Faculty Bibliography

Voltage-gated potassium (Kv) channels regulate many physiological functions and represent important therapeutic targets in the treatment of several clinical disorders. Although some of these channels have been well characterized, the study of others, such as Kv3 channels, has been hindered because of limited pharmacological tools. The current study was initiated to identify potent blockers of the Kv3.2 channel. CHO-K1 cells stably expressing human Kv3.2b (CHO-K1.hKv3.2b) were established and characterized. ShK, a peptide isolated from Stichodactyla helianthus venom, and a known high affinity blocker of Kv1.1 and Kv1.3 channels, was found to potently inhibit (86)Rb(+) efflux from CHO-K1.hKv3.2b (IC50 of ~ 0.6 nM). In electrophysiological recordings of Kv3.2b channels expressed in Xenopus oocytes or in planar patch clamp studies, ShK inhibited hKv3.2b channels with IC50s of ~ 0.3 and 6 nM, respectively. Despite the presence of Kv3.2 protein in human pancreatic beta cells, ShK has no effect on the Kv current of these cells, suggesting that it is unlikely that homotetrameric Kv3.2 channels contribute significantly to the delayed rectifier current of insulin-secreting cells. In mouse cortical GABAergic fast-spiking interneurons, however, application of ShK produced effects consistent with blockade of Kv3 channels, i.e., an increase in action potential half-width, a decrease in the amplitude of the action potential afterhyperpolarization, and a decrease in maximal firing frequency in response to depolarizing current injections. Taken together these results indicate that ShK is a potent inhibitor of Kv3.2 channels and may serve as a useful pharmacological probe for studying these channels in native preparations

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

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/

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

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

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

Voltage-gated potassium channels containing the K.v.3.2 subunit are expressed in specific neuronal populations such as thalamocortical neurons and fast spiking GABAergic interneurons of the neocortex and hippocampus. These K(+)-channels play a major role in the regulation of firing properties in these neurons. We investigated whether the K.v.3.2 subunit contributes to the generation of the sleep electroencephalogram (EEG). The EEG of a frontal and occipital derivation of K.v.3.2-deficient mice and littermate controls was recorded during a 24-h baseline, 6-h sleep deprivation (SD) and subsequent 18-h recovery to assess also the effects of the K.v.3.2 subunit deficiency under physiological sleep pressure. The K.v.3.2-deficient mice had lower EEG power density in the frequencies between 3.25 and 6 Hz in nonREM (NREM) sleep and 3.25-5 Hz in REM sleep. These differences were more prominent in the frontal derivation than in the occipital derivation. The waking EEG spectrum was not affected by the deletion. In both genotypes SD induced a prominent increase in slow-wave activity in NREM sleep (mean EEG power density between 0.75 and 4.0 Hz), and a concomitant decrease in sleep fragmentation. The effects of SD did not differ significantly between the genotypes. The results indicate that K.v.3.2 channels may be involved in the generation of EEG oscillations in the high delta and low theta range in sleep. They support the notion that GABA-mediated synchronization of cortical activity contributes to the electroencephalogram

Voltage-gated K(+) channels containing pore-forming subunits of the Kv3 subfamily have specific roles in the fast repolarization of action potentials and enable neurons to fire repetitively at high frequencies. Each of the four known Kv3 genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K(+) channel subunits differing only in their C-terminal sequence. The alternative splicing does not affect the electrophysiological properties of the channels, and its physiological role is unknown. It has been proposed that one of the functions of the alternative splicing of Kv3 genes is to produce subunit isoforms with differential subcellular membrane localizations in neurons and differential modulation by signaling pathways. We investigated the role of the alternative splicing of Kv3 subunits in subcellular localization by examining the brain distribution of the two alternatively spliced versions of the Kv3.1 gene (Kv3.1a and Kv3.1b) with antibodies specific for the alternative spliced C-termini. Kv3.1b proteins were prominently expressed in the somatic and proximal dendritic membrane of specific neuronal populations in the mouse brain. The axons of most of these neurons also expressed Kv3.1b protein. In contrast, Kv3.1a proteins were prominently expressed in the axons of some of the same neuronal populations, but there was little to no Kv3.1a protein expression in somatodendritic membrane. Exceptions to this pattern were seen in two neuronal populations with unusual targeting of axonal proteins, mitral cells of the olfactory bulb, and mesencephalic trigeminal neurons, which expressed Kv3.1a protein in dendritic and somatic membrane, respectively. The results support the hypothesis that the alternative spliced C-termini of Kv3 subunits regulate their subcellular targeting in neurons