With its billions of neurons firing at frequencies of hundreds of hertz, the brain is stunningly complex. Our research focuses on the workings of a single central neuron, the pyramidal neuron from the CA1 region of the hippocampus, a region of the brain important for learning and memory and among the first affected in Alzheimer’s disease and epilepsy. In the dendrites of hippocampal CA1 pyramidal neurons, non-uniformly dense, subthreshold, rapidly inactivating potassium channels regulate signal propagation. The non-uniform distribution (with higher expression in the dendrites than in the soma) means that the electrical properties of the dendrites differ markedly from those of the soma. Incoming synaptic signals are shaped by the activity of the potassium channels, and action potentials, once initiated in the axon, progressively decrease in amplitude as they propagate back into the dendrites. Combining patch clamp recording with molecular biology, we investigate the electrophysiological properties and molecular nature of the voltage-gated channels expressed in CA1 dendrites, how their expression is regulated, and the nature of their role in a cellular analogue of learning and memory.
Kv4.2 control of action potential repolarization and frequency-dependent broadening in hippocampal CA1 pyramidal neurons
Kim, Wei, Hoffman
Using a modified Sindbis virus, we expressed either Kv4.2 tagged with enhanced green fluorescent protein (EGFP) or an EGFP-tagged dominant negative mutant of Kv4.2 (Kv4.2gW362F) in CA1 pyramidal neurons of organotypic slice cultures. Compared with control neurons, neurons expressing Kv4.2gW362F displayed broader action potentials with an increase in frequency-dependent AP broadening during a train. In addition, Ca2+ imaging of Kv4.2gW362F-expressing dendrites revealed enhanced AP back-propagation compared with control neurons. Conversely, neurons expressing an increased A-type current through overexpression of Kv4.2 displayed narrower APs with less frequency-dependent broadening and decreased dendritic propagation. The results point to Kv4.2 as the major contributor to the A-current in hippocampal CA1 neurons and suggest a prominent role for Kv4.2 in regulating AP shape and dendritic signaling. As Ca2+ influx occurs primarily during AP repolarization, Kv4.2 activity can regulate cellular processes involving Ca2+-dependent second-messenger cascades such as gene expression and synaptic plasticity.
Kim J, Wei DS, Hoffman DA. Kv4 potassium channel subunits control action potential repolarization and frequency dependent broadening in hippocampal CA1 pyramidal neurons. J Physiol 2005;569:41-57.
Kv4.2 trafficking in CA1 pyramidal neuron dendrites
Kim
We are attempting to characterize the mechanisms that govern the cellular distribution (e.g., dendritic localization) and trafficking of Kv4.2 at both the protein and mRNA levels. To visualize Kv4.2 protein distribution, we tagged Kv4.2 with EGFP at the cytoplasmic C-terminus. EGFP-tagged Kv4.2 (Kv4.2g) showed no kinetic differences from wild-type Kv4.2 when expressed in HEK 293 cells. Moreover, Kv4.2g mimics endogenous Kv4.2 distribution when expressed in cultured hippocampal neurons. We have found that neuronal stimulation results in an activity-dependent redistribution of Kv4.2g away from synaptic sites to the dendritic shaft. The redistribution shares common requirements (NMDA receptor activation and calcium influx) with long-term potentiation (LTP), a cellular mechanism for learning and memory. As the average miniature excitatory postsynaptic current (mEPSC) amplitude was reduced by Kv4.2g expression, removal of Kv4.2 from the dendritic spine seemed a possible mechanism for LTP. To investigate such a possibility, we used a brief application of glycine to induce chemical long-term potentiation (cLTP), which caused not only GluR1 insertion but also Kv4.2g internalization. We observed a significant decrease in endogenous A-type transient K+ current amplitude during cLTP. Thus, activity-dependent trafficking of Kv4.2 in hippocampal neurons appears to provide a mechanism by which neurons dynamically modulate intrinsic excitability. We are currently determining the mechanisms by which Kv4.2 is internalized upon neuronal stimulation.
It is now believed that dendrites are capable of locally translating mRNA into proteins. Messenger RNA exists in hippocampal dendrites as highly dense RNA granules. We detected endogenous Kv4.2 mRNA from dendritic RNA granule fractions of hippocampal neurons by RT-PCR, suggesting that Kv4.2 may be locally translated in hippocampal dendrites. To visualize and track Kv4.2 mRNA, we fused reporter gene mRNA (beta-galactosidase and EGFP) with the 5´ and/or 3´ untranslated region (UTR) of Kv4.2 mRNA. We observed that 3´ but not 5´ UTR-fused reporter gene products were detected throughout dendrites in the form of granule-like puncta. Using live imaging, we are currently investigating the mechanisms of activity-dependent trafficking of both the GFP-tagged Kv4.2 protein and 3´ UTR-fused reporter mRNA.
Krestel HE, Mihaljevic AL, Hoffman DA, Schneider A. Neuronal co-expression of EGFP and beta-galactosidase in mice causes neuropathology and premature death. Neurobiol Dis 2004;17:310-318.
Creation and characterization of Kv4.2 transgenic mice
Hoffman; in collaboration with Anderson
We are currently characterizing a transgenic mouse expressing a dominant negative pore mutation in the voltage-gated potassium channel subunit Kv4.2, the likely molecular identity of transient currents recorded in CA1 dendrites. The mouse expresses the mutant Kv4.2 channel along with GFP under the control of a tetracycline transactivator (tTA)–responsive promoter. Expression is spatially controlled by a new line of tTA-expressing mice that limit tTA activity to the CA1 and dentate gyrus regions of the hippocampus. Expression can be controlled temporally by administrating doxycycline. Experiments in acute hippocampal slices from the mice will investigate Kv4.2’s role in the regulation of AP back-propagation into CA1 dendrites and in synaptic integration and plasticity. In collaboration with Anne Anderson, we are investigating seizure susceptibility in the mice.
Role of voltage-gated potassium channels in synaptic plasticity
Jung
Potassium channels have been shown to regulate the back-propagation of action potentials into CA1 dendrites. Although the function of back-propagation of action potentials is still unclear, recent work has suggested that such back-propagation may provide the depolarization necessary to unblock NMDA receptors, allowing for the induction of synaptic plasticity. We are currently investigating the effect of potassium channel mutations on back-propagation of action potentials and on the induction of LTP in organotypic slice cultures from wild-type and transgenic mice.
Role of voltage-gated potassium channels in synaptic integration
Wei
We are using Ca2+ imaging to examine the propagation of action potentials and synaptic responses between control and mutant Kv4.2-expressing organotypic slices. We are interested in the role of Kv4.2 in dendritic integration and have therefore implemented a localized photolysis technique that selectively activates oblique dendrite terminals. Using such a technique, we will determine if Kv4.2 channel microdomain expression patterns (e.g., dendritic trunk, branch points, and terminals) show different effects on dendritic signal integration.
Role of auxiliary proteins in regulating Kv4.2 expression and function
Yazdani, Kim, Hoffman; in collaboration with Rudy
Kv4 channel–accelerating factor DPPX facilitates Kv4.2 surface expression and reconstitutes the properties of the neuronal currents in heterologous expression systems. In collaboration with Bernardo Rudy’s laboratory, we are using virus-mediated expression of RNAi sequences against DPPX to look at the effect on Kv4.2 trafficking and synaptic signaling.
Collaborators
Anne Anderson, MD, Baylor College of Medicine, Houston, TX
Bernardo Rudy, MD, PhD, New York University Medical Center, New York, NY
For further information, contact hoffmand@mail.nih.gov.