Molecular mechanism for pharmacological activation of KCa2 channels - ABSTRACT Small-conductance Ca2+-activated K+ channels, KCa2 (also called SK), are important for regulating neuronal excitability in the central nervous system. These channels are gated by a Ca2+/calmodulin (CaM)-mediated mechanism. Small molecule KCa2 activators are highly sought-after as potential therapeutics for substance use disorders, cerebellar ataxia, and essential tremor. Based on our recent pharmacological studies, we propose the recategorization of KCa2 activators into two groups, classic positive allosteric modulators (PAMs) and superagonists. CyPPA was the first classic PAM described to positively modulate KCa2.2 and KCa2.3 channels, while being inactive on KCa2.1 and KCa3.1. We have developed novel, subtype-selective classic PAMs of KCa2.2 and KCa2.3, respectively. Superagonism has not been observed with these classic PAMs. NS309 was initially the most potent activator of KCa2 and KCa3.1 channels. We have developed a more drug-like compound, SKA-31, which potentiates both KCa2 and KCa3.1, and a subtype-selective KCa3.1 activator SKA-111. We discovered that this group of activators can produce maximal responses greater than the endogenous agonist (Ca2+) for the KCa2 and KCa3.1 channels and renamed them superagonists. In Aim 1, we will compare the mechanisms of action for the classic PAMs and superagonists of KCa2. We will define the interactions of classic PAMs and superagonists with KCa2 channels. We will compare the mechanisms of superagonism versus classic positive allosteric modulation. In Aim 2, we will search for novel KCa2 activators using both virtual high-throughput screening and Artificial Intelligence/Machine Learning approaches, followed by experimental testing using electrophysiology assays to determine their potency and selectivity. We will evaluate the efficacy of the most potent and selective classic PAMs and superagonists to rescue pathogenic loss-of-function KCa2.2 mutants. Loss-of-function KCa2.2 mutations are associated with neurodevelopmental disorders including cerebellar ataxia and tremor in humans.
