genetics / Q000502:physiology; D013649:Taste; D050051:Transient Receptor Potential Channels / Q000235:genetics / Q000502:physiology*; D015854:Up-Regulation
D015220:Calcium Channels; D017879:Isothiocyanates; D009419:Nerve Tissue Proteins; D000074025:TRPA1 Cation Channel; C473350:TRPA1 protein, human; D050053:TRPM Cation Channels; C404409:TRPM5 protein, human; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; D050051:Transient Receptor Potential Channels; C004471:allyl isothiocyanate; D002118:Calcium