Physiological and pharmacological modulation of kv7 channels

  1. Alicia de la Cruz Fernández
Supervised by:
  1. Teresa Gonzalez Gallego Director
  2. Carmen Valenzuela Miranda Director

Defence university: Universidad Autónoma de Madrid

Year of defence: 2017

Committee:
  1. Lisardo Boscá Chair
  2. Rubén Vicente García Secretary
  3. Ángel Luis Cogolludo Torralba Committee member
  4. Teresa Giraldez Fernandez Committee member
  5. Antonio Rodríguez Artalejo Committee member

Type: Thesis

Teseo: 556153 DIALNET

Abstract

Abstract Voltage-dependent potassium channels are spanning-membrane integral proteins involved in several and diverse physiological and pathological processes such as immune response or cardiovascular function. The first goal of this study was to elucidate the role of KV7.1/KV7.5, as a heterotetramer complex, in vascular smooth muscle. To that end, we studied the expression levels of both channels in rat vascular myocytes, as well as their interaction in both a heterologous system and in aorta artery cells. KV7.1 channels targets lipid-enriched domains in plasma membrane. The presence of KV7.5 channels modulates KV7.1 channels-targeting and, indeed, their physiological regulation. In those tissues where KV7.1 channels are the only ones expressed, as in the heart, KV7.1 channels are targeting lipid rafts. However, when both isoforms are expressed together, as in aorta artery and cava vein, the distribution of both channels throughout the membrane changes. Using patch-clamp technique we demonstrated that KV7.1/KV7.5 channels were functional, and we studied their electrophysiological and pharmacological properties. The activation of the channels generated a phenotypically intermediate current, which properties were between both the homotetrameric channels alone. In addition, electrophysiological characteristics of the channels were studied in the presence of different regulatory subunits (KCNE1 and KCNE3). In the presence of regulatory subunits KV7.1/KV7.5 channels showed their own electrophysiological properties. Retigabine is an activator drug of KV7.2-7.5 channels. KV7.1/KV7.5 channels can be regulated by retigabine as KV7.5 channels. We studied the possible role of these channels in the control of the vascular tone. To that end, we measured the relaxant response of coronary arteries induced by retigabine in constricted arteries by different blockers of KV7 channels. We demonstrated that KV7.1/KV7.5 are involved in the control of the vascular tone. Otherwise, we studied the KV7 first member, the KV7.1 channel, with the KCNE1 regulatory subunit. Together, they form one of the most important current of the repolarization phase of the cardiac action potential (IKs). We analyzed, using the patch-clamp technique, the acute and chronic exposition of polyunsaturated fatty acids (PUFAs), DHA and AA, in KV7.1/KCNE1 channels expressed in COS-7 cells. Also, in DHA analysis, we used western blot and isolation lipid rafts techniques. Acute DHA and AA exposition increased the magnitude of KV7.1/KCNE1 currents. DHA but not AA slowed the activation kinetics. However, both PUFAs accelerated the deactivation kinetics. Chronic exposition to these compounds did not modify the magnitude of the currents. However, DHA shifted the activation curve toward more negative membrane potentials and accelerated the activation and deactivation kinetics of the channels. On the contrary, AA slowed the activation whereas it accelerated the deactivation process. Chronic exposure to DHA, decreased the KV7.1 but not KCNE1 protein expression. Also, it induced a redistribution of KV7.1 channels in the plasma membrane. PUFAs effects on KV7.1/KCNE1 channels depends on both the compound’s chemical nature, n-3 or n-6 PUFAs, as well as the time of exposure, which might be acute or chronic. It has been described that enriched PUFAs diet has anti-inflammatory properties. Moreover, n-3 or n-6 PUFAs lipid-derived mediators such as lipoxins and resolvins (which levels are increased by the consumption of aspirin), have been emerged as potent anti-inflammatory agents that promote the resolution phase of the inflammation process. Ion channels, and specially potassium ion channels, play an essential role in macrophages physiology. Accordingly, we studied the lipid-derived mediators effects on both bone marrow derived macrophages (BMDM), which LPS-dependent activation are related with the increased of KV1.3/KV1.5 expression, and in KV7.1/KCNE1 channels where the PUFAs effects have been studied. Lipoxins, but not resolvins, could reverse the LPS-activated macrophages. However, resolvins KV7.1/KCNE1 block were much more potent.