Date of Award
Doctor of Philosophy (PhD)
Cell Biology and Physiology
Jonathan H. Jaggar, Ph.D.
Adebowale Adebiyi, Ph.D. William Earl Armstrong, Ph.D. Julio Cordero-Morales, Ph.D. Elena Parfenova, Ph.D.
Rationale: Large-conductance calcium (Ca2+)-activated potassium channels (BK) are expressed in arterial myocytes to control arterial contractility. It is composed of pore- forming BKα and auxiliary β1 subunits. Auxiliary β1 subunits associate with BKα which modulate Ca2+ sensitivity of BK channel. Previous data showed that BKα locates at cell membrane, whereas β1 subunits are primarily intracellular which regulated by Rab11A- positive recycling endosomes. Endothelin-1 (ET-1), a vasoconstrictor, induces contraction of myocytes. ET-1 inhibits BK channel but mechanisms are not fully understood. It is unclear that vasoconstrictors regulate the cellular distribution of BK channels. Furthermore, BK channels are involved in hypertension. Hypertension increases risk of major cardiovascular and cerebrovascular events, such as stroke and mental dysfunction. During hypertension, cerebral arteries have high myogenic tone and are less responsive to vasodilators, including nitric oxide (NO). The regulation of arterial contractility by BK channels is altered during hypertension, although mechanisms involved are also unclear.
Objective: Test the hypothesis that ET-1 inhibits β1 surface trafficking in myocytes via activation of PKC. Test the hypothesis that activation of PKC directly modulates Rab11A through phosphorylation. Furthermore, test the hypothesis that trafficking of pore- forming BK channel (BKα) and auxiliary β1 subunits contributes to pathological changes in contractility in cerebral arteries of stroke-prone spontaneously hypertensive rats (SP- SHRs).
Methods and Results: ET-1 decreased NO-induced or depolarization-induced surface β1 expression and association with BKα in myocytes through activation of protein kinase C (PKC). Total β1, total BKα proteins or surface BKα was not altered by ET-1. Rab11A regulates β1 protein trafficking in Rab11A-positive recycling endosome. ET-1 reduced Rab11 activity via phosphorylation. Five probable phosphorylated sites on Rab11A were identified, among which Ser177 has highest probability. A phosphorylation-mute Rab11A construct (Rab11A S177A) or wild-type Rab11A construct similarly increased total Rab11A protein in transfected myocytes. Rab11A S177A inhibited ET-1-reduced Rab11A activity and decreased β1 protein trafficking. Rab11A S177A reversed PKC- dependent block of single BK channels and transient BK currents in myocytes. Rab11A S177A partially blocked ET-1-induced vasoconstriction. In contrast, NO-induced surface-trafficking of β1 subunits, BK current activity and vasodilation did not involve Rab11A S177. Our data also indicate that the amounts of total and surface BKα and β1 subunits were similar in unstimulated arteries of SP-SHRs and age-matched, normotensive Wistar-Kyoto rat controls. In contrast, the stimulated surface-trafficking of β1 subunits by either NO (sodium nitroprusside, SNP) or membrane depolarization was inhibited in SP-SHR arteries. BIM, a PKC inhibitor, and overexpression of a mutant Rab11A construct that cannot be phosphorylated by PKC at serine 177 (Rab11A S177A) restored the stimulated surface-trafficking of β1 subunits. PKC-mediated inhibition of β1 trafficking prevented BK channel activation by NO in arterial myocytes of SP-SHRs and this was restored by the expression of Rab11A S177A, but not by Rab11A. Vasodilation to NO and lithocholate, an activator of β1 subunit-containing BK channels, was inhibited in pressurized arteries of SP-SHRs. Vasodilation to these agents was reestablished by BIM in SP-SHR arteries.
Conclusions: In smooth muscle cell, ET-1 activates protein kinase C which phosphorylates Rab11A at Ser177 to reduce Rab11A activity. Inhibition of Rab11A blocks anterograde trafficking of β1 subunits to associate with BKα on cell surface. Less β1 subunits reduces Ca2+ sensitivity of BK channel and transient BK channel currents which leads to vasoconstriction. Spontaneously active PKC inhibits β1 subunit trafficking in arterial myocytes and is responsible for dysfunctional NO-induced BK channel activation and vasodilation in cerebral arteries of SP-SHRs.
Available for download on Wednesday, July 08, 2020