Faculty Bibliography

The gene encoding the dipeptidyl peptidase-like protein DPP6 (also known as DPPX) has been associated with human neural disease. However, until recently no function had been found for this protein. It has been proposed that DPP6 is an auxiliary subunit of neuronal Kv4 K(+) channels, the ion channels responsible for the somato-dendritic A-type K(+) current, an ionic current with crucial roles in the regulation of firing frequency, dendritic integration and synaptic plasticity. This view has been supported mainly by studies showing that DPP6 is necessary to generate channels with biophysical properties resembling the native channels in some neurons. However, independent evidence that DPP6 is a component of neuronal Kv4 channels in the brain, and whether this protein has other functions in the CNS is still lacking. We generated antibodies to DPP6 proteins to compare their distribution in brain with that of the Kv4 pore-forming subunits. DPP6 proteins were prominently expressed in neuronal populations expressing Kv4.2 proteins and both types of protein were enriched in the dendrites of these cells, strongly supporting the hypothesis that DPP6 is an associated protein of Kv4 channels in brain neurons. The observed similarity in the cellular and subcellular patterns of expression of both proteins suggests that this is the main function of DPP6 in brain. However, we also found that DPP6 antibodies intensely labeled the hippocampal mossy fiber axons, which lack Kv4 proteins, suggesting that DPP6 proteins may have additional, Kv4-unrelated functions

Sensory signals of widely differing dynamic range and intensity are transformed into a common firing rate code by thalamocortical neurons. While a great deal is known about the ionic currents, far less is known about the specific channel subtypes regulating thalamic firing rates. We hypothesized that different K(+) and Ca(2+) channel subtypes control different stimulus-response curve properties. To define the channels, we measured firing rate while pharmacologically or genetically modulating specific channel subtypes. Inhibiting Kv3.2 K(+) channels strongly suppressed maximum firing rate by impairing membrane potential repolarization, while playing no role in the firing response to threshold stimuli. By contrast, inhibiting Kv1 channels with alpha-dendrotoxin or maurotoxin strongly increased firing rates to threshold stimuli by reducing the membrane potential where action potentials fire (V(th)). Inhibiting SK Ca(2+)-activated K(+) channels with apamin robustly increased gain (slope of the stimulus-response curve) and maximum firing rate, with minimum effects on threshold responses. Inhibiting N-type Ca(2+) channels with omega-conotoxin GVIA or omega-conotoxin MVIIC partially mimicked apamin, while inhibiting L-type and P/Q-type Ca(2+) channels had small or no effects. EPSC-like current injections closely mimicked the results from tonic currents. Our results show that Kv3.2, Kv1, SK potassium and N-type calcium channels strongly regulate thalamic relay neuron sensory transmission and that each channel subtype controls a different stimulus-response curve property. Differential regulation of threshold, gain and maximum firing rate may help vary the stimulus-response properties across and within thalamic nuclei, normalize responses to diverse sensory inputs, and underlie sensory perception disorders

Kv3.3 proteins are pore-forming subunits of voltage-dependent potassium channels, and mutations in the gene encoding for Kv3.3 have recently been linked to human disease, spinocerebellar ataxia 13, with cerebellar and extracerebellar symptoms. To understand better the functions of Kv3.3 subunits in brain, we developed highly specific antibodies to Kv3.3 and analyzed immunoreactivity throughout mouse brain. We found that Kv3.3 subunits are widely expressed, present in important forebrain structures but particularly prominent in brainstem and cerebellum. In forebrain and midbrain, Kv3.3 expression was often found colocalized with parvalbumin and other Kv3 subunits in inhibitory neurons. In brainstem, Kv3.3 was strongly expressed in auditory and other sensory nuclei. In cerebellar cortex, Kv3.3 expression was found in Purkinje and granule cells. Kv3.3 proteins were observed in axons, terminals, somas, and, unlike other Kv3 proteins, also in distal dendrites, although precise subcellular localization depended on cell type. For example, hippocampal dentate granule cells expressed Kv3.3 subunits specifically in their mossy fiber axons, whereas Purkinje cells of the cerebellar cortex strongly expressed Kv3.3 subunits in axons, somas, and proximal and distal, but not second- and third-order, dendrites. Expression in Purkinje cell dendrites was confirmed by immunoelectron microscopy. Kv3 channels have been demonstrated to rapidly repolarize action potentials and support high-frequency firing in various neuronal populations. In this study, we identified additional populations and subcellular compartments that are likely to sustain high-frequency firing because of the expression of Kv3.3 and other Kv3 subunits.

Transient subthreshold-activating somato-dendritic A-type K(+) currents (I(SA)s) have fundamental roles in neuronal function. They cause delayed excitation, influence spike repolarization, modulate the frequency of repetitive firing, and have important roles in signal processing in dendrites. We previously reported that DPPX proteins are key components of the channels mediating these currents (Kv4 channels) (Nadal, M.S., Ozaita, A., Amarillo, Y., Vega-Saenz, E., Ma, Y., Mo, W., Goldberg, E.M., Misumi, Y., Ikehara, Y., Neubert, T.A., Rudy, B., 2003. The CD26-related dipeptidyl aminopeptidase-like protein DPPX is a critical component of neuronal A-type K+ channels. Neuron 37, 449-461). The DPPX gene encodes alternatively spliced transcripts that generate single-spanning transmembrane proteins with a short, divergent intracellular domain and a large extracellular domain. We characterized the modulatory effects on Kv4.2-mediated currents and the rat brain distribution of three splice variants of the DPPX subfamily of proteins. These three splice isoforms--DPPX-S, DPPX-L, and DPPX-K--are expressed in adult rat brain and modify the voltage dependence and kinetic properties of Kv4.2 channels expressed in Xenopus oocytes. Analysis of a deletion mutant that lacks the variable N-terminus showed that the N-terminus is not necessary for the modulation of Kv4 channels. Using in situ hybridization analysis, we found that the three splice variants are prominently expressed in brain regions where Kv4 subunits are also expressed. DPPX-K and DPPX-S mRNAs have a widespread distribution, whereas DPPX-L transcripts are concentrated in few specific areas of the rat brain. The emerging diversity of DPPX splice variants, differing only in the N-terminus of the protein, opens up intriguing possibilities for the modulation of Kv4 channels

One class of spinal interneurons, the Renshaw cells, is able to discharge at very high frequencies in adult mammals. Neuronal firing at such high frequencies requires voltage-gated potassium channels to rapidly repolarize the membrane potential after each action potential. We sought to establish the pattern of expression of calbindin and potassium channels with Kv3.1b and Kv3.2 subunits in Renshaw cells at different developmental stages of postnatal mice. The pattern of expression of calbindin changed dramatically during early postnatal development. An adult pattern of calbindin reactive neurons started to emerge from postnatal day 10 to postnatal day 14, with cells in laminae I and II of superficial dorsal horn and the ventral lamina VII. Renshaw cells were identified immunohistochemically by their expression of calbindin and their location in the ventral horn of the spinal cord. Western blot results of the lumbar spinal cord showed that Kv3.1b expression became faintly evident from postnatal day 10, reached a maximum at postnatal day 21 and was maintained through postnatal day 49. Double labeling results showed that all Renshaw cells expressed Kv3.1b weakly from postnatal day 14, and strongly at postnatal day 21. Western blot results showed that Kv3.2 expression became detectable in the lumbar cord from postnatal day 12, and increased steadily until reaching an adult level at postnatal day 28. In contrast to the Kv3.1b results, Kv3.2 was not expressed in Renshaw cells, although some neurons located at laminae VIII and VI expressed Kv3.2. We conclude that Renshaw cells express Kv3.1b but not Kv3.2 from postnatal day 14.

Using patch clamp techniques we investigated the characteristics of the spontaneous oscillatory activity displayed by starburst amacrine cells in the mouse retina. At a holding potential of -70 mV, oscillations appeared as spontaneous, rhythmic inward currents with a frequency of ~3.5 Hz and an average maximal amplitude of ~120 pA. Application of TEA, a potassium channel blocker, increased the amplitude of oscillatory currents by more than 70%, but reduced their frequency by about 17%. The TEA effects did not appear to result from direct actions on starburst cells, but rather a modulation of their synaptic inputs. Oscillatory currents were inhibited by CNQX, an antagonist of AMPA/kainate receptors, indicating that they were dependent on a periodic glutamatergic input likely from presynaptic bipolar cells. The oscillations were also inhibited by the calcium channel blockers cadmium and nifedipine, suggesting that the glutamate release was calcium dependent. Application of AP4, an agonist of mGluR6 receptors on on-center bipolar cells, blocked the oscillatory currents in starburst cells. However, subsequent application of TEA overcame the AP4 blockade, suggesting that the periodic glutamate release from bipolar cells is intrinsic to the inner plexiform layer in that, under experimental conditions, it can occur independent of photoreceptor input. The GABA receptor antagonists picrotoxin and bicuculline enhanced the amplitude of oscillations in starburst cells pre-stimulated with TEA. Our results suggest that this enhancement was due to a reduction of a GABAergic feedback inhibition from amacrine cells to bipolar cells and the resultant increased glutamate release. Finally, we found that some ganglion cells and other types of amacrine cell also displayed rhythmic activity, suggesting that oscillatory behavior is expressed by a number of inner retinal neurons

Potassium (K+) channel subunits of the Kv3 subfamily (Kv3.1-Kv3.4) display a positively shifted voltage dependence of activation and fast activation/deactivation kinetics when compared with other voltage-gated K+ channels, features that confer on Kv3 channels the ability to accelerate the repolarization of the action potential (AP) efficiently and specifically. In the cortex, the Kv3.1 and Kv3.2 proteins are expressed prominently in a subset of GABAergic interneurons known as fast-spiking (FS) cells and in fact are a significant determinant of the fast-spiking discharge pattern. However, in addition to expression at FS cell somata, Kv3.1 and Kv3.2 proteins also are expressed prominently at FS cell terminals, suggesting roles for Kv3 channels in neurotransmitter release. We investigated the effect of 1.0 mM tetraethylammonium (TEA; which blocks Kv3 channels) on inhibitory synaptic currents recorded in layer II/III neocortical pyramidal cells. Spike-evoked GABA release by FS cells was enhanced nearly twofold by 1.0 mM TEA, with a decrease in the paired pulse ratio (PPR), effects not reproduced by blockade of the non-Kv3 subfamily K+ channels also blocked by low concentrations of TEA. Moreover, in Kv3.1/Kv3.2 double knock-out (DKO) mice, the large effects of TEA were absent, spike-evoked GABA release was larger, and the PPR was lower than in wild-type mice. Together, these results suggest specific roles for Kv3 channels at FS cell terminals that are distinct from those of Kv1 and large-conductance Ca2+-activated K+ channels (also present at the FS cell synapse). We propose that at FS cell terminals synaptically localized Kv3 channels keep APs brief, limiting Ca2+ influx and hence release probability, thereby influencing synaptic depression at a synapse designed for sustained high-frequency synaptic transmission

A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase IV (DPPIV) known as DPP10 was recently identified and linked to asthma susceptibility; however the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels. We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to the activity of DPPX, and significantly different than DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude and modified the voltage-dependence and kinetic properties of the current such that it resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization DPP10 was found to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10 and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity