Modulation of the MaxiK Potassium Channel by the Coexpression of its β1 and β2 subunits
The high conductance, calcium-activated potassium (MaxiK) channel is widely expressed among mammalian tissues. The activity of the channel is increased by membrane depolarization and by increases in intracellular calcium concentration. The channel is assembled in the membrane as a homotetramer of it...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2004 |
| País: | Chile |
| OAI Identifier: | oai:repositorio.anid.cl:10533/232857 |
| Acceso en línea: | https://hdl.handle.net/10533/232857 |
| Access Level: | acceso abierto |
| Palabra clave: | Ciencias Naturales Otras Ciencias Naturales |
| Sumario: | The high conductance, calcium-activated potassium (MaxiK) channel is widely expressed among mammalian tissues. The activity of the channel is increased by membrane depolarization and by increases in intracellular calcium concentration. The channel is assembled in the membrane as a homotetramer of its pore-forming α-subunit –coded by the Slo gene– and in some tissues its properties are modified by the presence of accessory proteins called β-subunits. Four β-subunits have been cloned in mammals, named β1 to β4. β1-subunit increases the apparent calcium sensitivity of the channel and slows down its activation and deactivation kinetics. It also allows the channel to be activated by 17-β-estradiol. In the presence of the β2-subunit the channel shows a fast inactivation process, which is removed by deleting the N-terminus of the protein. The β2-subunit without the fast inactivation domain (β2IR) has similar effects as the β1 subunit,increasing the apparent calcium sensitivity of the channel. The objective of this thesis work is to define molecular basis for the interaction of β1- and β2-subunits with the MaxiK channel, related to the modulation of the calcium- and voltage sensitivity and to the activation of the channel by estrogens. Heterologous expression systems were used to study several activation parameters of the MaxiK channel, alone or in combination with the β1- and β2IR-subunits.The data were analyzed in the context of an allosteric activation model that has been proposed for the MaxiK channel. The results indicate that the increase of the apparent calcium sensitivity in the presence of both β-subunits can not be explained by an increase in the affinity o the calcium binding sites. Instead, the β1- and β2-subunits increase the functional coupling between calcium binding and channel opening. An increase of the functional coupling between voltage sensor activation and channel opening was also observed, with some quantitative differences between both β-subunits. Therefore, β1- and β2-subunits must –directly or indirectly– interact with the domains of the α-subunit responsible for this functional coupling. For the voltage-activation, these are the linkers between the S4 and S5 transmembrane domains; whereas for the calcium-activation, these are the linkers between the S6 domain and the RCK domain –a domain adjacent to the inner mouth of the conduction pathway and that has been proposed to control the open-close state of the pore. The β1-subunit also decreases the voltage-dependence of the voltage sensor of the channel, located in the S4 segment of the α-subunit. This effect was not observed in the presence of the β2IR subunit. The transition between closed and open states of the channel has intrinsic voltage dependence, unrelated to voltage sensor movement, whose molecular nature is unclear. However, none of the β-subunits affected this process. Channel activation by 17-β-estradiol (17βE) and Tamoxifen (Tx) was studied in the absence and presence of different β-subunits. 5 μM 17βE activates the channel only in the presence of the β1- or β4-subunits, but not in their absence of in presence of β2- or β2IR-subunits. The antiestrogen Tx has different effects depending on concentration. When the concentration is lower than 1 μM, Tx activates the channel but only in the presence of the β1-subunit. When the Tx concentration is higher than 1 μM, there is an inhibition of MaxiK currents independent of the presence of any β-subunit. These results show that there are main differences in the pharmacology of MaxiK channel activation by estrogens and the pharmacology of the nuclear estrogen receptor. On the other hand, Tx, would have two interaction sites with the channel: an inhibition site present in the α-subunit and an activation site expressed only in the presence of the β1-subunit. In order to define functional domains in the β-subunits, the extracellular domains of the β1- and β2IR-subunit were exchanged, and the resulting quimerical β-subunits were studied. The effects of the quimerical β-subunits showed that all the differences between β1- and β2IR-subunits can be completely associated to the transmembrane and/or intracellular domains. This raises the conclusion that the transmembrane and/or intracellular domains of the β1-subunit interact with the voltage sensor of the channel, though a role of this domains on the other effects of the β-subunits is not discarded. Moreover, the transmembrane and/or intracellular domains of the β1-subunits are also needed for the activation of the channel by 17βE. All these results are an important step towards the molecular definition of the interaction networks between α- and β-subunits of the MaxiK channel. |
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