Maxik Channel Beta1 Subunit Interacts and Regulates Thromboxane A2 Receptor Function

Abstract

MaxiK channel, composed of the pore-forming α (MaxiKα) and regulatory β1 subunits, controls vascular tone via the activation or inhibition of its pore conducting activity. Thromboxane A2 receptor (TPR), a G-protein coupled receptor, induces potent vasoconstriction mediated by its agonist, thromboxane A2. Previously, we demonstrated that U46619, the stable analogue of thromboxane A2 inhibits MaxiK channel in vascular smooth muscle cell contributing to U46619-induced vasoconstriction. In this study, we report that this inhibition is reversed by nanomolar dehydrosoyasaponin I (DHS-I), a pharmacological tool that indicates MaxiKα and β1 association and functional coupling. The reversing effect of DHS-I indicated that the MaxiK channels inhibited by U46619 could be coupled with regulatory β1 subunits. Co-immunoprecipitation and double immunolabeling in co-expressing cells showed that TPR form a complex with β1 on the plasma membrane. To identify the interacting sites in β1 responsible for TPR-β1 complex formation, we prepared serial carboxyl-terminal deletions of β1 and analyzed their interaction properties with TPR in co-immunoprecipitation experiments. β1 lacking amino acids 103-191 reduces the TPR-β1 association by 44 ± 14% (p<0.01), while deletion of residues 73-191 completely reduces the TPR-β1 interaction, suggesting that amino acids 73 to 191 predominantly contribute to the TPR-β1 interaction. To further investigate how β1 regulates TPR-MaxiKα functional coupling, inside-out patch clamp experiments were performed in HEK293T cells expressing TPR + MaxiKα +/- β1 subunit. We found that the β1 subunit reduces U46619-induced MaxiKα inhibition in a dose-dependent manner. In summary, β1 interacts with TPR forming a tripartite complex with MaxiKα and opposes to TPR agonist-induced MaxiK channel inhibition serving as a buffer to vasoconstriction. Thus, in pathological situations like hypertension or aging where β1 expression is compromised the TPR-MaxiK complex would induce severe vasoconstriction. Supported by NIH and AHA.