Faculty Bibliography

The Ca(2+)-binding protein, K(+) channel-interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N-terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K(+) channels

1.Voltage-gated K+ channels containing Kv3 subunits play specific roles in the repolarization of action potentials. Kv3 channels are expressed in selective populations of CNS neurons and are thought to be important in facilitating sustained and/or repetitive high frequency firing. Regulation of the activity of Kv3 channels by neurotransmitters could have profound effects on the repetitive firing characteristics of those neurons. 2.Kv3 channels are found in several neuronal populations in the CNS that express nitric oxide synthases (NOSs). We therefore investigated whether Kv3 channels are modulated by the signalling gas nitric oxide (NO). 3.We found that Kv3.1 and Kv3.2 currents are potentially suppressed by D-NONOate and other NO donors. The effects of NO on these currents are mediated by the activation of guanylyl cyclase (GC), since they are prevented by Methylene Blue, an inhibitor of GC, and by ODQ, a specific inhibitor of the soluble form of GC. Moreover, application of 8-Br-cGMP, a permeant analogue of cGMP, also blocked Kv3.1 and Kv3.2 currents. 4.KT5283, a cGMP-dependent protein kinase (PKG) blocker, prevented the inhibition of Kv3.1 and Kv3.2 currents by D-NONOate and 8-Br-cGMP. This indicates that activation of PKG as a result of the increase in intracellular cGMP levels produced by D-NONOate or 8-Br-cGMP is necessary for channel block. 5.Although the effects of NO on Kv3.1 and Kv3.2 channels require PKG activity, two observations suggest that they are not mediated by phosphorylation of channel proteins: (a) the reagents affect both Kv3.2 and Kv3.1 channels, although only Kv3.2 proteins have a putative PKA-PKG phosphorylation site, and (b) mutation of the PKA-PKG phosphorylation site in Kv3.2 does not interfere with the effects of NO or cGMP. 6.The inhibitory effects of NO and cGMP on Kv3.1 and Kv3.2 currents appear to be mediated by the activation of serine-threonine phosphatase, since they are blocked by low doses of okadaic acid. Furthermore, direct intracellular application of the catalytic subunit of protein phosphatase 2A inhibited Kv3.2 currents, indicating that activity of PKG-induced phosphatase is necessary and sufficient to inhibit these channels. 7.The results suggest that basal phosphorylation of Kv3 channel proteins is required for proper channel function. Activation of phosphatases via NO or other signals that increase cGMP might be a potent mechanism to regulate Kv3 channel activity in neurons

Synchronized gamma band oscillations have been implicated in a number of neural processes including, attention, and sensory coding. The mechanism(s) for generation and synchronization of these oscillations are unknown. A central question is how distant ensembles of neurons are brought into synchrony despite conduction delays. One recent model demonstrates that such synchrony can be attained by evoking spike doublets (high frequency pairs of action potentials) in hippocampal interneurons, (Traub et al. Nature: 383, 621 1996). The duration of doublets are modulated by the phase relationships between the activity of distant and local excitatory populations, and serve as an error signal to bring these populations into synchrony. It has recently been demonstrated, (Lau et al. J. Neurosci: 15, 9071 2000), that such high frequency firing in cortical interneurons in part depends upon the presence of a particular voltage gated potassium channel (Kv3.2). This channel has properties that allow for a rapid repolarization of the membrane potential necessary for the generation of spike doublets. We show that Kv3.2 knockout mice are impaired in their ability to temporally organize gamma frequency oscillations in the cerebral cortex. Using arrays of electrodes spanning 2.5 mm of cortex we recorded field activity in anesthetized, and awake behaving mice. Spatiotemporal analysis performed across our array of electrodes revealed a significant reduction in the ability of Kv3.2-/- mice to synchronize distant neuronal populations at gamma frequencies. Such deficits were not evident when comparing local ensembles nor for slow oscillations

Neurotransmitters regulate synaptic transmission by activating K+ channels (GIRK channels) and inhibiting Ca2+ channels via a membrane-delimited action of subunits of activated G proteins. Several examples of inhibition of synaptic transmission as a result of the activation of post-synaptic GIRK channels have been documented in the mammalian CNS. However, pre-synaptic inhibition initiated by metabotropic receptors is thought to be mediated mainly through the inhibition of Ca2+ channels, rather than by the activation of K+ channels. In fact the presence of presynaptic GIRK channels has remained controversial. We now present data suggesting that GIRK channels mediate presynaptic inhibition of GABA release. Utilizing whole cell recording methods, we measured spontaneous IPSC's in pyramidal neurons in acute slices. The recording pipette contained QX314 and Cs+ to block post-synaptic voltage-gated Na+ and K+ channels. QX314 also blocks GIRK channels. Bath application of tertiapin a drug known to block GIRK channels (as well as ROMK1 inward rectifier K+ channels) produced an increase in IPSC frequency without effect on mean amplitude. Similar effects were produced by bath application of 200 uM Ba2+, a concentration known to block GIRK and other inward rectifier K+ channels. Immunohistochemistry with antibodies to GIRK1 proteins demonstrated punctate staining surrounding cortical pyramidal neurons, suggestive of GIRK1 expression in the basket axo-somatic terminals of GABAergic interneurons. Together, the results suggest that presynaptic inward rectifier K+ channels, probably GIRK-type, can regulate GABAergic transmitter release

Subthreshold-operating transient A-type currents (ISA) cause delayed excitation, may affect action potential repolarization, and influence the duration of the interspike interval. Antisense hybrid arrest of rat brain mRNA and other gene elimination methods have shown that subunits of the voltage-gated potassium subfamily Kv4 are the pore-forming subunits of native ISA channels. Expression of Kv4 proteins in Xenopus oocytes produces a current that resembles ISA, yet native ISAs produced by poly(A) RNA from rat brain have different kinetics. This discrepancy can be explained by the presence of auxiliary subunits. However, co-expression of members of a group of such auxiliary subunits, named KChIPs, and Kv4s does not render A-currents with the kinetics of the native currents. We size-fractionated poly(A)+ RNA from rat cerebellum and found a 4-7 kb RNA fraction that encodes a factor(s) that modifies the kinetics of A-currents expressed by Kv4.2 cRNA. Co-injection of Kv4.2 with this fraction accelerates the activation and inactivation of Kv4 channels, produces a 20 mV negative shift in the conductance voltage relation and accelerates recovery from inactivation. This activity cannot be eliminated by hybrid arrest with antisense degenerated oligonucleotides against Kv4 subunits, suggesting that the novel factor is not a new member of the Kv4 subfamily. Antisense treatment with oligonucleotides complementary to KChIP sequences also failed to arrest the activity. We propose that this factor is a novel component of native A-type currents, which contributes to generating subthreshold-operating A-channel functional diversity

Kv3 K+ channels have been shown to play critical roles in fast spike repolarization and in enabling high frequency firing in cortical interneurons and in brainstem auditory neurons. We examined the cellular and subcellular distribution of Kv3.1a, Kv3.1b and Kv3.2 subunits in mouse retina. Expression of Kv3.1b was detected in few ganglion cells and two types of amacrine cells. One type had small somata and were distributed in mirror symmetry in the inner nuclear layer (INL) and the ganglion cell layer (GCL), in neurons projecting to strata 2 and 4 of the inner plexiform layer (IPL). These cells coexpressed Kv3.1b, calretinin and choline acetyltransferase. This group of Kv3.1b expressing neurons is both morphologically and neurochemically identical to the starburst amacrine cells. Interestingly, Kv3.1a immunoreactivity was restricted to the dendritic processes of the starburst cells in strata 2 and 4. Kv3.2 subunits were detected in the somata of a few ganglion cells, in scattered amacrine cell bodies and proximal dendrites located in the INL and in diffuse fiber-like structures throughout the IPL. Thus, Kv3.1b and Ky3.2 showed both somatic and dendritic localization in retinal amacrine and ganglion cells whereas Kv3.1a subunits were restricted to the processes in the IPL. These results indicate that Kv3.1 and Kv3.2 subunits are located in specific cell types in the retina. The subcellular segregation of the three subunits may underlie a physiological disparity in the spiking activity or modulation of different parts of the neuron. Future studies will investigate the functional roles of Kv3 channels in the retina

Voltage-gated K(+) channels of the Kv3 subfamily have unusual electrophysiological properties, including activation at very depolarized voltages (positive to -10 mV) and very fast deactivation rates, suggesting special roles in neuronal excitability. In the brain, Kv3 channels are prominently expressed in select neuronal populations, which include fast-spiking (FS) GABAergic interneurons of the neocortex, hippocampus, and caudate, as well as other high-frequency firing neurons. Although evidence points to a key role in high-frequency firing, a definitive understanding of the function of these channels has been hampered by a lack of selective pharmacological tools. We therefore generated mouse lines in which one of the Kv3 genes, Kv3.2, was disrupted by gene-targeting methods. Whole-cell electrophysiological recording showed that the ability to fire spikes at high frequencies was impaired in immunocytochemically identified FS interneurons of deep cortical layers (5-6) in which Kv3.2 proteins are normally prominent. No such impairment was found for FS neurons of superficial layers (2-4) in which Kv3.2 proteins are normally only weakly expressed. These data directly support the hypothesis that Kv3 channels are necessary for high-frequency firing. Moreover, we found that Kv3.2 -/- mice showed specific alterations in their cortical EEG patterns and an increased susceptibility to epileptic seizures consistent with an impairment of cortical inhibitory mechanisms. This implies that, rather than producing hyperexcitability of the inhibitory interneurons, Kv3.2 channel elimination suppresses their activity. These data suggest that normal cortical operations depend on the ability of inhibitory interneurons to generate high-frequency firing