Ca2+ homeostasis is vital for cellular function. It is maintained by signals that initiate Ca2+ current into the cell and by negative feedback pathways that limit Ca2+ entry so that only the necessary functions are carried out. In one essential negative feedback mechanism, Ca2+ directly activates Ca2+ activated K+ channels. SK2 is a member of this family. Its activation changes the cell's membrane potential in a way that inactivates voltage dependent Ca2+ channels, thus inhibiting Ca2+ entry. The rapid activation of SK2 requires a ubiquitous Ca2+ sensing protein, calmodulin (CaM). Although it is known that CaM binds to SK2 channels in the presence or absence of Ca2+, the strength of binding in either state has not been investigated. To fully understand the gating mechanisms of these channels, the thermodynamic binding parameters of Ca2+ to CaM and CaM to SK2 must be determined. The emphasis of the proposed research is on the latter, i.e.
the aim i s to measure the affinity of SK2 for CaM by determining the state dependent macroscopic dissociation constant (KD). The potential influence of the environment and modulators will be researched in two specific aims. First, recombinant peptides representing the CaM binding site of SK are purified and then analyzed for CaM binding using fluorescence spectroscopy and microcalorimetry. The state dependent binding parameters will be determined. The KD will then be analyzed for influences by changes in peptide sequence or by mutations in CaM that alter its ability to bind Ca2+. Second, full-length SK2 channels are heterologously expressed in insect cells using the baculovirus expression system. Plasma membranes from infected cells are isolated and tested for binding to CaM metabolically labeled with 35S. In the heterologous system, mutations in the channel or in CaM will be made that may affect the KD. Once KDs have been determined, a study will commence to relate the effect of KD on SK2 opening. SK2 currents will be measured to determine how changes in KD, affect SK2 open probability and sensitivity to activation by Ca2+. Finally, several agonists have been reported to increase the sensitivity of channel to activation by Ca2+. In both aims above, the KDs of SK2 for CaM will be measured in the presence of agonists to determine what role drugs play in modulating CaM binding. This is important because of great interest in SK channels as therapeutic drug targets in prevalent diseases such as cardiovascular disease and epilepsy. The complete mechanism of drug action on the channel sensitivity to Ca2+ should be investigated for better drug development in the future.
When calcium increases inside a cell, calmodulin, a protein calcium sensor, opens SK channels as part of a negative feedback mechanism to control the rate of calcium entry. This mechanism is a potential therapeutic target for prevalent diseases such as epilepsy and cardiovascular disease. The proposed research aims to measure the strength of binding between SK2 channels and calmodulin and to relate this binding to channel function, which is crucial for designing better therapeutic strategies to target SK2 channels.
|Halling, D Brent; Kenrick, Sophia A; Riggs, Austen F et al. (2014) Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution. J Gen Physiol 143:231-52|