Faculty Bibliography

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

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

The pinceau is a cerebellar structure formed by descending GABA-ergic basket cell axonal terminals converging on the initial axonal segment of Purkinje cell. Although basket cells exert a powerful inhibitory influence on the output of the cerebellar cortex, the function and mode of action of the pinceau are not understood because the majority of basket cell axons fail to make identifiable synaptic contacts with the Purkinje cell axon. Several proteins were previously reported to cluster specifically in this area, including a number of voltage-activated potassium channel subunits. In this study, we used immunohistochemistry, electron microscopy, and electron tomography to examine the ultrastructural localization of a novel voltage-gated potassium channel subunit, Kv3.2, in the pinceau. We found strong, selective localization of Kv3.2 to basket cell axons. Additionally, because potassium buffering is often conducted through water channels, we studied the extent of a brain-specific water channel, aquaporin-4 (AQP4), using confocal and electron microscopy. As expected, we found AQP4 was heavily localized to astrocytic processes of the pinceau. The abundance of potassium channels and AQP4 in this area suggests rapid ionic dynamics in the pinceau, and the unusual, highly specialized morphology of this region implies that the structural features may combine with the molecular composition to regulate the microenvironment of the initial segment of the Purkinje cell axon

Subthreshold-activating somatodendritic A-type potassium channels have fundamental roles in neuronal signaling and plasticity which depend on their unique cellular localization, voltage dependence, and kinetic properties. Some of the components of A-type K(+) channels have been identified; however, these do not reproduce the properties of the native channels, indicating that key molecular factors have yet to be unveiled. We purified A-type K(+) channel complexes from rat brain membranes and found that DPPX, a protein of unknown function that is structurally related to the dipeptidyl aminopeptidase and cell adhesion protein CD26, is a novel component of A-type K(+) channels. DPPX associates with the channels' pore-forming subunits, facilitates their trafficking and membrane targeting, reconstitutes the properties of the native channels in heterologous expression systems, and is coexpressed with the pore-forming subunits in the somatodendritic compartment of CNS neurons

Ether-a-go-go related gene (erg) K+ channels contribute to the membrane repolarization during cardiac action potentials. Pharmacological blockage of erg K+ channels prevents firing frequency adaptation and unveils a contribution of these type of channels to the M current in cell lines with neuronal pedigree. Although erg K+ channel mRNAs are expressed prominently and widespread in the central nervous system (CNS), the presence of functional erg K+ channels in CNS neurons has not been demonstrated. We investigated the effects of selectively blocking erg K+ channels on the firing properties of somatosensory cortical neurons using whole-cell current clamp in acute mouse brain slices. Application of the erg K+ channel blocker WAY-123.398 (5-10 mM) (WAY) to pyramidal neurons of layers 2/3 (which prominently express erg3 mRNA) significantly increases the number of spikes fired by the neuron upon current injection. It also reduces spike frequency adaptation and afterhyperpolarization. In contrast, WAY has no effect on layer 6 pyramidal neurons (which do not express any erg channel mRNA) under the same conditions. Similar results are obtained with the use of the erg K+ channel blockers E-4031 and ergtoxin. We conclude that erg K+ channels are functionally expressed in cortical neurons and actively modulate their excitability

Somatodendritic A-type K+ currents mediated by Kv4 channels (ISA) exhibit preferential closed-state inactivation, which may be modulated by specific agents. DPPX accelerates the development of inactivation in Kv4 channels and is thought to be a key molecular component that is necessary to reconstitute the physiological properties of ISA. Here, we investigated the effect of DPPX on closed-state inactivation of heterologously expressed Kv4 channels (Kv4.1 and Kv4.3 in Xenopus oocytes). While macroscopic inactivation at positive membrane potentials (e.g., +70 mV) was modestly accelerated by DPPX, direct measurements of the development of closed-state inactivation in the presence of DPPX at hyperpolarized membrane potentials revealed dramatically reduced time constants for Kv4.1 and Kv4.3 (Fig. 1): 1.4+-0.2 s (control Kv4.1, at V1/2= -69 mV) and 0.2+-0.01 s (DDPX+Kv4.1, at V1/2= -81 mV), N=3; 5.2+-0.5 s (control Kv4.3, at V1/2= -67 mV) and 0.4+-0.08 s (DDPX+Kv4.3, at V1/2= -76 mV), N=4. Earlier studies from our laboratory suggested that internal sites in Kv4 channels control closed-state inactivation. Thus, DPPX may modulate Kv4 closed-state inactivation at an internal site

Kv4 proteins are thought to be the pore-forming subunits of the channels mediating most of the subthreshold-operating somatodendritic A-type K+ current (ISA) in neurons. Two types of proteins (KChIPs and DPPX) have been found that associate with Kv4 subunits and modulate channel properties. Both KChIPs and DPPX increase current magnitude, probably by facilitating the trafficking of Kv4 channel complexes to the plasma membrane. In addition, both subunits accelerate the recovery from inactivation and have effects on the voltage dependence of Kv4.2 channels, yet DPPX produces a more pronounced negative shift in both the conductance-voltage relation and in the voltage dependence of steady-state inactivation. The two types of protein differ on their effects on inactivation, while KChIP slows down the inactivation time course of Kv4 channels, DPPX accelerates the rate of channel inactivation, thus reproducing the fast kinetics of ISA channels in many neurons. The association of Kv4 proteins and KChIPs and DPPX has been demonstrated using co-immunoprecipitation assays. Quantitative immunoprecipitations show that only a small fraction of the KChIP or DPPX protein in brain is associated with Kv4.2 proteins, suggesting that KChIP and DPPX proteins may have Kv4 unrelated functions.We are currently investigating the effects of KChIPs and DPPX on the modulation of Kv4 channels by phorbol ester-mediated activation of PKC

This summary article presents an overview of the molecular relationships among the voltage-gated potassium channels and a standard nomenclature for them, which is derived from the IUPHAR Compendium of Voltage-Gated Ion Channels. The complete Compendium, including data tables for each member of the potassium channel family can be found at http://www.iuphar-db.org/iuphar-ic/