| Description |
Slo2.1 is an intracellular Na+-regulated K+ channel that is abundantly expressed in the brain and the heart. Although Slo2.1 channels are believed to provide cardioprotection under ischemic conditions in the heart, the exact physiological or pathophysiological significance of these channels is still unclear, in part because of the lack of pharmacological probes. While the fenamate niflumic acid (NFA) is the first compound shown to activate Slo2.1 channels, its low potency and reduced specificity limits its usefulness as a tool to study these channels in physiological conditions. In this study, I report that fenamates other than NFA, including flufenamic acid, mefenamic acid, tolfenamic acid, meclofenamic acid, and a phenyl acetic acid derivative, diclofenac, are low-potency modulators of Slo2.1 channels. In addition, N-phenylanthranilic acid was established as the minimal pharmacophore for the activation of Slo2.1. The structural basis of the activation gate in Slo channels is controversial. Emerging evidence suggests that the selectivity filter (SF) functions as the activation gate in Ca2+-activated Slo1 and cyclic nucleotide-gated (CNG) channels, unlike Kv channels where the S6 bundle crossing forms the canonical activation gate. Based on my studies, the SF also appears to serve as a gate in Slo2.1 channels. Two residues in the S6 segment, Pro271 and Glu275, were found to be vital for maintaining the intracellular bundle crossing in an open configuration. iv The pore helix residue Phe240 exhibited constitutive channel activity when substituted with polar residues. Further mutational analysis suggests that Phe240 forms a hydrophobic interaction with Leu209 in the S5 segment and Phe258, Met262, and Ala266 in the S6 segment. It is proposed that these interactions favor the closed state of the SF gate under normal low [Na+]i conditions. Collectively, these findings suggest that similar to CNG and Slo1 channels, dynamic rearrangement of S5 and S6 segments in Slo2.1 in response to Na+ binding is allosterically coupled via the pore helix to conformational changes at the SF gate that mediates channel opening. Finally, a long standing controversy prevails regarding the [ATP]i regulation of Slo2.1 channels. This dissertation provides both direct and indirect evidence that intracellular ATP has no effect on Slo2.1 channels. |
