ML213

Voltage-gated K v 7.4 channels have been implicated in vascular smooth muscle cells’ activity because they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are downregulated in several arterial beds in different models of hypertension. Angiotensin II (Ang II) is a key player in hypertension that affects the expression of several classes of ion channels. In this study, we evaluated the effects of Ang II on the expression and function of vascular K v 7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased K v 7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (K v 7)–sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the K v 7 activator ML213. Ang II also reduced K v 7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II–induced decrease of K v 7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of K v 7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of K v 7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased K v 7.4 ubiquitination. Similar alterations were found in mesenteric vascular smooth muscle cells isolated from Ang II–infused mice. The effect of Ang II was emulated by 17-AAG (17-demethoxy-17-(2-propenylamino) geldanamycin) that inhibits HSP90 interactions with client proteins. These results show that Ang II downregulates K v 7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and K v 7.4 ubiquitination and degradation via the proteasome.

Chronic peripheral neuropathic pain (PNP) is a debilitating condition that is associated with many types of injury/diseases, including diabetes mellitus. Patients with longstanding diabetes develop diabetic PNP (DPNP), which is resilient to currently available drugs. The underlying molecular mechanisms of DPNP are still illusive, but K_v7 channels that have been implicated in the pathogenesis of various types of chronic pain are likely to be involved. Indeed, using the streptozotocin (STZ) rat model of DPNP, we have previously shown that K_v7 activation with their non-selective activator retigabine attenuated neuropathic pain behavior suggesting that these channels are implicated in DPNP pathogenesis. Here, we evaluated, in the same STZ model, whether the more potent and more selective K_v7 channel openers flupirtine and ML213 attenuate STZ-induced pain hypersensitivity.

Male Sprague Dawley rats (250-300 g) were used. The STZ model involved a single injection of STZ (60 mg/kg, i.p.). Behavioral testing for mechanical and heat pain sensitivity was performed using a dynamic plantar aesthesiometer and Hargreaves analgesiometer, respectively.

STZ rats exhibited behavioral signs of mechanical and heat hypersensitivity as indicated by significant decreases in the mean paw withdrawal threshold (PWT) and mean paw withdrawal latency (PWL), respectively, at 35 days post-STZ treatment. Single injections of flupirtine (10 mg/kg, i.p.) and ML213 (5 mg/kg, i.p.) to STZ rats (35-days after STZ treatment) caused significant increases in the mean PWT, but not PWL, indicating attenuation of mechanical, but not heat hypersensitivity. Both flupirtine and ML213 were as effective as the positive control gabapentin (10/kg, i.p.), and their anti-allodynic effects were prevented by the K_v7 channel-specific blocker XE991 (3 mg/kg, i.p.).

The findings suggest that K_v7 channels are involved in the mechanisms of mechanical but not heat hypersensitivity associated with DPNP, and that their activation may prove to be effective in alleviating DPNP symptoms.