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W B Thornhill

Publications and source records attributed to W B Thornhill.

At least 19 recordsLinked to original sources
PubMed2003

Molecular cloning and expression of a Kv1.1-like potassium channel from the electric organ of Electrophorus electricus.

Electrocytes from the electric organ of Electrophorus electricus exhibited sodium action potentials that have been proposed to be repolarized by leak currents and not by outward voltage-gated potassium currents. However, patch-clamp recordings have suggested that electrocytes may contain a very low density of voltage-gated K(+) channels. We report here the cloning of a K(+) channel from an eel electric organ cDNA library, which, when expressed in mammalian tissue culture cells, displayed delayed-rectifier K(+) channel characteristics. The amino-acid sequence of the eel K(+) channel had the highest identity to Kv1.1 potassium channels. However, different important functional regions of eel Kv1.1 had higher amino-acid identity to other Kv1 members, for example, the eel Kv1.1 S4-S5 region was identical to Kv1.5 and Kv1.6. Northern blot analysis indicated that eel Kv1.1 mRNA was expressed at appreciable levels in the electric organ but it was not detected in eel brain, muscle, or cardiac tissue. Because electrocytes do not express robust outward voltage-gated potassium currents we speculate that eel Kv1.1 channels are chronically inhibited in the electric organ and may be functionally recruited by an unknown mechanism.

Amino Acid Sequence↗
PubMed2001

Determinants involved in Kv1 potassium channel folding in the endoplasmic reticulum, glycosylation in the Golgi, and cell surface expression.

Kv1.1 and Kv1.4 potassium channels are expressed as mature glycosylated proteins in brain, whereas they exhibited striking differences in degree of trans-Golgi glycosylation conversion and high cell surface expression when they were transiently expressed as homomers in cell lines. Kv1.4 exhibited a 70% trans-Golgi glycosylation conversion, whereas Kv1.1 showed none, and Kv1.4 exhibited a approximately 20-fold higher cell surface expression level as compared with Kv1.1. Chimeras between Kv1.4 and Kv1.1 and site-directed mutants were constructed to identify amino acid determinants that affected these processes. Truncating the cytoplasmic C terminus of Kv1.4 inhibited its trans-Golgi glycosylation and high cell surface expression (as shown by Li, D., Takimoto, K., and Levitan, E. S. (2000) J. Biol. Chem. 275, 11597-11602), whereas truncating this region on Kv1.1 did not affect either of these events, indicating that its C terminus is not a negative determinant for these processes. Exchanging the C terminus between these channels showed that there are other regions of the protein that exert a positive or negative effect on these processes. Chimeric constructs between Kv1.4 and Kv1.1 identified their outer pore regions as major positive and negative determinants, respectively, for both trans-Golgi glycosylation and cell surface expression. Site-directed mutagenesis identified a number of amino acids in the pore region that are involved in these processes. These data suggest that there are multiple positive and negative determinants on both Kv1.4 and Kv1.1 that affect channel folding, trans-Golgi glycosylation conversion, and cell surface expression.

Amino Acid Sequence↗
PubMed2001

Cloning and characterization of G protein-gated inward rectifier K+ channel (GIRK1) isoforms from heart and brain.

A G protein-gated inward rectifier potassium (K+) channel (GIRK1a) has been cloned from different tissues (Kubo et al., 1993b; Dascal et al., 1993). Here we report the cloning of three additional novel isoforms of GIRK1a from rat atria and and one from human brain. These isoform cDNAs code for proteins that have identical N-termini, M1-H5-M2 (predicted transmembrane and pore domains), and post-M2 amino acid regions to GIRK1a (1-501 amino acids), but they have shorter C-termini (GIRK1b (1-309), GIRK1c (1-308), GIRK1d (1-235), and GIRK1e (1-253). These results indicated that isoforms were generated by alternative splicing and partial genomic analysis confirmed the presence of exons and introns in the rat GIRK1 gene. RNase protection analysis and immunoblot analysis indicated that the isoforms were expressed in both rat atria and brain but at lower levels versus GIRK1a. The physiological role that the isoforms may play in atrial and brain physiology remains to be determined.

Alternative Splicing↗
PubMed2000

Glycosylation of GIRK1 at Asn119 and ROMK1 at Asn117 has different consequences in potassium channel function.

GIRK (G protein-gated inward rectifier K(+) channel) proteins play critical functional roles in heart and brain physiology. Using antibodies directed to either GIRK1 or GIRK4, site-directed mutagenesis, and specific glycosidases, we have investigated the effects of glycosylation in the biosynthesis and heteromerization of these proteins expressed in oocytes. Both GIRK1 and GIRK4 have one extracellular consensus N-glycosylation site. Using chimeras between GIRK1 and GIRK4 as well as a GIRK1 N-glycosylation mutant, we report that GIRK1 was glycosylated at Asn(119), whereas GIRK4 was not glycosylated at Asn(132). GIRK1 membrane-spanning domain 1 was required for optimal glycosylation at Asn(119) because a chimera that contained GIRK4 membrane-spanning domain 1 significantly reduced the addition of a carbohydrate structure at this site. This finding may partly account for the reason that GIRK4 is not glycosylated at Asn(132), either as a homomer or when coexpressed with GIRK1. When the GIRK1(N119Q) mutant was coexpressed with GIRK4, the biophysical properties of the heteromeric channel and the magnitude of the agonist-induced currents were similar to those of controls. Thus, N-glycosylation of GIRK1 at Asn(119) does not appear to affect its physical association with GIRK4, the routing of the heteromer to the cell surface, or heteromeric channel function, unlike the dramatic functional effects of N-glycosylation of ROMK1 at Asn(117) (Schwalbe, R. A., Wang, Z., Wible, B. A., and Brown, A. M. (1995) J. Biol. Chem. 270, 15336-15340).

Amidohydrolases↗
PubMed2000

CNS voltage-dependent Na(+) channel expression and distribution in an undifferentiated and differentiated CNS cell line.

Upon serum removal, CAD-R1 cells undergo neurite outgrowth and an increase in voltage-dependent Na(+) current (VDNaC) density without changing their activation and inactivation properties. Insulin and endothelial cell growth supplement inhibited the increase in VDNaC density but not the neurite outgrowth. RI, RII, RIII Na(+) channel proteins were expressed in CAD-R1 cells. These proteins exhibited both similar and different distribution and clustering patterns which suggested the channel's structural differences play a role in channel distribution.

Cell Culture Techniques↗
PubMed1999

Fast inactivation of a brain K+ channel composed of Kv1.1 and Kvbeta1.1 subunits modulated by G protein beta gamma subunits.

Modulation of A-type voltage-gated K+ channels can produce plastic changes in neuronal signaling. It was shown that the delayed-rectifier Kv1.1 channel can be converted to A-type upon association with Kvbeta1.1 subunits; the conversion is only partial and is modulated by phosphorylation and microfilaments. Here we show that, in Xenopus oocytes, expression of Gbeta1gamma2 subunits concomitantly with the channel (composed of Kv1.1 and Kvbeta1.1 subunits), but not after the channel's expression in the plasma membrane, increases the extent of conversion to A-type. Conversely, scavenging endogenous Gbetagamma by co-expression of the C-terminal fragment of the beta-adrenergic receptor kinase reduces the extent of conversion to A-type. The effect of Gbetagamma co-expression is occluded by treatment with dihydrocytochalasin B, a microfilament-disrupting agent shown previously by us to enhance the extent of conversion to A-type, and by overexpression of Kvbeta1.1. Gbeta1gamma2 subunits interact directly with GST fusion fragments of Kv1.1 and Kvbeta1.1. Co-expression of Gbeta1gamma2 causes co-immunoprecipitation with Kv1.1 of more Kvbeta1.1 subunits. Thus, we suggest that Gbeta1gamma2 directly affects the interaction between Kv1.1 and Kvbeta1.1 during channel assembly which, in turn, disrupts the ability of the channel to interact with microfilaments, resulting in an increased extent of A-type conversion.

Actin Cytoskeleton↗
PubMed1998

Activation of a metabotropic glutamate receptor and protein kinase C reduce the extent of inactivation of the K+ channel Kv1.1/Kvbeta1.1 via dephosphorylation of Kv1.1.

Various brain K+ channels, which may normally exist as complexes of alpha (pore-forming) and beta (auxiliary) subunits, were subjected to regulation by metabotropic glutamate receptors. Kv1.1/Kvbeta1.1 is a voltage-dependent K+ channel composed of alpha and beta proteins that are widely expressed in the brain. Expression of this channel in Xenopus oocytes resulted in a current that had fast inactivating and noninactivating components. Previously we showed that basal and protein kinase A-induced phosphorylation of the alpha subunit at Ser-446 decreases the fraction of the noninactivating component. In this study we investigated the effect of protein kinase C (PKC) on the channel. We showed that a PKC-activating phorbol ester (phorbol 12-myristate 13-acetate (PMA)) increased the noninactivating fraction via activation of a PKC subtype that was inhibited by staurosporine and bisindolylmaleimide but not by calphostin C. However, it was not a PKC-induced phosphorylation but rather a dephosphorylation that mediated the effect. PMA reduced the basal phosphorylation of Ser-446 significantly in plasma membrane channels and failed to affect the inactivation of channels having an alpha subunit that was mutated at Ser-446. Also, the activation of coexpressed mGluR1a known to activate phospholipase C mimicked the effect of PMA on the inactivation via induction of dephosphorylation at Ser-446. Thus, this study identified a potential neuronal pathway initiated by activation of metabotropic glutamate receptor 1a coupled to a signaling cascade that possibly utilized PKC to induce dephosphorylation and thereby to decrease the extent of inactivation of a K+ channel.

Amino Acid Sequence↗
PubMed1997

Changes in sodium channel function during postnatal brain development reflect increases in the level of channel sialidation.

Developmental changes of forebrain sodium channels were studied at three postnatal ages: P0, P15 and adult (P30/P180). Electrophysiological analysis determined that the midpoint potential of activation was -64, -75 and -81 mV for P0, P15 and adult channels, respectively. At negative potentials, gating state changes were observed in all channels; at positive potentials they were observed in most P0 (72%) and to a lower extent in older channels (25%). A long non-conductive state was displayed with a higher frequency in P0 than in older channels. Immunoblot analysis determined that the apparent molecular weight was approximately 227, approximately 241 and approximately 246 kDa for P0, P15 and adult channels, respectively. Upon neuraminidase treatment, which cleaves sialic acids, these differences in molecular weight were abolished. The data suggest that these developmental changes in the function of forebrain sodium channels correlate with changes in the channel's sialidation level.

Animals↗
PubMed1997

Inactivation of a voltage-dependent K+ channel by beta subunit. Modulation by a phosphorylation-dependent interaction between the distal C terminus of alpha subunit and cytoskeleton.

Kv1.1/Kvbeta1.1 (alphabeta) K+ channel expressed in Xenopus oocytes was shown to have a fast inactivating current component. The fraction of this component (extent of inactivation) is increased by microfilament disruption induced by cytochalasins or by phosphorylation of the alpha subunit at Ser-446, which impairs the interaction of the channel with microfilaments. The relevant sites of interaction on the channel molecules have not been identified. Using a phosphorylation-deficient mutant of alpha, S446A, to ensure maximal basal interaction of the channel with the cytoskeleton, we show that one relevant site is the end of the C terminus of alpha. Truncation of the last six amino acids resulted in alphabeta channels with an extent of inactivation up to 2.5-fold larger and its further enhancement by cytochalasins being reduced 2-fold. The wild-type channels exhibited strong inactivation, which could not be markedly increased either by cytochalasins or by the C-terminal mutations, indicating that the interaction of the wild-type channels with microfilaments was minimal to begin with, presumably because of extensive basal phosphorylation. Since the C-terminal end of Kv1.1 was shown to participate in channel clustering via an interaction with members of the PSD-95 family of proteins, we propose that a similar interaction with an endogenous protein takes place, contributing to channel connection to the oocyte cytoskeleton. This is the first report to assign a modulatory role to such an interaction: together with the state of phosphorylation of the channel, it regulates the extent of inactivation conferred by the beta subunit.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of a K+ channel alpha subunit modulates the inactivation conferred by a beta subunit. Involvement of cytoskeleton.

Voltage-gated K+ channels isolated from mammalian brain are composed of alpha and beta subunits. Interaction between coexpressed Kv1.1 (alpha) and Kvbeta1.1 (beta) subunits confers rapid inactivation on the delayed rectifier-type current that is observed when alpha subunits are expressed alone. Integrating electrophysiological and biochemical analyses, we show that the inactivation of the alphabeta current is not complete even when alpha is saturated with beta, and the alphabeta current has an inherent sustained component, indistinguishable from a pure alpha current. We further show that basal and protein kinase A-induced phosphorylations at Ser-446 of the alpha protein increase the extent, but not the rate, of inactivation of the alphabeta channel, without affecting the association between alpha and beta. In addition, the extent of inactivation is increased by agents that lead to microfilament depolymerization. The effects of phosphorylation and of microfilament depolymerization are not additive. Taken together, we suggest that phosphorylation, via a mechanism that involves the interaction of the alphabeta channel with microfilaments, enhances the extent of inactivation of the channel. Furthermore, phosphorylation at Ser-446 also increases current amplitudes of the alphabeta channel as was shown before for the alpha channel. Thus, phosphorylation enhances in concert inactivation and current amplitudes, thereby leading to a substantial increase in A-type activity.

Animals↗
PubMed1996

Expression of Kv1.1 delayed rectifier potassium channels in Lec mutant Chinese hamster ovary cell lines reveals a role for sialidation in channel function.

Kv1.1 potassium (K+) channels contain significant amounts of negatively charged sialic acids. To examine the role of sialidation in K+ channel function, Chinese hamster ovary cell lines deficient in glycosylation (Lec mutants) were transfected with rat brain Kv1.1 cDNA. The K+ channel was functionally expressed in all cell lines, but the voltage dependence of activation (V1/2) was shifted to more positive voltages and the activation kinetics were slower in the mutant cell lines compared with control. A similar positive shift in V1/2 was recorded in control cells expressing Kv1.1 following treatment with sialidase or by raising extracellular Ca2+. In contrast, these treatments had little or no effect on the Lec mutants, which indicates that channel sialic acids appear to be the negative surface charges sensitive to Ca2+. The data suggest that sialic acid addition modifies Kv1.1 channel function, possibly by influencing the local electric field detected by its voltage sensor, but that these carbohydrates are not required for cell surface expression.

Animals↗
PubMed1996

G-protein-gated inward rectifier K+ channel proteins (GIRK1) are present in the soma and dendrites as well as in nerve terminals of specific neurons in the brain.

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.

Amino Acid Sequence↗
PubMed1996

Modulation by protein kinase C activation of rat brain delayed-rectifier K+ channel expressed in Xenopus oocytes.

The modulation by protein kinase C (PKC) of the RCK1 K+ channel was investigated in Xenopus oocytes by integration of two-electrode voltage clamp, site-directed mutagenesis and SDS-PAGE analysis techniques. Upon application of beta-phorbol 12-myristate 13-acetate (PMA) the current was inhibited by 50-90%. No changes in the voltage sensitivity of the channel, changes in membrane surface area or selective elimination of RCK1 protein from the plasma membrane could be detected. The inhibition was mimicked by 1-oleoyl-2-acetyl-rac-glycerol (OAG) but not by alphaPMA, and was blocked by staurosporine and calphostin C. Upon deletion of most of the N-terminus a preceding enhancement of about 40% of the current was prominent in response to PKC activation. Its physiological significance is discussed. The N-terminus deletion eliminated 50% of the inhibition. However, phosphorylation of none of the ten classical PKC phosphorylation sites on the channel molecule could account, by itself or in combination with others, for the inhibition. Thus, our results show that PKC activation can modulate the channel conductance in a bimodal fashion. The N-terminus is involved in the inhibition, however, not via its direct phosphorylation.

Alkaloids↗
PubMed1995

Regulation of RCK1 currents with a cAMP analog via enhanced protein synthesis and direct channel phosphorylation.

We have recently shown that the rat brain Kv1.1 (RCK1) voltage-gated K+ channel is partially phosphorylated in its basal state in Xenopus oocytes and can be further phosphorylated upon treatment for a short time with a cAMP analog (Ivanina, T., Perts, T., Thornhill, W. B., Levin, G., Dascal, N., and Lotan, I. (1994) Biochemistry 33, 8786-8792). In this study, we show, by two-electrode voltage clamp analysis, that whereas treatments for a short time with various cAMP analogs do not affect the channel function, prolonged treatment with 8-bromoadenosine 3',5'-cyclic monophosphorothioate ((Sp)-8-Br-cAMPS), a membrane-permeant cAMP analog, enhances the current amplitude. It also enhances the current amplitude through a mutant channel that cannot be phosphorylated by protein kinase A activation. The enhancement is inhibited in the presence of (Rp)-8-Br-cAMPS, a membrane-permeant protein kinase A inhibitor. Concomitant SDS-polyacrylamide gel electrophoresis analysis reveals that this treatment not only brings about phosphorylation of the wild-type channel, but also increases the amounts of both wild-type and mutant channel proteins; the latter effect can be inhibited by cycloheximide, a protein synthesis inhibitor. In the presence of cycloheximide, the (Sp)-8-Br-cAMPS treatment enhances only the wild-type current amplitudes and induces accumulation of wild-type channels in the plasma membrane of the oocyte. In summary, prolonged treatment with (Sp)-8-Br-cAMPS regulates RCK1 function via two pathways, a pathway leading to enhanced channel synthesis and a pathway involving channel phosphorylation that directs channels to the plasma membrane.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

Phosphorylation by protein kinase A of RCK1 K+ channels expressed in Xenopus oocytes.

Phosphorylation-mediated regulation of voltage-gated K+ channels has been implicated in numerous electrophysiological studies; however, complementary biochemical studies have so far been hampered by the failure to isolate and characterize any K+ channel proteins of distinct molecular identity. We used the Xenopus oocyte expression system to study the biosynthesis and phosphorylation by protein kinase A (PKA) of rat brain RCK1 (Kv1.1) K+ channel protein. RCK1 protein was isolated by immunoprecipitation from oocytes injected with RCK1 cRNA and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The channel protein was expressed in the form of several polypeptides. The 57-kDa polypeptide, usually the major constituent, resided both in the cytosol and in the plasma membrane. Its levels were correlated with RCK1 current amplitudes (IRCK1) and upon incubation of the cRNA-injected oocytes with tunicamycin, its molecular weight was decreased and at the same time IRCK1 was reduced. These results suggest that the membranal 57-kDa polypeptides represent functional channels that are N-glycosylated. Furthermore, a study of the phosphorylation of the RCK1 polypeptides revealed that the 57-kDa polypeptide was specifically targeted for phosphorylation by PKA. It could be phosphorylated in vitro by the catalytic subunit of PKA (PKA-CS). In its native state in intact oocytes, the 57-kDa polypeptide was partially phosphorylated and could be further phosphorylated in vivo by addition of a membrane-permeant cAMP analog. Site-directed mutagenesis demonstrated that phosphorylation of a single site on the C-terminus of the channel molecule fully accounts for these phosphorylations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Detection of 180 kDa proteins in electroplax sodium channel preparations.

1. Co-isolating proteins (M(r) 170,000-220,000) from sodium channel preparations made from the electric organ of the electric eel (Electrophorus electricus) were detected on Western blots using monoclonal antibodies. 2. Similar protein patterns were seen on immunoblots containing immunoprecipitated protein from eel muscle and brain tissues but not heart. 3. These co-isolating proteins could be separated from the mature TTX-sensitive channel protein (M(r) 280,000) using a lentil lectin-Sepharose column. 4. The 180 kDa proteins do not appear to be channel-related and can be detected as contaminants in electroplax sodium channel preparations using the monoclonal antibodies described here.

Amino Acid Sequence↗
PubMed1991

Monoclonal antibodies raised against post-translational domains of the electroplax sodium channel.

Eleven monoclonal antibodies were identified that recognized eel electroplax sodium channels. All the monoclonal antibodies specifically immunostained the mature TTX-sensitive sodium channel (Mr 265,000) on immunoblots. None of the monoclonal antibodies would precipitate the in vitro translated channel core polypeptide in solution. One monoclonal antibody, 3G4, was found to bind to an epitope involving terminal polysialic acids. Extensive digestion of the channel by the exosialidase, neuraminidase, or partial polysialic acid removal by the endosialidase, endo-N-acetylneuraminidase, destroy the 3G4 epitope. 3G4 is, therefore, a highly selective probe for the post-translationally attached polysialic acids. Except for this monoclonal antibody, the epitopes recognized by the remaining antibodies were highly resistant to extensive N-linked deglycosylation. Thus, the monoclonal antibodies may be directed against unique post-translationally produced domains of the electroplax sodium channel, presumably sugar groups that are abundant on this protein (Miller, J.A., Agnew, W.S., Levinson, S.R. 1983, Biochemistry 22:462-470). These monoclonal antibodies should prove useful as tools to study discrete post-translational processing events in sodium channel biosynthesis.

Animals↗