In the mammalian organism, electrical signals control important processes such as nerve impulses, muscle contraction, and hormone secretion. The advantage of this method of communication is that the temporal resolution is higher than that of chemical signalling. Changes in transmembrane voltage occur when different ionic species flow from one side of the membrane to the other through specialist integral membrane proteins - ion channels. For example, sodium flux is responsible for the propagated action potential along a nerve axon, and the flow of calcium ions into heart cells will trigger a heartbeat. Potassium (K+) currents are a foot on the break for most cellular electrical events, and when coupled with intrinsic voltage and chemical sensing mechanisms provide negative feedback for many cell types. Thus there is a large biotechnological drive for identifying chemical agents that modulate K+-selective channels. Activators (nicorandil, minoxidil) of one particular class are used clinically to relax vascular smooth muscle and thus lower blood pressure, whilst an inhibitor (gliclazide) is one of the most wide-spread treatments for type-2 diabetes. The ion channels that are the topic of the proposed project are a family of intracellular ion-regulated K+ channels, which are formed by the assembly of four a subunits. The first member of the family (Sloa1) assembles to form a well known and characterized calcium-activated K+ channel with a unique pharmacological profile. Its siblings Sloa2.2, a2.2, and a3 are poorly understood, and over the last six years there have been only as many research publications describing their behaviour. Unlike other multi-gene families of K+ channels the individual members have quite different properties with respect to which intracellular ions modulate their behaviour, the rate of ion flow through the channel pore, the ability to reach the cell membrane following synthesis, and the chemical agents that alter activity. The aim of the project is to demonstrate that even more ion channel diversity can be achieved by the co-assembly of more than one type of Sloa subunit. The properties that each individual subunit can confer to the protein complex with be determined by recording K+ currents from cells that have been engineered to synthesise the proteins from foreign nucleic acids. By using this approach modified ion channel DNA can also be introduced, which will allow us to identify which domains of the proteins are responsible for the different behaviour. Because the molecular structures of the different Sloa subunits are similar, domains can be swapped by transplanting segments of DNA from one gene to the other. Electrophysiological experiments will determine whether the functional properties have also been transferred. A separate family of 4 membrane proteins Slob1-4 are known to co-assemble with channels comprised of 4 Sloa1 subunits, altering the functional and pharmacological properties of the channel to different extents. It is not know if they are able to assemble with and modulate the other Sloa subunits, and this will also be addressed. In summary, the aim is to investigate the range of functional and pharmacological K+ channel properties that can be obtained by different combinations of Sloa and b subunits. This will allow us to make predictions of the membrane currents when it is known which of these 8 genes a particular cell-type expresses. On the other hand, we will be able to predict which molecular subunits are likely to be present by studying the properties of the K+ current from a native mammalian cell. Furthermore, by understanding the pharmacology of heteromeric (mixture of subunits) Slo channels we will be able to define more cell-specific drug targets. This is because the chance of other cell types having the same combination of subunits will be lower than the chance of them having just the one subunit, if a drug targets a homomeric assembly of one subunit alone.

Technical Abstract:
The proposed research involves the investigation of the assembly and properties of all four Sloa potassium channel subunits as homomultimers and heteromultimers. This family of membrane proteins is ideal for investigating multiprotein assemblies since each subunit is sensitive to unique stimuli: Sloa1 to intracellular Ca2+ and Mg2+ ions, Sloa2.1 and a2.2 to Na+ and Cl- ions with a2.1 inhibited by ATP4-, and Sloa3 by changes in pH between 7.0 and 8.0. Heteromultimerisation between Sloa1 (BKCa) and Sloa2.2 (Slack) has already been demonstrated (Joiner et al., 1998) but I hypothesise that this can occur between each of the four a subunits via a conserved tetramerisation domain. Cloned channels will be expressed in Xenopus oocytes and/or HEK cells and channels studied by the inside-out patch clamp technique or whole oocyte two-electrode voltage clamp. Heteromultimerisation will be demonstrated by dominant-negative analysis and by co-immunoprecipitation of epitope-tagged constructs. The biophysical and pharmacological properties of heteromeric channels will be compared with those of the corresponding homomultimers to assess whether the properties are intermediate, additive, or lost. These biophysical and pharmacological profiles can potentially be used to characterize currents recorded from native cells. Unlike Sloa1 which produces significant ionic currents when 0.1ng RNA is injected into Xenopus oocytes, the other 3 a subunits express poorly and require more than 50ng RNA per oocytes for comparable currents. The trafficking of heteromeric channels will be studied and chimeras will be constructed with Sloa1 domains to identify the molecular basis of restricted membrane translocation Trafficking will be assessed by electrophysiology and surface biotinylation assays. It is not known whether the Slob1-4 BKCa channel accessory subunits are able to assemble and modulate the other a-subunits. This will be assessed by co-expression and biophysical analysis of currents in inside-out patches. Co-immunniprecipitation and the ability of an inactivating ball-peptide present on the N-terminus of a Slob subunit to confer an inactivating current will also be indications of co-assembly.