Yeast genetic systems have been exploited in various accessions for large scales screening in functional clonings, structural studies and in revelation of protein interactions. Systems based on the signal transduction involving Son of Sevenless (Sos) around yeast plasma membrane, have been used in identifying novel protein interactions. The reverse Ras recruitment system (RRS), a modified system of Sos has been developed specifically to study membrane protein interacting partners. We have been able to visualize that Slo family potassium (K) channels could be expressed in the yeast host strain CDC25-2, and be delivered to the plasma membrane. After adoption of the reverse RRS, we have modified the system to make it user-friendlier, and been to recapitulate two known interactions, the Slo1 Core/Tail and the SK/Calmodulin, from application of the system. We propose to search for the novel regulatory proteins with two physiological important K channel, Ca2+-activated, voltage-gated K+ channel (BK channel or Slo1) and another Slo channel family member Slo2.1. Both of these K channels have very large intracellular domains that are likely involved in ligand activations and functional modulations.
The first aim i s to identify modulators of BK channel.
The second aim i s to identify possible missing subunit(s) of Slo2.1 that prevents channel from being in normal function in heterologous expression systems. A successful reverse RRS protocol can be widely applied to analyze signal transduction across cell membrane, as well as to study the mechanisms of diseases of public interests. ? ? ?
