Small conductance Ca2+-activated potassium channels (SK channels) are voltage-independent and activated by submicromolar concentrations of Ca2+. SK channel activity underlies the prolonged afterhyperpolarization (AHP) which follows an action potential. In CA1 hippocampal pyramidal neurons (HPNs) the AHP has two kinetic components. The medium AHP is apamin-sensitive and is not affected by neurotransmitter-induced second messengers, while the slow AHP is insensitive to apamin, and is modulated by neurotransmitter-induced second messengers that act in a convergent manner and exert strong effects on neuronal excitability. A sustained stimulus elicits a train of action potentials and with each action potential the AHP increases in depth and duration, lengthening interspike intervals until the cell is no longer able to reach action potential threshold. This phenomenon, termed spike-frequency adaptation, regulates burst frequency and is essential for normal, integrative neurotranmission. We have cloned three distinct SK channel subunits that exhibit the essential functional features of native SK channels in heterologous expression systems. The cloned SK subunits have remarkably homologous primary sequences except in their structurally divergent intracellular N- and C-terminal domains. The three SK subunits are expressed in overlapping but distinct patterns in the mammalian CNS, and all three cloned subunits are expressed in CA1 HPNs. The central hypothesis underlying this application is that the different SK subunits make distinct contributions to CA1 neuronal physiology. Specifically, a) the different SK subunits underlie the two AHP components, b) the different kinetics result from spatial and functional coupling between the medium AHP channels and voltage-gated Ca2+ channels, while the slow AHP channels are localized such that Ca2+ released from intracellular stores serves to activate them, and c) heterologous proteins associate with the SK subunits and influence subcellular distribution, regulation by second messengers, and pharmacology. First, we will construct transgenic mice through homologous recombination in which expression each of the SK subunit may be acutely regulated in vivo. We will record CA1 HPNs in brain slices and acutely dispersed cultures examining the AHP, interspike interval, spike-frequency adaptation, and coupling with L-type Ca2+ channels. Second, we will use pharmacological approaches to determine the sources of Ca2+ that activate the medium and slow AHPs in CA1 HPNs. Third, we will investigate the composition of the SK channel microdomain using the 2-hybrid system and a CA1-specific cDNA library to isolate proteins that associate with the pore-forming SK channel subunits and influence SK channel assembly, distribution, regulation by second messengers, and pharmacology.
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