Regulation of K+ channels encoded by KCNQ and ether-a-go-go related genes in smooth muscle cells.

K+ channels expressed by KCNQ gene family (KCNQ1-5) have recently been shown to be crucial regulators of smooth muscle contraction by Dr Greenwood and collaborators. However, nothing is known about the molecular stoichiometry in smooth muscle cells, how the membrane abundance is regulated nor how intracellular signals such as cAMP control activity. As smooth muscle contraction underlines all involuntary activity and poorly regulated contractility underpins a number of clinical disorders such as hypertension, impotence, urinary incontinence and bowel disease this information is crucial. The planned project will be separated into the following areas with readily identifiable outcomes: 1.The initial aim will be to determine the molecular constituents of the native KCNQ channels in different smooth muscles (vascular and non-vascular). KCNQ encoded channels form heteromultimers and associate with auxillary proteins encoded by KCNE genes to produce cell type-specific functional complexes, which increase K channel diversity. The expression of KCNQ1-5, as well as known protein partners, will be assessed in each smooth muscle by RT-PCR and western blot analysis using antibodies characterised previously. Specific protein-protein interactions will be identified by western blot analysis with antibodies against specific KCNE expression products following co-immunoprecipitation using antibodies against certain KCNQ channels under non-denaturing conditions. KCNQ1 has also been shown to interact physically with the proteins encoded by ether-a-go-go related gene (ERG)1 in mammalian expression systems but not in native cells so far. As ERG1 is also expressed in the vascular smooth muscle shown to express KCNQ genes a set of experiments will specifically determine whether an ERG-KCNQ complex exists in these cells. All these studies will be supported by heterologous expression work using mammalian expression systems (eg CHOs). 2.KCNQ channels have been shown to be regulated by ubiquitination, membrane phospholipids and protein kinase A. Consequently, molecular experiments will be undertaken to determine whether smooth muscle KCNQ channels are similarly regulated. These molecular studies will be undertaken in conjunction with single cell electrophysiology (whole cell patch clamp techniques). These single cell studies will characterise currents generated by KCNQ expression products and will assess how channel regulators impact on channel activity. NeuroSearch has synthesised a number of KCNQ channel modulators that will be applied to single smooth muscle cells in the absence and presence of different cellular regulators. 3.A dominant negative approach will be utilised to assess the functional impact of specific KCNQ-encoded proteins to cellular K+ currents, membrane potential and whole tissue function. The generation of dominant negative plasmids will be undertaken with the Industrial partner and will be assessed by heterologous expression in mammalian systems. Dominant negative plasmids will be loaded into smooth muscles using a reversible permeabilization process used currently by Dr Greenwood and collaborators. Experiments similar to those described in aims 1 and 2 will then be performed on smooth muscle incubated with the KCNQ plasmid. From the initial studies by the applicant the most commonly expressed KCNQ gene in vascular smooth muscle is KCNQ4 so this gene will be the focus of these dominant negative studies. NeuroSearch run state-of-the-art facilities for studying ion channel function including high throughput screening, recombinant ion channel technology and mutagenesis expertise.They have a drugs pipeline containing a number of modulators for KCNQ, ERG and other ion channels. Prof Olesen, the co-supervisor, has published a considerable amount of high quality papers on KCNQ channel pharmacological, regulatory and biophysical properties and is considered to be a leading researcher in this field.