Abstract
Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses.
Highlights
Voltage-dependent potassium channels (Kv)10 have a crucial function in excitable cells of determining resting membrane potential and controlling action potentials [1]
Kv1.5. Because Shaker subunits (Kv1).3, the major Kv channel in leukocytes, is associated with the n-type channel and Kv3.1 accounts for the l-type, the proteins responsible for the nЈ-type are unknown [2]. Electrophysiological properties such as activation and inactivation of Kv1.3 expressed in T-cells and heterologous expression systems (Refs. 9 and 10 and references ) are significantly different from those described in macrophages [8, 10, 11]
Our results suggest that Kv1.5 co-associates with Kv1.3, generating functional Kv1.3/Kv1.5 heterotetrameric channels
Summary
Animals and Cell Culture—BMDM and Raw 264.7 macrophages, human embryonic kidney 293 (HEK-293) cells, EL-4 T cell line, and Xenopus laevis oocytes were used. Cryosections were incubated at room temperature on drops of 2% gelatin in phosphate buffer for 20 min at 37 °C followed by 50 mM glycine in PBS for 15 min and 5% normal goat serum in PBS for 10 min. They were incubated with anti-Kv1.5 or and anti-Kv1.3 polyclonal antibodies in 5% normal goat serum in PBS for 30 min. After 3 washes with drops of 5% normal goat serum in PBS for 20 min, sections were incubated for 60 min using IgG anti-rabbit coupled to 10-nm or to 15-nm diameter colloidal gold particles (Aurion) using a 1:60 dilution in 5% normal goat serum in PBS. A value of p Ͻ 0.05 was considered significant
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