Slow Traffic Makes for Bad Circulation

Hypertension is the dominant risk factor for cardiovascular and cerebrovascular disease. The primary feature of hypertension is a rise in total peripheral resistance in arteries including the cerebral vasculature¹. The diameter of arteries and arterioles is largely determined by the tone and thickness of the vascular smooth muscle cell (VSMC) layer. Plasma membrane calcium (Ca²⁺) channels primarily regulate VSMC tone through activation of the cytosolic contractile apparatus. Membrane depolarization activates voltage-gated L-type Ca²⁺ (Ca_v1.2) causing Ca²⁺ influx and vasoconstriction². As a part of a negative feedback regulatory loop, ryanodine receptor-mediated cytosolic Ca²⁺ sparks activate plasma membrane potassium (K⁺) channels known as the large conductance Ca²⁺-activated K⁺ channels (BK). This leads to K⁺ efflux, hyperpolarization, and vasodilation. The BK channel is a homotetramer of the pore-forming α subunit, which is encoded by a single gene. BK channels possess auxiliary subunits, which include four β (β1–4) and four known γ subunits (γ1–4). VSMCs predominantly express β1 and γ1 accessory subunits^{3, 4}. The β1 and γ1 subunits have a profound regulatory role by promoting BK channel activation and vasodilation. The β1 subunit associates with BKα to enhance the open probability of BK channels by increasing their Ca²⁺-sensitivity, whereas the γ1 subunit enhances BK channel voltage-sensitivity.

In this issue of Hypertension, Leo et al., report that hypertensive VSMCs are defective in trafficking of the β1 subunit from recycling endosomes to the plasma membrane⁵. Cerebral arteries of hypertensive patients are resistant to vasodilation, and studies have speculated that dysregulation of BK channels may contribute to the pathogenesis of hypertension. Some studies showed that VSMC BK channels are upregulated in hypertension as a compensatory mechanism⁶, while others proposed that BK channels are downregulated, and this downregulation contributes to hypertension⁷. The expression of BKα and the open probability of Ca²⁺-activated K⁺ currents were increased in aortic and cerebral VSMCs isolated from genetically induced spontaneously hypertensive rats (SHR)⁶, and similar finding were reported in renal interlobar arteries and aortas from the aldosterone-salt hypertensive rat model⁶. However, a conflicting study showed that β1 subunit protein expression and BK currents were decreased in cerebral VSMCs in an angiotensin II-infused rat model of hypertension⁷. It seems therefore that the regulation of BK channel subunit expression in hypertension is complex, and may be specific to the particular vascular bed and the rodent model of hypertension considered.

Interestingly, Leo et al., moved beyond this controversy to report defective trafficking of the β1 subunit in VSMCs from stroke-prone SHR rats as they failed to detect any difference in total BK channel expression in both α and β1 subunits between wild-type and the stroke-prone SHR rats⁵. While the earlier studies discussed above focused mainly on mechanisms that control the activity of plasma membrane BK channels, more recent studies from Jagger and co-workers investigated the physiological stimuli that regulate trafficking of BK channel subunits to the plasma membrane as means to control VSMC contractility⁸. At resting conditions, the BKα subunit is primarily located at the plasma membrane. The β1 subunit is sequestered in recycling endosomes that are positive for the endosomal GTPase, Rab11A. Jaggar and co-workers showed that the β1 subunit traffics to the cell surface in response to membrane depolarization and NO⁸. Through activation of protein kinase G (PKG), NO caused rapid trafficking and increased surface expression of β1. Membrane depolarization caused Ca²⁺-dependent activation of Rho kinase (ROCK) 1 and 2 and ROCK1/2 activated trafficking of β1 subunits to the plasma membrane through activation of Rab11A on recycling endosomes⁸. This Increased β1 trafficking to the cell surface enhanced the Ca²⁺ sensitivity of BKα subunit, thus promoting vasodilation^{4, 8}. The same group also demonstrated that the vasoconstrictor Endothelin-1 was able to activate PKC and subsequently inhibit β1 subunit surface trafficking in rat VSMCs to promote vasoconstriction⁸. Activation of PKC by Endothelin-1 led to phosphorylation of Rab11A at Serine 177, which prevented the endosomal trafficking of the β1 subunit to the plasma membrane⁸.

Building on their previous findings, here Leo et al. describe a disruption in the trafficking of the β1 subunit of the BK channel in the stroke-prone SHR model⁵. Through cell surface biotinylation on vessel preparations, the authors convincingly showed that cerebral VSMCs isolated from SHR rats had reduced trafficking of the β1 subunit to the plasma membrane after stimulation with either NO or membrane depolarization. This reduced trafficking was also documented functionally using inside-out patch clamp recordings of BK currents and whole cerebral vessel myography, demonstrating reduced BK channel activity and vessel dilation respectively in the SHR model. These are significant findings that may explain many of the functional changes seen in previous studies and represent a critical step towards resolving the discrepancy regarding BK channel function in hypertension. The results from Leo et al. also suggest there is a pathological aberration in hypertensive VSMCs in the cellular signaling that retains β1 subunits within recycling endosomes, which would consequently prevent activation of BK channels and vasodilation. Here, Leo et al. demonstrate that β1 subunit retention is dependent on the activation of protein kinase C (PKC). They show that the hypertensive SHR model is dysregulated in the PKC/Rab11A/β1 trafficking signaling pathway⁵. Specifically, they show that VSMC from SHR rats had impaired β1 trafficking due to increased activation and expression of PKCα and PKCβII. This constitutive activation of PKC induced inhibitory phosphorylation of Rab11A in recycling endosomes and prevented translocation of β1 subunits to the plasma membrane (Figure 1). Thus, PKCα and PKCβII isoforms could represent novel targets in hypertension. PKC inhibitors have already been proposed for treatment of hypertension⁹, but studies have mostly focused on the role of PKC inhibitors in targeting VSMC remodeling. The study by Leo et al. suggests that targeting PKC may also help in acutely relieving VSMC contractility in hypertension. Systematic targeting of PKC is clearly not a good idea due to the ubiquitous role of PKC signaling in various aspects of cellular physiology. Although further mechanistic understanding is needed, the indication from this study that Rab11A, PKCα and PKCβII isoforms are specifically dysregulated in the local cerebral vasculature might offer a therapeutic window for targeting these isoforms in cerebrovascular disease.

Figure 1. Dysregulation of BK β1 subunit trafficking in hypertension.

Top panel illustrates normal trafficking of the BK β1 subunit in VSMC of normotensive animals, promoting VSMC hyperpolarization and vasodilation. Bottom panel shows that aberrant PKC signaling causes dysregulation in trafficking of BK β1 subunit to the VSMC surface in hypertensive animals, which leads to decrease Ca²⁺ sensitivity of BKα channels and vasoconstriction.

The dysregulation of β1 subunit trafficking in SHR rats may apply to human cerebrovascular disease. Previous studies have described single nucleotide polymorphisms (SNPs) in the BKα gene (KCa1.1) and BKβ1 subunit gene (KCNMB1) that correlated with risks for developing cardiovascular disease¹⁰. For example, the E65K polymorphism in the β1 subunit is a “gain of function” mutation that increases the activity of the β1 subunit and decreases the risk of hypertension and cerebrovascular disease¹⁰. More Genome-Wide Association Studies (GWAS) are necessary to characterize the physiological consequences of SNPs in BK channels as well as potential SNPs affecting the trafficking of the BK channel β1 subunit. It would be particularly interesting to identify SNPs associated with specific PKC isoform dysregulation. In light of these findings, the impaired trafficking of the BK channel β1 subunit in the SHR model contributes to our understanding of the elusive pathogenesis of idiopathic hypertension and cerebrovascular disease. Further studies are necessary to determine if these mechanisms are conserved in humans and what particular SNPs would either protect or predispose an individual to this form of dysregulation. It would also be particularly useful to further enhance our knowledge of the mechanisms of dysregulation of recycling endosomes in hypertension and the molecular pathways involved in specific hypertensive models. As we toil to better understand these mechanisms, this will bring us closer to better treatments of hypertension and cerebrovascular disease.