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

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

Frequenin, a Ca(2+)-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K(+) currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca(2+)-dependent manner. It also accelerates recovery from inactivation. Closely related Ca(2+)-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca(2+)-sensitive regulatory component of native A-type K(+) currents

Analysis of the Kv3 subfamily of K(+) channel subunits has lead to the discovery of a new class of neuronal voltage-gated K(+) channels characterized by positively shifted voltage dependencies and very fast deactivation rates. These properties are adaptations that allow these channels to produce currents that can specifically enable fast repolarization of action potentials without compromising spike initiation or height. The short spike duration and the rapid deactivation of the Kv3 currents after spike repolarization maximize the quick recovery of resting conditions after an action potential. Several neurons in the mammalian CNS have incorporated into their repertoire of voltage-dependent conductances a relatively large number of Kv3 channels to enable repetitive firing at high frequencies - an ability that crucially depends on the special properties of Kv3 channels and their impact on excitability

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

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

The members of the three subfamilies (eag, erg, and elk) of the ether-a-go-go (EAG) family of potassium channel pore-forming subunits express currents that, like the M-current (I(M)), could have considerable influence on the subthreshold properties of neuronal membranes, and hence the control of excitability. A nonradioactive in situ hybridization (NR-ISH) study of the distribution of the transcripts encoding the eight known EAG family subunits in rat brain was performed to identify neuronal populations in which the physiological roles of EAG channels could be studied. These distributions were compared with those of the mRNAs encoding the components of the classical M-current (Kcnq2 and Kcnq3). NR-ISH was combined with immunohistochemistry to specific neuronal markers to help identify expressing neurons. The results show that each EAG subunit has a specific pattern of expression in rat brain. EAG and Kcnq transcripts are prominent in several types of excitatory neurons in the cortex and hippocampus; however, only one of these channel components (erg1) was consistently expressed in inhibitory interneurons in these areas. Some neuronal populations express more than one product of the same subfamily, suggesting that the subunits may form heteromeric channels in these neurons. Many neurons expressed multiple EAG family and Kcnq transcripts, such as CA1 pyramidal neurons, which contained Kcnq2, Kcnq3, eag1, erg1, erg3, elk2, and elk3. This indicates that the subthreshold current in many neurons may be complex, containing different components mediated by a number of channels with distinct properties and neuromodulatory responses

1. Subthreshold-operating transient (A-type) K(+) currents (I(SA)s) are important in regulating neuronal firing frequency and in the modulation of incoming signals in dendrites. It is now known that Kv4 proteins are the principal, or pore-forming, subunits of the channels mediating I(SA)s(.) In addition, accessory subunits of Kv4 channels have also been identified. These either have no effect or slow down the inactivation kinetics of Kv4 channels. However, in many neuronal populations the I(SA) is faster, not slower, than the current generated by channels containing only Kv4 proteins. 2. Evidence is presented for the presence in rat cerebellar mRNA of transcripts encoding a molecular factor, termed KAF, that accelerates the kinetics of Kv4 channels. Size-fractionation of cerebellar mRNA in sucrose gradients separated the high molecular weight mRNAs (4-7 kb) encoding KAF from the low molecular weight ones (1.5-3 kb) encoding factors that slow down the inactivation kinetics of Kv4 channels. The latter were identified as KChIPs using anti-KChIP antisense oligonucleotides. 3. Both anti-KChIP and anti-Kv4 antisense oligonucleotides failed to eliminate KAF's activity from the high molecular weight mRNA fraction, thus suggesting that KAF might be a novel subunit(s) that can contribute to generating native I(SA) channel diversity. 4. The time course of the currents expressed by KAF-modified Kv4 channels resembles more closely the time course of the native I(SA) in cerebellar granule cells

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

Several two pore K+ channels (KT) have been identified in mammals. Amino acid sequence and phylogenetic analysis suggest the existence of 8 distinct subfamilies, with 3 subfamilies containing at least two members. Eight KT genes are expressed in the CNS. In heterologous expression systems KT genes produce currents at resting membrane potentials and therefore, are likely to play important roles in neuronal subthreshold properties. The diversity of these channels may be associated with a high degree of cellular or subcellular specificity. The diversity also provides multiple ways to regulate the subthreshold behavior of neurons. In situ hybridization experiments revealed that TASK-1 and KT3.2 are co-expressed in cerebellar granule cell layer, the olfactory bulb and in several brainstem nuclei like the ambigual, motor trigeminal, facial, vagal and hypoglossal nuclei. These two channels belong to the same subfamily (75% identity and 86% similarity in the core region) but differ in their pH dependence, and modulation by second messengers. TASK-1 is very sensitive to changes in pH in the physiological range while KT3.2 is almost insensitive to pH changes in the pH 7.0 to 8.0 range. KT3.2 is modulated by the phorbol ester PMA, a potent protein kinase C stimulator, but TASK-1 is insensitive to this treatment. If these channels are expressed in the same cells they may form heteromultimeric channels. Therefore the ability to form heteromeric complexes in vitro was investigated