Date of Award
Doctor of Philosophy (PhD)
Chun Jiang - Chair
Contractility of vascular smooth muscles (VSMs) in resistance arteries determines systemic blood pressure and blood supplies to local tissues, in which ATP sensitive K+ (KATP) channels play a role. The KATP channels that couple metabolic state to cellular activity are activated by multiple hormonal vasodilators and inhibited by vasoconstrictors. To understand the molecular mechanisms for the channel regulation by vasodilators, we studied the effects of β-adrenergic receptors on Kir6.1/SUR2B in HEK cells. Stimulation of β-adrenergic receptors activated the channels, which relied on the GS-protein, adenylyl cyclase, cAMP and PKA system. Using mutational analysis, we scanned all the putative PKA sites on Kir6.1 and SUR2B subunits and identified two residues (Ser1351 and Ser1387) in SUR2B critical for channel activation. In vitro phosphorylation experiments confirmed that Ser1387 but not Ser1351 was phosphorylated in isolated SUR2B peptides. Molecular modeling and molecular dynamics simulations reveal that phosphorylation at Ser1387 causes interdomain movements in SUR2B subunit. Blockage of the movements by engineering a disulfide bond across NBD2 and TMD1 eliminated the PKA-dependent channel activation. We also studied the molecular basis for the inhibition of vascular KATP channels by PKC. In the HEK expression system, we found that the Kir6.1/SUR2B channel but not the Kir6.2/SUR2B was drastically inhibited by PKC stimulation. We constructed Kir6.1/Kir6.2 chimeras and identified two critical protein domains for the Kir6.1 channel inhibition by PKC. The distal C-terminus was the direct target of PKC where multiple phosphorylation sites were identified. These phosphorylation sites were located in a short sequence with stereotypical sequence repeats. Mutation of any decreased the effects of PKC. Joint mutation of all of them prevented the channel inhibition by PKC. The proximal N-terminus is also involved in PKC effects without phosphorylation sites, suggesting it may play a role in channel gating. Thus, this thesis provides experimental evidence for the vascular KATP channel modulation by PKA and PKC. Phosphorylation of the Kir6.1 and SUR2B subunits by PKC and PKA produce inhibition and activation of the vascular KATP channel, respectively, which appears to be one of the molecular bases contributing to vascular tone regulation by both vasoconstricting and vasodilating hormones and neurotransmitters.
Shi, Yun, "KATP Channel Phosphorylation: Mechanisms and Contribution to Vascular Tone Regulation by Vasodilating and Vasoconstricting Hormones and Neurotransmitters." Dissertation, Georgia State University, 2007.