Pattern of Kvβ Subunit Expression in Macrophages Depends upon Proliferation and the Mode of Activation

Abstract

Voltage-dependent potassium channels (Kv) in leukocytes are involved in the immune response. In bone marrow-derived macrophages (BMDM), proliferation and activation induce delayed rectifier K+ currents, generated by Kv1.3, via transcriptional, translational, and posttranslational controls. Furthermore, modulatory Kvβ subunits coassociate with Kvα subunits, increasing channel diversity and function. In this study we have identified Kvβ subunits in mouse BMDM, studied their regulation during proliferation and activation, and analyzed K+ current parameters influenced by these proteins. BMDM express all isoforms of Kvβ1 (Kvβ1.1, Kvβ1.2, and Kvβ1.3) and Kvβ2 (Kvβ2.1), but not Kvβ4, the alternatively spliced murine Kvβ3 variant. M-CSF-dependent proliferation induced all Kvβ isoforms. However, LPS- and TNF-α-induced activation differentially regulated these subunits. Although LPS increased Kvβ1.3, reduced Kvβ1.2, and maintained Kvβ1.1 mRNA levels constant, TNF-α up-regulated Kvβ1.1, down-regulated Kvβ1.2, and left Kvβ1.3 expression unchanged. Moreover, in contrast to TNF-α, M-CSF- and LPS- up-regulated Kvβ2.1. K+ currents from M-CSF- and LPS-stimulated BMDM exhibited faster inactivation, whereas TNF-α increased τ values. Although in M-CSF-stimulated cells the half-inactivation voltage shifted to more positive potentials, the incubation with LPS and TNF-α resulted in a hyperpolarizing displacement similar to that in resting BMDM. Furthermore, activation time constants of K+ currents and the kinetics of the tail currents were different depending upon the mode of activation. Our results indicate that differential Kvβ expression modifies the electrical properties of Kv in BMDM, dependent upon proliferation and the mode of activation. This could determine physiologically appropriate surface channel complexes, allowing for greater flexibility in the precise regulation of the immune response.

Kv1.5 Association Modifies Kv1.3 Traffic and Membrane Localization

“…Transient transfection was performed using Lipofectamine 2000 (Invitrogen) at nearly 80% confluence. Twenty-four hours after transfection, the cells were washed in phosphate-buffered saline (PBS), fixed, and mounted with Aqua Poly/Mount from Polysciences, Inc. Murine bone marrow-derived macrophages from 6 -10-week-old BALB/c mice (Charles River laboratories) were isolated and cultured as described elsewhere (7,12,19). Briefly, the animals were killed by cervical dislocation, and both femurs were dissected with the adherent tissue removed.…”

“…This is important in leukocytes where Kv1.3 and rafts concentrate at the immunological synapse, and raft composition regulates Kv1.3-related cellular functions (16 -18, 28). Unlike T-lymphocytes, macrophages express Kv1.3 and Kv1.5 channels (7,12,13,19). Raft extractions showed that, although Kv1.3 colocalized partially with caveolin (12 Ϯ 2%), Kv1.5 did not target to lipid rafts (Fig.…”

“…Thus, Kv1.3 activity could be regulated by kinases, modulatory ␤ subunits, and assemblies of other Shaker (Kv1) isoforms. Although considerable progress has been made in the first two subjects, less is known about the heterotetrameric structure of the functional channel (3)(4)(5)(6)(7). The Kv subunit composition determines the activity and the pharmacology of the channel and affects surface expression and membrane localization (8,9).…”

Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1.3 voltage-dependent potassium channels

“…Murine bone marrow-derived macrophages (BMDM) were isolated as described elsewhere [2,21]. Cells were cultured in DMEM containing 20% FBS and 30% supernatant of L-929 fibroblast (L-cell) conditioned media as a source of MacrophageColony Stimulating Factor (MCSF).…”

Electrical Membrane Properties in the Model Leishmania-Macrophage

“…The differences in the timing of mRNA transcription resulted in electrophysiological differences. Furthermore, they recorded evidence of the impact of differential expression of Kv subunits in macrophages during proliferation and differential activation between LPS and TNF- (Vicente et al, 2005). The expression of Kv1.5 resulted in associations with Kv1.3 to conform hetero-dimers to distinct biophysical properties (Vicente et al, 2006;Vicente et al, 2007).…”