Differential Expression ofI_AChannel Subunits Kv4.2 and Kv4.3 in Mouse Visual Cortical Neurons and Synapses

Abstract

In cortical neurons, pore-forming ␣-subunits of the Kv4 subfamily underlie the fast transient outward K ϩ current (I A ). Considerable evidence has accumulated demonstrating specific roles for I A channels in the generation of individual action potentials and in the regulation of repetitive firing. Although I A channels are thought to play a role in synaptic processing, little is known about the cell typeand synapse-specific distribution of these channels in cortical circuits. Here, we used immunolabeling with specific antibodies against Kv4.2 and Kv4.3, in combination with GABA immunogold staining, to determine the cellular, subcellular, and synaptic localization of Kv4 channels in the primary visual cortex of mice, in which subsets of pyramidal cells express yellow fluorescent protein. The results show that both Kv4.2 and Kv4.3 are concentrated in layer 1, the bottom of layer 2/3, and in layers 4 and 5/6. In all layers, clusters of Kv4.2 and Kv4.3 immunoreactivity are evident in the membranes of the somata, dendrites, and spines of pyramidal cells and GABAergic interneurons. Electron microscopic analyses revealed that Kv4.2 and Kv4.3 clusters in pyramidal cells and interneurons are excluded from putative excitatory synapses, whereas postsynaptic membranes at GABAergic synapses often contain Kv4.2 and Kv4.3. The presence of Kv4 channels at GABAergic synapses would be expected to weaken inhibition during dendritic depolarization by backpropagating action potentials. The extrasynaptic localization of Kv4 channels near excitatory synapses, in contrast, should stabilize synaptic excitation during dendritic depolarization. Thus, the synapse-specific distribution of Kv4 channels functions to optimize dendritic excitation and the association between presynaptic and postsynaptic activity.

DPP10 splice variants are localized in distinct neuronal populations and act to differentially regulate the inactivation properties of Kv4-based ion channels

“…However, given what we do know about functional effects of known Kv4 channel subunits in heterologous expression we can likely rule out differences in Kv4 subunits or KChIPs as being responsible. Although cortical pyramidal cells and interneurons express both Kv4.2 and Kv4.3, the impact of Kv4.2/Kv4.3 heterotetramer formation on inactivation kinetics is likely to be minimal (Burkhalter et al, 2006;Guo et al, 2002). Furthermore, we have shown that although different KChIPs, such as KChIP3 and KChIP4a, have distinct effects on channel functional properties the robust DPP10a effect on inactivation properties overrides these differences.…”

Postnatal development of A-type and Kv1- and Kv2-mediated potassium channel currents in neocortical pyramidal neurons

“…The anesthetized animals were transcardially perfused with 0.01 M sodium phosphate buffer plus 0.89% NaCl (PBS) followed by PBS-buffered 4% paraformaldehyde and 0.2% picric acid. (For Kv4.2, we also used 4% paraformaldehyde without picric acid, postfixed for ϳ12 h, and washed overnight in 0.1 M PBS; Burkhalter et al 2006). Brains were removed, postfixed for ϳ12 h at 4°C, and then blocked and postfixed for ϳ24 h. Sections through the cortex were taken at 50 m on a vibrating microtome (Leica VT1000S), rinsed in PBS, and incubated in 5% normal goat serum with 3% H 2 O 2 for 1-2 h to reduce background staining.…”

“…Brains were removed, postfixed for ϳ12 h at 4°C, and then blocked and postfixed for ϳ24 h. Sections through the cortex were taken at 50 m on a vibrating microtome (Leica VT1000S), rinsed in PBS, and incubated in 5% normal goat serum with 3% H 2 O 2 for 1-2 h to reduce background staining. For Kv4.2, we also tried treatment with 0.5% sodium borohydride, followed by 0.3% H 2 O 2 and then 50% ethanol (Burkhalter et al 2006). All antibodies used were monoclonal (NeuroMab, Univ.…”

“…We previously tested these pharmacological agents and found them to be selective at the doses used (Guan et al 2006(Guan et al , 2007; however, this remains a concern. The monoclonal antibodies used were all reported not to stain cortex of knockout animals, suggesting their specificity (Burkhalter et al 2006). Our voltage protocols for isolating A-type current are biased toward channel types with relatively rapid recovery from inactivation; thus our findings are likely biased toward detection of Kv4-mediated versus Kv1-mediated Protocol is identical to that in A and B except that the recovery voltage was varied in 10 mV increments (step B in protocol).…”

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells

“…There are over 80 VGIC pore-forming subunits encoded in the human genome (5), and many of these are coexpressed within the same cell (3), making it difficult to dissect specific channel contributions to electrical signaling. In neurons, the precise trafficking of VGIC subtypes to distinct cell regions such as the axon hillock, presynaptic terminals, and distal dendrites allows specific subtypes to control voltage changes in subcellular compartments (6)(7)(8)(9). However, the presence of an ion channel subunit in a cell does not indicate whether it is activated under physiological conditions (10)(11)(12)(13).…”