We have cloned, expressed, purified and crystallised the first DH GEF domain of Kalirin (gene: KALRN) in complex with RAC1 (gene: RAC1) as part of a programme to explore a new way of targeting GTPases. Fragment screening and X-ray crystallography has identified binders within the orthosteric binding site. A nucleotide exchange assay has been developed and GEF activity established.
The TWIK related acid-sensitive K+ channel 1 (TASK-1) belongs to the family of two-pore domain potassium (K2P) channels. It regulates resting membrane potential and is expressed in cardiomyocytes, neurons and vascular smooth muscle cells. Loss of function mutations in TASK-1 lead to primary pulmonary hypertension type 4 (PPH4) which is often fatal in mid-life (1). We have produced TASK-1 and determined structures of this protein alone and in complex with two highly potent inhibitors, BAY 1000493 and BAY 2341237, with EC50 values of 9.5 nM and 7.6 nM, respectively. We have used a two-electrode voltage clamp assay to measure the effect of mutations in TASK-1 and the effect of inhibitors. The native structure of TASK-1 also allowed us to map the six known disease mutations leading to PPH4.
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