Yimy Amarillo

Molecular Diversity of K+ Channels

ABSTRACT: K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates.

Contributions of Kv3 Channels to Neuronal Excitability

ABSTRACT: Four mammalian Kv3 genes have been identified, each of which generates, by alternative splicing, multiple protein products differing in their C‐terminal sequence. Products of the Kv3.1 and Kv3.2 genes express similar delayed‐rectifier type currents in heterologous expression systems, while Kv3.3 and Kv3.4 proteins express A‐type currents. All Kv3 currents activate relatively fast at voltages more positive than −10 mV, and deactivate very fast. The distribution of Kv3 mRNAs in the rodent CNS was studied by in situ hybridization, and the localization of Kv3.1 and Kv3.2 proteins has been studied by immunohistochemistry. Most Kv3.2 mRNAs (∼90%) are present in thalamic‐relay neurons throughout the dorsal thalamus. The protein is expressed mainly in the axons and terminals of these neurons. Kv3.2 channels are thought to be important for thalamocortical signal transmission. Kv3.1 and Kv3.2 proteins are coexpressed in some neuronal populations such as in fast‐spiking interneurons of the cortex and hippocampus, and neurons in the globus pallidus. Coprecipitation studies suggest that in these cells the two types of protein form heteromeric channels. Kv3 proteins appear to mediate, in native neurons, similar currents to those seen in heterologous expression systems. The activation voltage and fast deactivation rates are believed to allow these channels to help repolarize action potentials fast without affecting the threshold for action potential generation. The fast deactivating current generates a quickly recovering afterhyperpolarization, thus maximizing the rate of recovery of Na+ channel inactivation without contributing to an increase in the duration of the refractory period. These properties are believed to contribute to the ability of neurons to fire at high frequencies and to help regulate the fidelity of synaptic transmission. Experimental evidence has now become available showing that Kv3.1‐Kv3.2 channels play critical roles in the generation of fast‐spiking properties in cortical GABAergic interneurons.

Morphology and physiology of the polyaxonal amacrine cells in the rabbit retina

We examined the morphology and physiological response properties of the axon‐bearing, long‐range amacrine cells in the rabbit retina. These so‐called polyaxonal amacrine cells all displayed two distinct systems of processes: (1) a dendritic field composed of highly branched and relatively thick processes and (2) a more extended, often sparsely branched axonal arbor derived from multiple thin axons emitted from the soma or dendritic branches. However, we distinguished six morphological types of polyaxonal cells based on differences in the fine details of their soma/dendritic/axonal architecture, level of stratification within the inner plexiform layer (IPL), and tracer coupling patterns. These morphological types also showed clear differences in their light‐evoked response activity. Three of the polyaxonal amacrine cell types showed on‐off responses, whereas the remaining cells showed on‐center responses; we did not encounter polyaxonal cells with off‐center physiology. Polyaxonal cells respected the on/off sublamination scheme in that on‐off cells maintained dendritic/axonal processes in both sublamina a and b of the IPL, whereas processes of on‐center cells were restricted to sublamina b. All polyaxonal amacrine cell types displayed large somatic action potentials, but we found no evidence for low‐amplitude dendritic spikes that have been reported for other classes of amacrine cell. The center‐receptive fields of the polyaxonal cells were comparable to the diameter of their respective dendritic arbors and, thus, were significantly smaller than their extensive axonal fields. This correspondence between receptive and dendritic field size was seen even for cells showing extensive homotypic and/or heterotypic tracer coupling to neighboring neurons. These data suggest that all polyaxonal amacrine cells are polarized functionally into receptive dendritic and transmitting axonal zones. J. Comp. Neurol. 440:109–125, 2001.

Ternary Kv4.2 channels recapitulate voltage‐dependent inactivation kinetics of A‐type K+ channels in cerebellar granule neurons

Kv4 channels mediate most of the somatodendritic subthreshold operating A‐type current (ISA) in neurons. This current plays essential roles in the regulation of spike timing, repetitive firing, dendritic integration and plasticity. Neuronal Kv4 channels are thought to be ternary complexes of Kv4 pore‐forming subunits and two types of accessory proteins, Kv channel interacting proteins (KChIPs) and the dipeptidyl‐peptidase‐like proteins (DPPLs) DPPX (DPP6) and DPP10. In heterologous cells, ternary Kv4 channels exhibit inactivation that slows down with increasing depolarization. Here, we compared the voltage dependence of the inactivation rate of channels expressed in heterologous mammalian cells by Kv4.2 proteins with that of channels containing Kv4.2 and KChIP1, Kv4.2 and DPPX‐S, or Kv4.2, KChIP1 and DPPX‐S, and found that the relation between inactivation rate and membrane potential is distinct for these four conditions. Moreover, recordings from native neurons showed that the inactivation kinetics of the ISA in cerebellar granule neurons has voltage dependence that is remarkably similar to that of ternary Kv4 channels containing KChIP1 and DPPX‐S proteins in heterologous cells. The fact that this complex and unique behaviour (among A‐type K+ currents) is observed in both the native current and the current expressed in heterologous cells by the ternary complex containing Kv4, DPPX and KChIP proteins supports the hypothesis that somatically recorded native Kv4 channels in neurons include both types of accessory protein. Furthermore, quantitative global kinetic modelling showed that preferential closed‐state inactivation and a weakly voltage‐dependent opening step can explain the slowing of the inactivation rate with increasing depolarization. Therefore, it is likely that preferential closed‐state inactivation is the physiological mechanism that regulates the activity of both ternary Kv4 channel complexes and native ISA‐mediating channels.

Differential characterization of three alternative spliced isoforms of DPPX

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.

Evidence for the presence of a novel Kv4‐mediated A‐type K+ channel‐modifying factor

1 Subthreshold‐operating transient (A‐type) K+ currents (ISAs) 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 ISAs. 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 ISA 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 KAFs activity from the high molecular weight mRNA fraction, thus suggesting that KAF might be a novel subunit(s) that can contribute to generating native ISA channel diversity. 4 The time course of the currents expressed by KAF‐modified Kv4 channels resembles more closely the time course of the native ISA in cerebellar granule cells.

Modulation of Kv3 potassium channels expressed in CHO cells by a nitric oxide-activated phosphatase

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.

The interplay of seven subthreshold conductances controls the resting membrane potential and the oscillatory behavior of thalamocortical neurons

The signaling properties of thalamocortical (TC) neurons depend on the diversity of ion conductance mechanisms that underlie their rich membrane behavior at subthreshold potentials. Using patch-clamp recordings of TC neurons in brain slices from mice and a realistic conductance-based computational model, we characterized seven subthreshold ion currents of TC neurons and quantified their individual contributions to the total steady-state conductance at levels below tonic firing threshold. We then used the TC neuron model to show that the resting membrane potential results from the interplay of several inward and outward currents over a background provided by the potassium and sodium leak currents. The steady-state conductances of depolarizing Ih (hyperpolarization-activated cationic current), IT (low-threshold calcium current), and INaP (persistent sodium current) move the membrane potential away from the reversal potential of the leak conductances. This depolarization is counteracted in turn by the hyperpolarizing steady-state current of IA (fast transient A-type potassium current) and IKir (inwardly rectifying potassium current). Using the computational model, we have shown that single parameter variations compatible with physiological or pathological modulation promote burst firing periodicity. The balance between three amplifying variables (activation of IT, activation of INaP, and activation of IKir) and three recovering variables (inactivation of IT, activation of IA, and activation of Ih) determines the propensity, or lack thereof, of repetitive burst firing of TC neurons. We also have determined the specific roles that each of these variables have during the intrinsic oscillation.

Voltage Gated Potassium Channels: Structure and Function of Kv1 to Kv9 Subfamilies

The nervous system contains a large diversity of potassium channels, the membrane proteins that regulate the movement of potassium ions across the cell membrane. All potassium channels have a similar ionic selectivity for potassium ions but vary in how, when, and for how long their pore is open. K+ channel diversity is one of the main factors contributing to the dynamic, electrophysiological identity of neurons and to the functional specificity of the modulatory actions of neurotransmitters and neuropeptides. This article reviews the K+ channels for which the opening of the pore is controlled by the electrical membrane potential and discusses what has been learned about the contribution of each channel type to neuronal function.

Analysis of the role of the low threshold currents IT and Ih in intrinsic delta oscillations of thalamocortical neurons.

Thalamocortical neurons are involved in the generation and maintenance of brain rhythms associated with global functional states. The repetitive burst firing of TC neurons at delta frequencies (1–4 Hz) has been linked to the oscillations recorded during deep sleep and during episodes of absence seizures. To get insight into the biophysical properties that are the basis for intrinsic delta oscillations in these neurons, we performed a bifurcation analysis of a minimal conductance-based thalamocortical neuron model including only the IT channel and the sodium and potassium leak channels. This analysis unveils the dynamics of repetitive burst firing of TC neurons, and describes how the interplay between the amplifying variable mT and the recovering variable hT of the calcium channel IT is sufficient to generate low threshold oscillations in the delta band. We also explored the role of the hyperpolarization activated cationic current Ih in this reduced model and determine that, albeit not required, Ih amplifies and stabilizes the oscillation.