Molecular determinants of beta-adrenergic signaling to voltage-gated K+ channels in the cerebral circulation

Abstract

Voltage-gated K⁺ (K_v ) channels are major determinants of membrane potential in vascular smooth muscle cells (VSMCs) and regulate the diameter of small cerebral arteries and arterioles. However, the intracellular structures that govern the expression and function of vascular K_v channels are poorly understood. Scaffolding proteins including postsynaptic density 95 (PSD95) recently were identified in rat cerebral VSMCs. Primarily characterized in neurons, the PSD95 scaffold has more than 50 known binding partners, and it can mediate macromolecular signaling between cell-surface receptors and ion channels. In cerebral arteries, Shaker-type K_v 1 channels appear to associate with the PSD95 molecular scaffold, and PSD95 is required for the normal expression and vasodilator influence of members of this K⁺ channel gene family. Furthermore, recent findings suggest that the β1-subtype adrenergic receptor is expressed in cerebral VSMCs and forms a functional vasodilator complex with K_v 1 channels on the PSD95 scaffold. Activation of β1-subtype adrenergic receptors in VSMCs enables protein kinase A-dependent phosphorylation and opening of K_v 1 channels in the PSD95 complex; the subsequent K⁺ efflux mediates membrane hyperpolarization and vasodilation of small cerebral arteries. Early evidence from other studies suggests that other families of K_v channels and scaffolding proteins are expressed in VSMCs. Future investigations into these macromolecular complexes that modulate the expression and function of K_v channels may reveal unknown signaling cascades that regulate VSMC excitability and provide novel targets for ion channel-based medications to optimize vascular tone.

Keywords: beta adrenergic receptors; potassium channels; scaffolding proteins; smooth muscle.

Figure 1.. PSD95 interaction with the K_v1 channel and disruption by K_v1-C peptide.

A) Schematic of the association of the K_v1 channel α1.2 subunit with the PSD95 scaffold via the PDZ1 binding domain. PSD95 contains three PDZ binding domains (PDZ1–3), and Src-homology (SH3) and guanylate kinase (GK) domains. B) The K_v1-C dominant negative peptide was designed to compete for the PDZ binding domain on PSD95. The last 10 amino acids of the C-terminus of the α1.2 pore protein were conjugated to HIV-tat (YGRKKRRQRRR) to confer cell-permeability. P is a spacer. LTDV is a class-1 PDZ binding motif on the α1.2 subunit. A peptide with same amino acid composition in a scrambled order (Scm) was used as control. C) Immunoprecipitation using anti-K_vα1.2 of rat cerebral arterial lysate treated with Scm or K_v1-C peptide for 30 min. The K_vα1.2 immunoprecipitate and column flow-through (Flow-through) were probed for PSD95 on a Western blot. Depicted is a representative scan from three similar experiments showing that K_v1-C peptide disrupted PSD95 association with α1.2. D) Biotinylation of rat cerebral arteries treated with Scm or K_v1-C peptide for 30 min. Cytosolic and surface fractions were probed for the K_v1 channel α1.2 subunit. Control lysate from freshly isolated cerebral arteries (CA) was loaded for size comparison. Depicted is a representative blot from five similar experiments. K_v1-C did not alter the surface expression of K_v1 channel α1.2 subunits, which appear as a doublet band at ~ 58 kD and 80 kD; the upper band represents the glycosylated form. Figures and legend from reference .

Figure 2.. Proposed association of the β1-subtype adrenergic receptor (β1AR) and K_v1 channel on a PSD95 scaffold to form a “vasodilator signalosome” in cVSMCs.

The carboxyl termini of the β1AR and α1.2 pore protein of the K_v1 channel bind to PDZ domains on the PSD95 scaffold to create a receptor-effector signaling complex or “signalosome”. Agonist binding to β1AR initiates the G_S-protein, adenylyl cyclase (AC), protein kinase A (PKA) cascade. This second-messenger cascade in cVSMCs culminates in PKA-phosphorylation (P) and activation of K_v1 channels, which promotes vasodilation of cerebral arteries. PDZ domains (yellow), SH3 domain (green octagon), guanylate kinase domain (purple), A-kinase anchoring protein (AKAP150), cyclic adenosine monophosphate (cAMP). Involvement of gray-colored proteins has not been tested directly in cVSMCs, but is speculated based on reported associations in other cell types. For simplicity, PSD95 is depicted linearly to illustrate proposed association of the receptor, signaling proteins and K_v1 channel. This two-dimensional schematic may not represent the actual relative positions of components in three-dimensional space. Figures and legend from reference .

Figure 3.. Isoproterenol-induced dilation of cerebral arteries in vivo.

A-B) Representative images of a rat middle cerebral artery branch (arrowhead) in a cranial window at (A) baseline and (B) in response to 10 μmol/L isoproterenol. Veins (asterisks) are unresponsive to isoproterenol. Figures and legend from reference . C-F) Image of a rat superior cerebellar artery cannulated and pressurized to 80 mmHg as performed in reference 101. After 1 hour of equilibration (C, Baseline), isoproterenol (Iso) was added to the bath in half-log concentrations between 1 nM and 10 μmol/L. The diameter response to the highest Iso concentration of 10 μmol/L is shown (D, Iso). After washout, the β1AR blocker CGP20712 was applied for 10 minutes (C, CGP) and then Iso was added cumulatively up to 10 μmol/L (D, CGP+Iso). Scale bar, 100 μm.