The overall objective of this application is to understand the molecular and kinetic gating mechanisms of small-conductance, calcium-activated potassium channels (SK channels). The SK channels participate in calcium signaling, electrical signaling and synaptic plasticity in the brain. They are potential molecular substrates for treating diseases of the nervous system such as Parkinson's disease and Schizophrenia. Understanding the gating mechanisms of the SK channels will provide specific knowledge about how these molecules can be manipulated in a therapeutic setting. ? ? I propose experiments for two specific aims.
The first aim i s to probe the inner pore properties and gate location of SK channels with intracellular blockers. I will characterize in detail dose-dependent activation of SK channels by calcium and dose- and voltage-dependent blockade by a series of quaternary ammonium blockers. Comparisons between blockers of different sizes and hydrophobicity will provide information about the properties and size of the SK channel pore. Correlation between doses of calcium (or open probability in single-channel recordings) and block efficiency will reveal possible links between the state of the channel (open or closed) and accessibility of the pore to the blockers. State-dependence of such accessibility would suggest an intracellular gate location. On the other hand, if the accessibility of the pore is state-independent, this would suggest a gate location above the blocking site.
The second aim i s to test the hypothesis that dimerization of the C termini of SK channels by calcium-bound calmodulin stabilizes the open state. I plan to make mutations of the SK channel with predictable results based on structural data. One set of mutations will be cysteine substitutions for residues with short inter-subunit distance in the dimmer structure. If the dimer structure is associated with an open channel, cross-linking these residues should lock the channel in the open state. Another set of mutations will target the hydrophobic interactions between the SK channel and the N-lobe of calmodulin in the dimer structure. Hydrophobic residues in the SK channel will be mutated to polar residues, which are expected to weaken the interaction and destabilize the dimer. This should lead to a reduced efficiency of calcium activation of the channel. ? ? ?
