YOUNG SCIENTISTS

Young Scientists Journal Club Article

Young Scientists Journal Club Article

Ins(1,4,5)P3 interacts with PIP2 to regulate activation of TRPC6/C7 channels by diacylglycerol in native vascular myocytes

Canonical transient receptor potential (TRPC) channels are non-selective cation channels which when stimulated allow Na+ and Ca2+ ions to enter cells. TRPC channels are widely expressed in vascular smooth muscle cells where they have been linked to various physiological and pathological responses, notably contraction, cell growth and proliferation (Inoue et al. 2006; Abramowitz & Birnbaumer, 2009).

An important question concerns the activation mechanism(s) of TRPC channels where there is growing interest in the role of membrane phospholipids. It is generally accepted that TRPC channels are activated by G-protein-coupled receptors linked to phosphoinositide-phospholipase C (PI-PLC, Clapham, 2003; Albert & Large, 2006; Hardie, 2007; Abramowitz & Birnbaumer, 2009). The present study focuses on native members of the TRPC3/C6/C7 subfamily in freshly dispersed vascular myocytes. In a study on the action of noradrenaline in rabbit portal vein myocytes, it was shown that diacylglycerol (DAG), one of the products of PI-PLC-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), stimulated a non-selective cation conductance (Helliwell & Large, 1997). Subsequently it was shown that TRPC6 was a component of this conductance (Inoue et al. 2001). However, it was also considered that other TRPC subunits might contribute to the ion channel (Inoue et al. 2001), and in light of our data we refer to this as a TRPC6-like channel. A significant observation was that DAG activated the TRPC6-like channel in portal vein myocytes by a protein kinase C (PKC)-independent mechanism (Helliwell & Large, 1997), which is considered to be a hallmark mechanism of heterologously expressed TRPC3/C6/C7 conductances (Hofmann et al. 1999; Okada et al. 1999; Trebak et al. 2003; Estacion et al. 2004; Shi et al. 2004), and this has been observed with diverse native TRPC conductances in several blood vessels (Albert et al. 2005, 2006; Peppiatt-Wildman et al. 2007).

Whole-cell and single cation channel currents were recorded in voltage-clamp mode using cell-attached and inside-out patch configurations with an AXOpatch 200B amplifier (Axon Instruments, USA) at room temperature (20-23°C). Patch pipettes were manufactured from borosilicate glass to produce pipettes with resistances of about 5 MO for whole-cell recording and 10 MO for single channel recording when filled with patch pipette solution. To reduce 'line' noise the recording chamber (vol. ca 150-200 µl) was perfused using two 20 ml syringes, one filled with external solution and the other used to drain the chamber, in a 'push and pull' technique. The external solution could be exchanged twice within 30 s.

The differential sensitivity to anti-TRPC antibodies indicates that both TRPC6 and TRPC7 proteins are constituents of the conductance in portal vein but that the channels in mesenteric artery myocytes are composed of only TRPC6 subunits. This proposal was further investigated using co-immunoprecipitation studies. Figure 2Da and b shows that in respectively portal vein and mesenteric artery tissue lysate immunoprecipitation (IP) with anti-TRPC7 followed by Western blotting (WB) with anti-TRPC7 antibodies produced predicted bands of ~90 ...