The Kv2.1 C terminus can autonomously transfer Kv2.1-like phosphorylation-dependent localization, voltage-dependent gating, and muscarinic modulation to diverse Kv channels

Abstract

Modulation of K+ channels is widely used to dynamically regulate neuronal membrane excitability. The voltage-gated K+ channel Kv2.1 is an abundant delayed rectifier K+ (IK) channel expressed at high levels in many types of mammalian central neurons where it regulates diverse aspects of membrane excitability. Neuronal Kv2.1 is constitutively phosphorylated, localized in high-density somatodendritic clusters, and has a relatively depolarized voltage dependence of activation. Here, we show that the clustering and voltage-dependent gating of endogenous Kv2.1 in cultured rat hippocampal neurons are modulated by cholinergic stimulation, a common form of neuromodulation. The properties of neuronal Kv2.1 are recapitulated in recombinant Kv2.1 expressed in human embryonic kidney 293 (HEK293) cells, but not COS-1 cells, because of cell background-specific differences in Kv2.1 phosphorylation. As in neurons, Kv2.1 in HEK293 cells is dynamically regulated by cholinergic stimulation, which leads to Ca2+/calcineurin-dependent dephosphorylation of Kv2.1, dispersion of channel clusters, and hyperpolarizing shifts in the voltage-dependent gating properties of the channel. Immunocytochemical, biochemical, and biophysical analyses of chimeric Kv channels show that the Kv2.1 cytoplasmic C-terminal domain can act as an autonomous domain sufficient to transfer Kv2.1-like clustering, voltage-dependent activation, and cholinergic modulation to diverse Kv channels. These findings provide novel mechanistic insights into cholinergic modulation of ion channels and regulation of the localization and voltage-dependent gating properties of the abundant neuronal Kv2.1 channel by cholinergic and other neuromodulatory stimuli.

Figure 1.

Carbachol-treatment of cultured rat hippocampal neurons leads to calcineurin-dependent dephosphorylation and disruption of Kv2.1 clusters and hyperpolarizing shifts in voltage-dependent gating of I_K currents. A, Calcineurin-dependent disruption of Kv2.1 clusters in cultured rat hippocampal neurons after carbachol treatment. Images of immunofluorescence staining of control and carbachol-treated (100 μm, 15 min) neurons, without or with pretreatment of FK520 (5 μm, 10 min) are shown. Neurons were immunostained with rabbit anti-Kv2.1 polyclonal antibody KC (green) and mouse anti-microtubule-associated protein 2 monoclonal antibody (red), as detailed in Materials and Methods. The arrows indicate high-density Kv2.1 clusters. Scale bar, 10 μm. B, Immunoblot analysis of native Kv2.1 from cultured rat hippocampal neurons (∼10 μg of crude lysate) without or with carbachol and FK520 treatments, as in A. Samples were size fractionated on 7.5% SDS-PAGE and analyzed by immunoblot/ECL using the anti-Kv2.1 mouse monoclonal antibody K89/41. Native Kv2.1 from rat brain [∼10 μg of crude rat brain membrane proteins (RBM)] without or with in vitro treatment with AP (100 U/ml) is presented to allow for a comparison of the extent of electrophoretic mobility shift obtained after in vivo carbachol treatment versus in vitro AP treatment. The numbers on left refer to the mobility of prestained molecular weight standards in kilodaltons. C, Voltage-dependent activation (filled symbols) and steady-state inactivation (open symbols) curves of outward currents from cultured rat hippocampal neurons without or with carbachol and FK520 treatments, as in A. See Table 1 for voltage-dependent activation and steady-state inactivation parameters. CCh, Carbachol. Error bars represent mean ± SEM.

Figure 2.

Phosphorylation-dependent differences in localization and function of recombinant Kv2.1 in different mammalian cell backgrounds. A, Immunofluorescence staining analysis of recombinant rat Kv2.1 expressed in COS-1 (left) and HEK293 (right) cells. The cells were immunostained with anti-Kv2.1 mouse monoclonal antibody K89/41, as detailed in Materials and Methods. The arrow indicates a high-density Kv2.1 cluster in an HEK293 cell; note the uniform localization in COS-1 cells. Scale bar, 10 μm. B, Immunoblot analysis of native Kv2.1 from rat brain [∼10 μg of crude rat brain membrane proteins (RBM)] and recombinant Kv2.1 in detergent extracts from COS-1 or HEK293 cells (∼5 μg of total protein each) without or with in vitro treatment with AP (100 U/ml), as detailed in Materials and Methods. Samples were size fractionated on 7.5% SDS-PAGE and analyzed by immunoblot using anti-Kv2.1 mouse monoclonal antibody K89/41, followed by ECL detection. The numbers on the left refer to the mobility of prestained molecular weight standards in kilodaltons. C, D, Representative whole-cell Kv2.1 current traces from COS-1 (C) and HEK293 (D) cells before and after 30 min intracellular dialysis of AP (100 U/ml) as per the shown pulse protocol. E, F, Voltage-dependent activation (squares) and steady-state inactivation (circles) relationships of Kv2.1 currents in COS-1 (E) and HEK293 (F) cells before (filled symbols) and after (open symbols) intracellular dialysis of AP for 30 min, as detailed in Materials and Methods. See Table 1 for voltage-dependent activation and steadystate inactivation parameters. Error bars represent mean ± SEM.

Figure 3.

Ionomycin and carbachol induce Ca²⁺/calcineurin-dependent dephosphorylation and disruption of Kv2.1 clusters in HEK293 cells. A, HEK293 cells expressing Kv2.1 were treated with 1 μm ionomycin or 100 μm carbachol. One aliquot each of the drug-treated cell lysates was treated with AP (100 U/ml). Approximately 5 μg of protein each from control and AP-treated samples were size fractionated on 7.5% SDS-PAGE and analyzed by immunoblot/ECL using anti-Kv2.1 mouse monoclonal antibody K89/41. B, HEK293 cells expressing Kv2.1 were treated with 1 μm ionomycin or 100 μm carbachol without or with pretreatment of 5 μm FK520, as detailed in Materials and Methods. Lysates from these drug-treated cells (∼5 μg of protein each) were size fractionated on 7.5% SDS-PAGE and analyzed by immunoblot/ECL using anti-Kv2.1 mouse monoclonal antibody K89/41. The numbers on the left refer to the mobility of prestained molecular weight standards in kilodaltons. C, Ca²⁺/calcineurin-dependent altered localization of Kv2.1 in HEK293 cells after ionomycin or carbachol treatment. Kv2.1-transfected cells were treated with ionomycin or carbachol, as in A and B, in either Ca²⁺-containing or Ca²⁺-free extracellular conditions and immunostained with anti-Kv2.1 antibody K89/41. The projected images (top) were reconstructed from ∼40 optical X-Z sections (0.35 μm) of the cells taken with the Zeiss ApoTome confocal microscope. The bottom panel in each cell is the cross-sectional view at the level of drawn line. The arrows indicate high-density Kv2.1 clusters. Scale bar, 10 μm. Quantitative analyses of clustering in these samples are presented in supplemental Table 2 (available at www.jneurosci.org as supplemental material). CCh, Carbachol; Inm, ionomycin.

Figure 4.

Ionomycin and carbachol treatment of HEK293 cells expressing Kv2.1 lead to Ca²⁺/calcineurin-dependent hyperpolarizing shifts in voltage-dependent channel-gating properties. A, Representative Kv2.1 current traces from control cells and from cells treated with ionomycin (1 μm) or carbachol (100 μm) and with or without previous intracellular dialysis of FK520 (5 μm) or BAPTA (10 μm). Currents were recorded following the same pulse protocol as used in Figure 2C. B, C, Voltage-dependent activation (B) and steady-state inactivation (C) relationship of Kv2.1 currents obtained in experiments performed as in A. See Table 1 for voltage-dependent activation and steady-state inactivation parameters. Error bars represent mean ± SEM.

Figure 5.

Immunocytochemical analyses of surface localization of the chimeric Kv2.2N-Kv2.1C and Kv1.5N-Kv2.1C channels in HEK293 cells without or with cholinergic stimulation. A, Schematic showing the construction of chimeric Kv2.2N-Kv2.1C and Kv1.5N-Kv1.5C channels. B, C, The chimeric Kv2.2N-Kv2.1C (B) and Kv1.5N-Kv2.1C (C) channels containing the cytoplasmic C-terminal domain of Kv2. 1 show clustered localizations that are disrupted after carbachol treatment. HEK293 cells expressing WT or chimeric channels were treated with 100 μm carbachol and immunostained with the Kv1.5e rabbit polyclonal antibody (A) and the anti-Kv2.2 mouse monoclonal antibody K37/89 (B). The projected images (top) were reconstructed from ∼40 optical X-Z sections (0.35 μm) of the cells taken with the Zeiss ApoTome confocal microscope. The bottom panel in each cell is the cross-sectional view at the level of drawn line. The arrows indicate high-density channel clusters. Scale bar, 10 μm. Quantitative analyses of clustering in these samples are presented in supplemental Table 2 (available at www.jneurosci.org as supplemental material). CCh, Carbachol.

Figure 6.

Voltage-dependent biophysical properties of Kv2.2N-Kv2.1C and Kv1.5N-Kv2.1C chimeric channels without or with carbachol treatment. Voltage-dependent activation (A, C) and steady-state inactivation (B, D) curves of WT Kv2.2 (A, B) and Kv1.5 (C, D) and of chimeric Kv2.2N-Kv2.1C (A, B) and Kv1.5N-Kv2.1C (C, D) channels, without or with the extracellular application of 100 μm carbachol (CCh) for 15 min. The voltage-dependent parameters are detailed in Table 2. Error bars represent mean ± SEM.