Pannexin-1 and other anion channels in platelets and megakaryocytes

Platelet ion channels are essential for Ca2+-influx, maintaining the resting membrane potential and cell volume regulation. Cation channels have been widely studied but few reports of platelet anion channels exist. A recent ‘channelome’ screen has suggested that human platelets express several anion-permeable channels of unknown function. This thesis explores the function of two such channels, Pannexin-1 and TMEM16F, in platelets, primary megakaryocytes (MKs) and related cell lines. Using pannexin-1 inhibitors, these channels were shown to open in response to stimulation by thrombin, contribute to Ca2+-influx and release cytosolic ATP following stimulation by threshold concentrations of platelet agonists. Experiments also suggested that ATP release by pannexin-1 channels contributes to Ca2+-influx via stimulation of ATP-gated P2X1 receptors. Anion-selective TMEM16F channels have been recorded in a variety of cell types and activate in response to sustained elevation of [Ca2+]i to 100 μM. Controversially, these channels were reported to be cation-selective in mouse MKs. Thus, whole cell patch clamp recordings were performed to assess the biophysical properties of TMEM16F channels in HEL cells and primary mouse and rat MKs. Elevating [Ca2+]i to 100 μM in HEL cells and rat MKs induced a Ca2+-dependent, outwardly rectifying anion-permeable conductance, which was blocked by the TMEM16F inhibitor A01. Recordings of mouse MKs identified an equally Ca2+-dependent, outwardly rectifying and A01-sensitive conductance, however this was predominantly permeable to cations. Thus, a major interspecies difference exists in the ionic selectivity of MK TMEM16F channels; possible explanations for this difference, such as mutations within the pore region, are discussed. In summary, this thesis has explored the biophysical properties and function of platelet and MK anion channels using in vitro assays. These studies have relied heavily upon pharmacological tools and future studies of platelet function would benefit from the use of transgenic models.