The role of tyrosine kinases and calcineurin in the calcium-mediated modulation of the voltage-gated potassium channel, Kv1.1.

摘要

The voltage-gated K+ channels strongly contribute to defining the electrophysiological properties and the functional roles of many different types of cells. Kv1.1 channels are functionally modulated through G protein coupled receptors by a Ca2+-mediated signal transduction pathway. Previous work in other laboratories has demonstrated that this signal transduction pathway involves protein kinase C (PKC), protein tyrosine kinases (PTKs) and Rho A. My investigations demonstrate that the Ca2+-activated serine/threonine phosphatase, calcineurin (CaN), is also involved in this process. Mouse Kv1.1 channels were expressed in Xenopus oocytes and the macroscopic Kv1.1 current was monitored by two-electrode voltage clamp (TEVC). The Kv1.1 current was strongly suppressed by three different methods; (1) application of 1 muM A23187, a Ca2+ ionophore, (2) 100 nM 4-beta-PMA, a PKC activator, and (3) the ionophoretic injection of orthovanadate ions (VO43-), a protein tyrosine phophatase inhibitor. The pre-treatment of oocytes with 10 muM cyclosporine A (CsA)), a specific CaN inhibitor, prevented or decreased the suppression of the Kv1.1 current by the three different methods. The involvement of PTKs in the Ca2+-mediated suppression of the Kv1.1 current was confirmed by the blockade of 4-beta-PMA-induced suppression by the pre-incubation of oocytes in 100 muM genistein, a broad-spectrum PTK inhibitor.; Because CaN plays a key role in endocytosis, the oocyte membrane capacitance was monitored in these experiments by examining the capacitive transients generated during TEVC below the threshold of activation of the Kv1.1 current. The application of 4-beta-PMA produced a strong decrease in oocyte membrane capacitance consistent with the internalization of Kv1.1 channels. The pre-incubation of oocytes in CsA reduced this decrease in the membrane capacitance. Additionally, the pre-incubation of oocytes in genistein blocked the 4-beta-PMA-induced decrease in the membrane capacitance. The ionophoretic injection of VO 43- did not change the oocyte membrane capacitance despite suppressing the Kv1.1 current. Together these results suggest that CaN is involved in the internalization of Kv1.1 channels and PTKs are involved in both the inactivation and internalization of Kv1.1 channels. Experiments performed with compounds (cytochalasin D, concanavalin A, methyl-beta-cylclodextrin, and monodansylcadaverine) that inhibit endocytosis further suggest the internalization of Kv1.1 channels occurs by clathrin-dependent endocytosis.