Identification of the Kv2.1 K+ channel as a major component of the delayed rectifier K+ current in rat hippocampal neurons

Abstract

Molecular cloning studies have revealed the existence of a large family of voltage-gated K+ channel genes expressed in mammalian brain. This molecular diversity underlies the vast repertoire of neuronal K+ channels that regulate action potential conduction and neurotransmitter release and that are essential to the control of neuronal excitability. However, the specific contribution of individual K+ channel gene products to these neuronal K+ currents is poorly understood. We have shown previously, using an antibody, "KC, " specific for the Kv2.1 K+ channel alpha-subunit, the high-level expression of Kv2.1 protein in hippocampal neurons in situ and in culture. Here we show that KC is a potent blocker of K+ currents expressed in cells transfected with the Kv2.1 cDNA, but not of currents expressed in cells transfected with other highly related K+ channel alpha-subunit cDNAs. KC also blocks the majority of the slowly inactivating outward current in cultured hippocampal neurons, although antibodies to two other K+ channel alpha-subunits known to be expressed in these cells did not exhibit blocking effects. In all cases the blocking effects of KC were eliminated by previous incubation with a recombinant fusion protein containing the KC antigenic sequence. Together these studies show that Kv2.1, which is expressed at high levels in most mammalian central neurons, is a major contributor to the delayed rectifier K+ current in hippocampal neurons and that the KC antibody is a powerful tool for the elucidation of the role of the Kv2.1 K+ channel in regulating neuronal excitability.

Fig. 1.

Immunofluorescence staining of recombinant K⁺ channel α-subunit polypeptides expressed in COS-1 cells. COS-1 cells were transfected with 8 μg/ml of Kv2.1/RBG4 (in A, D), Kv2.2/RBG4 (inB, E), and Kv1.5/RBG4 (inC, F). Then the transfected cells were fixed, permeabilized, and stained with affinity-purified KC IgG plus anti-Kv2.1 monoclonal D4/11 (in A,D), anti-Kv2.2 monoclonal K37/89 (in B,E), and anti-Kv1.5 monoclonal K7/45 (inC, F). Finally, the cells were incubated with fluorescein-conjugated goat-anti rabbit and Texas Red goat anti-mouse secondary antibodies. Photographs show fluorescein (A–C) and Texas Red (D–F) staining of double-labeled cells.

Fig. 2.

KC antibody block of voltage-dependent currents in transiently transfected COS-1 cells. Shown are typical membrane currents from COS-1 cells expressing recombinant Kv2.1 recorded 0 min (top) and 10 min (middle) after a seal was made in whole-cell patch-clamp configuration with 3 nmKC IgG in the pipette internal solution. The bottom trace shows the resultant subtracted current. The current traces that are shown were recorded by a step depolarization from a holding potential of −80 to +20 mV in 20 mV steps for 200 msec.

Fig. 3.

KC antibody block of voltage-dependent currents in the drk1CGN/l(tk⁻) stable cell line.A, Membrane currents were recorded at the time the seal was made in whole-cell patch-clamp configuration and at successive 1 min intervals thereafter for 10 min. Currents were recorded from drk1cgn/l(tk⁻) cells expressing recombinant Kv2.1 after a seal was made with pipette solution alone (Control, top trace) or 3 nmKC IgG (+ Antibody, bottom trace) in the pipette internal solution. The current traces that are shown were recorded by a step depolarization from a holding potential of −80 mV to a test potential of +20 mV for 200 msec. B, Time course of the effects of antibody treatment on current amplitude in drk1cgn/l(tk⁻) cells. The current amplitude in the cells was recorded with 3 nm KC IgG (filled circles) or no antibody (open circles) present in the patch pipette internal solution. Currents were evoked once every minute by a depolarization from −80 to +20 mV for 200 msec. Current amplitudes (I) are expressed relative to those obtained at time 0 (I₀); mean ± SEM of four cells for each treatment. *p < 0.05 versus time 0. C, Dose–response of KC block of current in drk1cgn/l(tk⁻) cells. Currents were recorded as in A and B at the time the seal was made in whole-cell patch-clamp configuration (time0) and 10 min later, using patch pipettes with different amounts of KC IgG in the patch pipette internal solution. Current amplitudes after 10 min (I) are expressed as relative to those obtained at time 0 (I₀); mean ± SEM of four cells for each treatment.

Fig. 4.

Kv2.1 expression in cultured hippocampal neurons. Shown is immunofluorescence staining of E19 hippocampal neurons after 14 d in culture, using 0.6 nm KC IgG and an anti-synaptophysin mouse monoclonal antibody. The cultured cells were fixed, permeabilized, and stained with primary antibodies, followed by incubation with fluorescein-conjugated goat anti-rabbit and Texas Red-conjugated goat anti-mouse secondary antibodies.

Fig. 5.

KC antibody block of slowly inactivating voltage-dependent currents in cultured hippocampal neurons.A, Typical membrane currents recorded from E19 rat hippocampal neurons after 14 d in culture. The current traces that are shown were recorded at the time the seal was made in whole-cell patch-clamp configuration (time 0) and 10 min later by step depolarizations from a holding potential of −30 mV to voltages ranging from −30 to +20 mV in increments of 10 mV for 200 msec.B, Time course of the effects of antibody treatment on current amplitude in hippocampal neurons. The current amplitude in cells was recorded with 3 nm KC IgG (filled circles), 3 nm Kv1.4E IgG (open squares), or no antibody (open circles) present in the patch pipette internal solution. Currents were evoked once every minute by a depolarization from −30 to +20 mV for 200 msec. Current amplitudes (I) are expressed relative to those obtained at time 0 (I₀); mean ± SEM of four cells for each treatment. *p < 0.05 versus time 0. C, Dose–response of KC block of neuronal outward currents. Currents were recorded as inA and B at 0 and 10 min after the seal was made in whole-cell patch-clamp configuration and with different amounts of KC IgG in the patch pipette internal solution. Current amplitudes after 10 min (I) are expressed as relative to those obtained at the time the seal was made (I₀); mean ± SEM of four cells for each treatment.