Faculty Bibliography

Current evidence suggests that members of the Kv4 subfamily may encode native cardiac transient outward current (I(to)). Antisense hybrid-arrest with oligonucleotides targeted to Kv4 mRNAs specifically inhibited rat ventricular I(to), supporting this hypothesis. To determine whether protein kinase C (PKC) affects I(to) by an action on these molecular components, we compared the effects of PKC activation on Kv4.2 and Kv4.3 currents expressed in Xenopus oocytes and rat ventricular I(to). Phorbol 12-myristate 13-acetate (PMA) suppressed both Kv4.2 and Kv4.3 currents as well as native I(to), but not after preincubation with PKC inhibitors (e.g., chelerythrine). An inactive stereoisomer of PMA had no effect. Phenylephrine or carbachol inhibited Kv4 currents only when coexpressed, respectively, with alpha1C-adrenergic or M1 muscarinic receptors (this inhibition was also prevented by chelerythrine). The voltage dependence and inactivation kinetics of Kv4.2 were unchanged by PKC, but small effects on the rates of inactivation and recovery from inactivation of native I(to) were observed. Thus Kv4.2 and Kv4.3 proteins are important subunits of native rat ventricular I(to), and PKC appears to reduce this current by affecting the molecular components of the channels mediating I(to).

Kv3 K+ channel genes encode multiple products by alternative splicing of 3' ends resulting in the expression of K+ channel proteins that differ only in their C-termini. This divergence does not affect the electrophysiological properties of the channels expressed by these proteins. A similar alternative splicing with unknown function is seen in K+ channel genes of other families. We have investigated the possibility that the alternative splicing serves to generate channel subunits with different membrane targeting signals by examining the sorting behavior of three alternatively-spliced Kv3.2 isoforms when expressed in polarized MDCK cells. Two Kv3.2 proteins, Kv3.2b and Kv3.2c were expressed predominantly in the apical membrane, while Kv3.2a was localized mainly to the basolateral side (thought to be equivalent to the axonal and somatodendritic compartments in neurons, respectively). The Kv3.2 mRNA transcripts used in these studies are identical except for their 3' sequence, encoding the extreme C-terminal domain of the protein and the 3'UTR of the mRNA. However, the proteins achieve the same localizations in MDCK cells when expressed from constructs containing or lacking the 3'UTR, indicating that the differential localization is due to targeting signals present in the C' terminal domain of the protein. These results suggest that the alternative splicing of Kv3 genes is involved in channel localization. Since the precise localization of any given ion channel on the neuronal surface has significant functional implications, the results shown here suggest an important function for the alternative splicing of 3' ends seen in many K+ channel genes

Inactivating and noninactivating Na+ conductances are known to generate, respectively, the rising phase and the prolonged plateau phase of cerebellar Purkinje cell (PC) action potentials. These conductances have different voltage activation levels, suggesting the possibility that two distinct types of ion channels are involved. Single Purkinje cell reverse transcription-PCR from guinea pig cerebellar slices identified two Na+ channel alpha subunit transcripts, the orthologs of RBI (rat brain I) and Nach6/Scn8a. The latter we shall name CerIII. In situ hybridization histochemistry in rat brain demonstrated broad CerIII expression at high levels in many neuronal groups in the brain and spinal cord, with little if any expression in white matter, or nerve tracts. RBII (rat brain II), the most commonly studied recombinant Na+ channel alpha subunit is not expressed in PCs. As the absence of Scn8a has been correlated with motor endplate disease (med), in which transient sodium currents are spared, RBI appears to be responsible for the transient sodium current in PC. Conversely, jolting mice with a mutated Scn8a message demonstrates PC abnormalities in rapid, simple spike generation, linking CerIII to the persistent sodium current

Human epithelial kidney cells (HEK) were prepared to coexpress alpha1A, alpha2delta with different beta calcium channel subunits and green fluorescence protein. To compare the calcium currents observed in these cells with the native neuronal currents, electrophysiological and pharmacological tools were used conjointly. Whole-cell current recordings of human epithelial kidney alpha1A-transfected cells showed small inactivating currents in 80 mM Ba2+ that were relatively insensitive to calcium blockers. Coexpression of alpha1A, betaIb, and alpha2delta produced a robust inactivating current detected in 10 mM Ba2+, reversibly blockable with low concentration of omega-agatoxin IVA (omega-Aga IVA) or synthetic funnel-web spider toxin (sFTX). Barium currents were also supported by alpha1A, beta2a, alpha2delta subunits, which demonstrated the slowest inactivation and were relatively insensitive to omega-Aga IVA and sFTX. Coexpression of beta3 with the same combination as above produced inactivating currents also insensitive to low concentration of omega-Aga IVA and sFTX. These data indicate that the combination alpha1A, betaIb, alpha2delta best resembles P-type channels given the rate of inactivation and the high sensitivity to omega-Aga IVA and sFTX. More importantly, the specificity of the channel blocker is highly influenced by the beta subunit associated with the alpha1A subunit

Voltage-gated potassium channels constitute the largest group of heteromeric ion channels discovered to date. Over 20 genes have been isolated, encoding different channel subunit proteins which form functional tetrameric K+ channels. We have analyzed the subcellular localization of subunit Kv3.1b, a member of the Kv3 (Shaw-like) subfamily, in rat brain at the light and electron microscopic level, using immunocytochemical detection. Detailed localization was carried out in specific neurons of the neocortex, hippocampus and cerebellum. The identity of Kv3.1b-positive neurons was established using double labeling with markers for specific neuronal populations. In the neocortex, the Kv3.1b subunit was expressed in most parvalbumin-containing bipolar, basket or chandelier cells, and in some bipolar or double bouquet neurons containing calbindin. In the hippocampus, Kv3.1b was expressed in many parvalbumin-containing basket cells, as well as in calbindin-positive neurons in the stratum oriens, and in a small number of interneurons that did not stain for either parvalbumin or calbindin. Kv3.1b protein was not present in pyramidal cells in the neocortex and the hippocampus, but these cells were outlined by labeled presynaptic terminals from interneuron axons that surround the postsynaptic cell. In the cerebellar cortex, granule cells were the only population expressing the channel protein. Careful examination of individual granule cells revealed a non-uniform distribution of Kv3.1 staining on the somata: circular bands of labeling were present in the vicinity of the axon hillock. In cortical and hippocampal interneurons, as well as in cerebellar granule cells, the Kv3.1b subunit was present in somatic and unmyelinated axonal membranes and adjacent cytoplasm, as well as in the most proximal portion of dendritic processes, but not throughout most of the dendrite. Labeling was also seen in the terminals of labeled axons, but not at a higher concentration than in other parts of the axon. The distribution in the cells analyzed supports a role in action potential transmission by regulating action potential duration

G-protein-gated inward rectifier potassium (GIRK) channels are coupled to numerous neurotransmitter receptors in the brain and can play important roles in modulating neuronal function, depending on their localization in a given neuron. Site-directed antibodies to the extreme C terminus of GIRK1 (or KGA1), a recently cloned component of GIRK channels, have been used to determine the relative expression levels and distribution of the protein in different regions of the rat brain by immunoblot and immunohistochemical techniques. We report that the GIRK1 protein is expressed prominently in the olfactory bulb, hippocampus, dentate gyrus, neocortex, thalamus, cerebellar cortex, and several brain stem nuclei. In addition to the expected localization in somas and dendrites, where GIRK channels may mediate postsynaptic inhibition, GIRK1 proteins were also found in axons and their terminal fields, suggesting that GIRK channels can also modulate presynaptic events. Furthermore, the distribution of the protein to either somatodendritic or axonal-terminal regions of neurons varied in different brain regions, which would imply distinct functions of these channels in different neuronal populations. Particularly prominent staining of the cortical barrels of layer IV of the neocortex, and the absence of this staining with unilateral kainate lesions of the thalamus, suggest that the GIRK1 protein is expressed in thalamocortical nerve terminals in which GIRK channels may mediate the actions of mu opiate receptors

1. Proteins of the Kv4 or Shal-related subfamily are key components of transient K+ channels (A channels) operating at subthreshold values of the membrane potential. We have cloned and characterized a new mammalian Kv4 or Shal-related cDNA (Kv4.3) that predicts a protein with strong sequence conservation with the other known members of this subfamily. 2. Injection of Kv4.3 transcripts into Xenopus oocytes generates an A type K+ current, with small but physiologically significant differences from the currents expressed by Kv4.2 and Kv4.1 mRNAs. Kv4.3 currents can be modified to resemble native A currents by coinjection with a low molecular weight mRNA fraction from rat brain which does not express detectable currents on its own. Particularly striking is a 7-to-10-fold increase in the rate of recovery from inactivation, a 5- to 10-fold increase in current magnitude and a 3- to 4-fold increase in sensitivity to 4-amino pyridine (4-AP). 3. In situ hybridization histochemistry was used to compare the expression of the three known Kv4 genes. Kv4.2 and Kv4.3 (but not Kv4.1) are abundant in the adult rat brain, with each displaying a specific, but sometimes overlapping pattern of expression. Moreover, a reciprocal gradient of expression of Kv4.2 and Kv4.3 transcripts is seen in some brain areas, such as in the pyramidal cell layers of the hippocampus and the granule cell layer of the cerebellum. Therefore Kv4 proteins may form heteromultimeric channels of distinct subunit composition in different neurons. Moreover, the results suggest that neurons such as pyramidal cells in the hippocampus and granule cells in the cerebellum represent heterogeneous cell populations in terms of their ISA, and hence in their firing patterns. Kv4.2 and Kv4.3 also display complementary expression in the heart, with Kv4.3 being more abundant in atria and Kv4.2 in ventricle. The existence of multiple Kv4 proteins forming channels of variable subunit combinations helps explain the diversity of ISA channels in neurons