Transient receptor potential M3 channels are ionotropic steroid receptors in pancreatic β cells

Abstract

Transient receptor potential (TRP) cation channels are renowned for their ability to sense diverse chemical stimuli. Still, for many members of this large and heterogeneous protein family it is unclear how their activity is regulated and whether they are influenced by endogenous substances. On the other hand, steroidal compounds are increasingly recognized to have rapid effects on membrane surface receptors that often have not been identified at the molecular level. We show here that TRPM3, a divalent-permeable cation channel, is rapidly and reversibly activated by extracellular pregnenolone sulphate, a neuroactive steroid. We show that pregnenolone sulphate activates endogenous TRPM3 channels in insulin-producing beta cells. Application of pregnenolone sulphate led to a rapid calcium influx and enhanced insulin secretion from pancreatic islets. Our results establish that TRPM3 is an essential component of an ionotropic steroid receptor enabling unanticipated crosstalk between steroidal and insulin-signalling endocrine systems.

Signal Transduction of Pregnenolone Sulfate in Insulinoma Cells

“…Pancreatic ␤-cells express synaptic vesicle proteins such as synapsin I, synaptophysin or synaptotagmin, neurotransmitters, and neurotransmitter-synthesizing enzymes. In line with this, it has recently been reported that the neurosteroid pregnenolone sulfate functions in insulinoma and ␤-cells by triggering a rapid Ca 2ϩ influx into the cells, leading to enhanced insulin secretion (12). Here, we describe the first comprehensive analysis of pregnenolone sulfate-induced signal transduction in insulinoma cells.…”

“…The sequence used to knock down rat TRPM3 has been described (12). The oligonucleotides for creating RNAi stem loops for pLL3.7 were designed as described (26).…”

“…Proteins were separated by SDS-PAGE, blotted, and incubated with antibodies directed against Egr-1 (Santa Cruz, Heidelberg, Germany, sc-189), HDAC-1 (Upstate Biotechnology, Lake Placid, NY, 05-100), TRPM3 (12), Calnexin (Stressgen), or Synapsin I (a kind gift of T. C. Südhof, Stanford University). The antibody directed against histone deacetylase-1 (HDAC1) was used as a loading control as previously described (36).…”

“…Recently it has been shown that insulin-secreting rat INS-1 insulinoma cells are responsive to pregnenolone sulfate stimulation, leading to a rapid influx of Ca 2ϩ ions into the cells (12). Therefore, we used INS-1 cells as a cellular model system to analyze the signal transduction pathway induced by pregnenolone sulfate.…”

Ligand-gated Ion Channels

“…Physical activators -Heat (Q10 = 7.2 between 15-25°C; Vriens et al, 2011), hypotonic cell swelling Chemical activators Pregnenolone sulphate (Lambert et al, 2011) Pregnenolone sulphate (Wagner et al, 2008), epipregnanolone sulphate (Majeed et al, 2010), nifedipine, D-erythro-sphingosine, dihydrosphingosine Blockers Zn 2+ (IC50 = 1 mM) Rosiglitazone, troglitazone, pioglitazone (independent of PPAR-g; Majeed et al, 2011), mefenamic acid, Klose et al, 2011), 2-APB, La 3+ , Gd 3+ , intracellar Mg 2+ , extracellular Na + (TRPM3a2 only)…”