Biomedical subjects

T C Holmes

Publications and source records attributed to T C Holmes.

15 recordsLinked to original sources

PubMed2001

A mechanism for combinatorial regulation of electrical activity: Potassium channel subunits capable of functioning as Src homology 3-dependent adaptors.

It is an open question how ion channel subunits that lack protein-protein binding motifs become targeted and covalently modified by cellular signaling enzymes. Here, we show that Src-family protein tyrosine kinases (PTKs) bind to heteromultimeric Shaker-family voltage-gated potassium (Kv) channels by interactions between the Src homology 3 (SH3) domain and the proline-rich SH3 domain ligand sequence in the Shaker-family subunit Kv1.5. Once bound to Kv1.5, Src-family PTKs phosphorylate adjacent subunits in the Kv channel heteromultimer that lack proline-rich SH3 domain ligand sequences. This SH3-dependent tyrosine phosphorylation contributes to significant suppression of voltage-evoked currents flowing through the heteromultimeric channel. These results demonstrate that Kv1.5 subunits function as SH3-dependent adaptor proteins that marshal Src-family kinases to heteromultimeric potassium channel signaling complexes, and thereby confer functional sensitivity upon coassembled channel subunits that are themselves not bound directly to Src-family kinases by allowing their phosphorylation. This is a mechanism for information transfer between subunits in heteromultimeric ion channels that is likely to underlie the generation of combinatorial signaling diversity in the control of cellular electrical excitability.

Action Potentials↗

PubMed2001

Is the molecular composition of K(ATP) channels more complex than originally thought?

ATP-sensitive K+ (K(ATP)) channels are abundantly expressed in the heart and may be involved in the pathogenesis of myocardial ischemia. These channels are heteromultimeric, consisting of four pore-forming subunits (Kir6.1, Kir6.2) and four sulfonylurea receptor (SUR) subunits in an octameric assembly. Conventionally, the molecular composition of K(ATP) channels in cardiomyocytes and pancreatic beta -cells is thought to include the Kir6.2 subunit and either the SUR2A or SUR1 subunits, respectively. However, Kir6.1 mRNA is abundantly expressed in the heart, suggesting that Kir6.1 and Kir6.2 subunits may co-assemble to form functional heteromeric channel complexes. Here we provide two independent lines of evidence that heteromultimerization between Kir6.1 and Kir6.2 subunits is possible in the presence of SUR2A. We generated dominant negative Kir6 subunits by mutating the GFG residues in the channel pore to a series of alanine residues. The Kir6.1-AAA pore mutant subunit suppressed both wt-Kir6.1/SUR2A and wt-Kir6.2/SUR2A currents in transfected HEK293 cells. Similarly, the dominant negative action of Kir6.2-AAA does not discriminate between either of the wild-type subunits, suggesting an interaction between Kir6.1 and Kir6.2 subunits within the same channel complex. Biochemical data support this concept: immunoprecipitation with Kir6.1 antibodies also co-precipitates Kir6.2 subunits and conversely, immunoprecipitation with Kir6.2 antibodies co-precipitates Kir6.1 subunits. Collectively, our data provide direct electrophysiological and biochemical evidence for heteromultimeric assembly between Kir6.1 and Kir6.2. This paradigm has profound implications for understanding the properties of native K(ATP)channels in the heart and other tissues.

Adenosine Triphosphate↗

PubMed2000

Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds.

A new type of self-assembling peptide (sapeptide) scaffolds that serve as substrates for neurite outgrowth and synapse formation is described. These peptide-based scaffolds are amenable to molecular design by using chemical or biotechnological syntheses. They can be tailored to a variety of applications. The sapeptide scaffolds are formed through the spontaneous assembly of ionic self-complementary beta-sheet oligopeptides under physiological conditions, producing a hydrogel material. The scaffolds can support neuronal cell attachment and differentiation as well as extensive neurite outgrowth. Furthermore, they are permissive substrates for functional synapse formation between the attached neurons. That primary rat neurons form active synapses on such scaffold surfaces in situ suggests these scaffolds could be useful for tissue engineering applications. The buoyant sapeptide scaffolds with attached cells in culture can be transported readily from one environment to another. Furthermore, these peptides did not elicit a measurable immune response or tissue inflammation when introduced into animals. These biological materials created through molecular design and self assembly may be developed as a biologically compatible scaffold for tissue repair and tissue engineering.

Amino Acid Sequence↗

PubMed1997

Expression of voltage-gated potassium channels decreases cellular protein tyrosine phosphorylation.

Protein tyrosine phosphorylation by endogenous and expressed tyrosine kinases is reduced markedly by the expression of functional voltage-gated potassium (Kv) channels. The levels of tyrosine kinase protein and cellular protein substrates are unaffected, consistent with a reduction in tyrosine phosphorylation that results from inhibition of protein tyrosine kinase activity. The attenuation of protein tyrosine phosphorylation is correlated with the gating properties of expressed wild-type and mutant Kv channels. Furthermore, cellular protein tyrosine phosphorylation is reduced within minutes by acute treatment with the electrogenic potassium ionophore valinomycin. Because tyrosine phosphorylation in turn influences Kv channel activity, these results suggest that reciprocal modulatory interactions occur between Kv channel and protein tyrosine phosphorylation signaling pathways.

Cells, Cultured↗

PubMed1997

Modulation of the Kv1.3 potassium channel by receptor tyrosine kinases.

The voltage-dependent potassium channel, Kv1.3, is modulated by the epidermal growth factor receptor (EGFr) and the insulin receptor tyrosine kinases. When the EGFr and Kv1.3 are coexpressed in HEK 293 cells, acute treatment of the cells with EGF during a patch recording can suppress the Kv1.3 current within tens of minutes. This effect appears to be due to tyrosine phosphorylation of the channel, as it is blocked by treatment with the tyrosine kinase inhibitor erbstatin, or by mutation of the tyrosine at channel amino acid position 479 to phenylalanine. Previous work has shown that there is a large increase in the tyrosine phosphorylation of Kv1.3 when it is coexpressed with the EGFr. Pretreatment of EGFr and Kv1.3 cotransfected cells with EGF before patch recording also results in a decrease in peak Kv1.3 current. Furthermore, pretreatment of cotransfected cells with an antibody to the EGFr ligand binding domain (alpha-EGFr), which blocks receptor dimerization and tyrosine kinase activation, blocks the EGFr-mediated suppression of Kv1.3 current. Insulin treatment during patch recording also causes an inhibition of Kv1.3 current after tens of minutes, while pretreatment for 18 h produces almost total suppression of current. In addition to depressing peak Kv1.3 current, EGF treatment produces a speeding of C-type inactivation, while pretreatment with the alpha-EGFr slows C-type inactivation. In contrast, insulin does not influence C-type inactivation kinetics. Mutational analysis indicates that the EGF-induced modulation of the inactivation rate occurs by a mechanism different from that of the EGF-induced decrease in peak current. Thus, receptor tyrosine kinases differentially modulate the current magnitude and kinetics of a voltage-dependent potassium channel.

Cell Line↗

PubMed1997

Tyrosine phosphorylation modulates current amplitude and kinetics of a neuronal voltage-gated potassium channel.

The modulation of the Kv1.3 potassium channel by tyrosine phosphorylation was studied. Kv1.3 was expressed in human embryonic kidney (HEK 293) cells, and its activity was measured by cell-attached patch recording. The amplitude of the characteristic C-type inactivating Kv1.3 current is reduced by >95%, in all cells tested, when the channel is co-expressed with the constitutively active nonreceptor tyrosine kinase, v-Src. This v-Src-induced suppression of current is accompanied by a robust tyrosine phosphorylation of the channel protein. No suppression of current or tyrosine phosphorylation of Kv1.3 protein is observed when the channel is co-expressed with R385A v-Src, a mutant with severely impaired tyrosine kinase activity. v-Src-induced suppression of Kv1.3 current is relieved by pretreatment of the HEK 293 cells with two structurally different tyrosine kinase inhibitors, herbimycin A and genistein. Furthermore, Kv1.3 channel protein is processed properly and targeted to the plasma membrane in v-Src cotransfected cells, as demonstrated by confocal microscopy using an antibody directed against an extracellular epitope on the channel. Thus v-Src-induced suppression of Kv1.3 current is not mediated through decreased channel protein expression or interference with its targeting to the plasma membrane. v-Src co-expression also slows the C-type inactivation and speeds the deactivation of the residual Kv1.3 current. Mutational analysis demonstrates that each of these modulatory changes, in current amplitude and kinetics, requires the phosphorylation of Kv1.3 at multiple tyrosine residues. Furthermore, a different combination of tyrosine residues is involved in each of the modulatory changes. These results emphasize the complexity of signal integration at the level of a single ion channel.

DNA, Complementary↗

PubMed1996

Association of Src tyrosine kinase with a human potassium channel mediated by SH3 domain.

The human Kv1.5 potassium channel (hKv1.5) contains proline-rich sequences identical to those that bind to Src homology 3 (SH3) domains. Direct association of the Src tyrosine kinase with cloned hKv1.5 and native hKv1.5 in human myocardium was observed. This interaction was mediated by the proline-rich motif of hKv1.5 and the SH3 domain of Src. Furthermore, hKv1.5 was tyrosine phosphorylated, and the channel current was suppressed, in cells coexpressing v-Src. These results provide direct biochemical evidence for a signaling complex composed of a potassium channel and a protein tyrosine kinase.

Amino Acid Sequence↗

PubMed1996

Tyrosine phosphorylation of the Kv1.3 potassium channel.

Kv1.3, a voltage-dependent potassium channel cloned from mammalian brain and T lymphocytes, contains multiple tyrosine residues that are putative targets for tyrosine kinases. We have examined the tyrosine phosphorylation of Kv1.3, expressed transiently in human embryonic kidney (or HEK) 293 cells, by endogenous and coexpressed tyrosine kinases. Tyrosine phosphorylation is measured by a strategy of immunoprecipitation followed by. Western blot analysis, using antibodies that specifically recognize Kv1.3 and phosphotyrosine. Coexpression of the constitutively active tyrosine kinase v-src, together with Kv1.3, causes a large increase in the tyrosine phosphorylation of the channel protein. This phosphorylation of Kv1.3 can be reversed by treatment with alkaline phosphatase before Western blot analysis. Coexpression with a receptor tyrosine kinase, the human epidermal growth factor receptor, also causes an increase in tyrosine phosphorylation of Kv1.3. The effects of endogenous tyrosine kinases were examined by treating Kv1.3-transfected cells with the specific membrane-permeant tyrosine phosphatase inhibitor pervanadate. Pervanadate treatment causes a time- and concentration-dependent increase in the tyrosine phosphorylation of Kv1.3. This increased tyrosine phosphorylation of Kv1.3 is accompanied by a time-dependent decrease in Kv1.3 current, measured by patch-clamp analysis with cell-attached membrane patches. The pervanadate-induced suppression of current and much of the channel tyrosine phosphorylation are eliminated by mutation of a specific tyrosine residue, at position 449 of Kv1.3, to phenylalanine. Thus, there is a continual phosphorylation and dephosphorylation of Kv1.3 by endogenous kinases and phosphatases, and perturbation of this constitutive phosphorylation/dephosphorylation cycle can profoundly influence channel activity.

Animals↗

PubMed1995

Effects of unilateral lesion of the nucleus basalis magnocellularis on carbachol- and serotonin-stimulated [3H]inositolmonophosphate accumulation in rat fronto-parietal cortex.

We examined the effects of ibotenic acid induced unilateral lesion of the nucleus basalis magnocellularis (nBM) on carbachol- and serotonin-stimulated phosphoinositide (PI) breakdown in miniprisms obtained from rat fronto-parietal cortex 1 week following the lesion. Lesion-side muscarinic and serotoninergic receptor responsivity to agonist increased linearly relative to the severity of the nBM lesion as measured by choline acetyltransferase (ChAT) activity. These results provide further evidence for an interaction between central cholinergic and serotoninergic neurons.

Animals↗

PubMed1995

Self-complementary oligopeptide matrices support mammalian cell attachment.

A new class of ionic self-complementary oligopeptides is described, two members of which have been designated RAD16 and EAK16. These oligopeptides consist of regular repeats of alternating ionic hydrophilic and hydrophobic amino acids and associate to form stable beta-sheet structures in water. The addition of buffers containing millimolar amounts of monovalent salts or the transfer of a peptide solution into physiological solutions results in the spontaneous assembly of the oligopeptides into a stable, macroscopic membranous matrix. The matrix is composed of ordered filaments which form porous enclosures. A variety of mammalian cell types are able to attach to both RAD16 and EAK16 membranous matrices. These matrices provide a novel experimental system for analysing mechanisms of in vitro cell attachment and may have applications in in vivo studies of tissue regeneration, tissue transplantation and would healing.

Amino Acid Sequence↗

PubMed1993

Phospholipid and phospholipid metabolites in rat frontal cortex are decreased following nucleus basalis lesions.

Membrane phospholipid metabolism is abnormal in Alzheimer's disease (AD) brain. Phosphatidylcholine and phosphatidylethanolamine levels are decreased as are choline and ethanolamine, while glycerophosphocholine (GPC) and glycerophosphoethanolamine are increased. To develop a rat model for these changes, we examined the effects of unilateral lesion of the cholinergic nucleus basalis (nBM) with ibotenic acid (10 mg/ml in PBS, 0.5 microliter) and sham lesion on frontocortical phospholipid, choline and GPC. After one week, choline acetyltransferase activity in frontal cortex was decreased (26%, p < 0.005, n = 14) on the nBM ibotenate-lesion side relative to the contralateral side, while there were no differences following the nBM sham-lesion. Levels of membrane phospholipids (nmol/mg protein) in adjacent frontal cortex sections exhibited concomitant decreases (13%, p < 0.05, n = 14) on the nBM ibotenate-lesion side, while there were no differences following the nBM sham-lesion. Tissue nBM ibotenate-lesion frontocortical choline and GPC levels were also decreased relative to those in control tissue (choline: 21%, p < 0.05, n = 14; GPC: 10%, p < 0.05, n = 14), while nBM sham-lesion showed no effect. Muscarinic receptor sensitivity in frontal cortex following nBM ibotenate-lesion was increased, as measured by carbachol-stimulated inositol phosphate production (p < 0.001, n = 12), indicating that increased receptor mediated phospholipid hydrolysis in cortex may occur following nBM ibotenate-lesion. These data suggest that impaired cholinergic transmission alters phospholipid metabolism in cholinergic target regions.

Animals↗

PubMed1991

Interactions of 3,4-diaminopyridine and choline in stimulating acetylcholine release and protecting membrane phospholipids.

We investigated the effects of 3,4-DAP on ACh release from rat striatal slices superfused with or without choline, at rest and during electrical stimulation. In a choline-free medium, 3,4-DAP increased basal and stimulated ACh release while lowering the net efflux of choline; thus while the sum of ACh plus choline released remained constant, the ratio of released ACh to that of choline was increased. The drug failed to affect tissue ACh, choline or membrane phospholipid levels (including those of phosphatidylcholine). In a choline-containing medium, 3,4-DAP potentiated the enhancement by choline of both basal and electrically stimulated ACh release. Electrical stimulation alone increased ACh release from the slices without altering choline efflux or depleting tissue choline or ACh stores; however, this treatment did deplete membranes of phosphatidylcholine and of other major phospholipids. Superfusion of the slices with 3,4-DAP protected the slices from stimulation-induced phospholipid depletion. Calcium-dependent activation of high-affinity choline uptake may underlie the observed effects of 3,4-DAP.

4-Aminopyridine↗

PubMed1991

Intensity-amplitude relationships in monkey event-related potentials: parallels to human augmenting-reducing responses.

In human, the amplitudes of specific event-related potential (ERP) components can increase or decrease in response to increasing stimulus intensity depending on the location of the recording site. Large increases characterize components presumably generated by modality-specific sites, while smaller increases or even decreases are associated with those originating in associational areas. Comparable data from non-human primates, which would permit invasive studies of the neural substrates underlying these intensity-amplitude differences, are limited. To more fully characterize these relationships, auditory ERPs were recorded from chronically implanted epidural electrodes in 5 squirrel monkeys (Saimiri sciureus) in response to tones (500 Hz, 300 msec duration) of varying intensities (50, 60, 70, 80 dB SPL). Squirrel monkey ERPs recorded at Fz exhibited 3 peaks during the 200 msec post-stimulus interval. These peaks included a positivity (P1), followed by a negativity (N1), and then another positivity (P2). At posterior sites, the frontal P1-N1 configuration was recorded as an N1-P1 complex. At these sites, a small negativity (N2) preceded the last positive peak (P2). Changes in polarity were independent of reference site and posterior N1-P1 peaks exhibited latencies similar to those of the frontal P1-N1 components. Amplitudes at Fz, Cz, and Pz increased substantially with increasing stimulus intensity ('augmenting'). In contrast, only small increases or even decreases in amplitude ('reducing') were evident at T3 and T4. On the other hand, peak latencies decreased with higher stimulus intensities at most sites. The site-specific amplitude responses exhibited considerable temporal stability. In one subject, for example, similar 'augmenting' profiles were recorded at Fz in 8 sessions over a 6-month period. The topography of monkey intensity-amplitude response profiles, their temporal stability, and peak latency shifts resemble observations made in humans. The data show that 'augmenting' characterizes monkey vertex potentials, which, like the analogous human potentials, may originate in primary auditory cortex. In contrast, potentials recorded over temporal cortex, which may originate in auditory association cortex, exhibit 'reducing.' Thus, the data support the hypothesis that differences in amplitude with increasing intensity may reflect differences in cortical origin.

Acoustic Stimulation↗

PubMed1989

Brain-stem auditory evoked potentials in squirrel monkey (Saimiri sciureus).

To more fully characterize brain-stem auditory evoked potentials (BAEPs) in non-human primates, BAEPs were recorded from chronically implanted epidural electrodes in 10 squirrel monkeys (Saimiri sciureus). The effects of stimulus intensity, repetition rate, and anesthesia (ketamine 20 mg/kg i.m.) on peak latencies and inter-peak intervals were evaluated. Monkey wave forms consisted of approximately 7 peaks (I-VII), each exhibiting similar latencies across sessions, with later peaks exhibiting greater variability. In some subjects, additional peaks (IIa, IIIa) and slow potentials were recorded. The slow potentials provided a substratum for peaks IV through VII. As with human, monkey peaks exhibited systematic changes in latency with changes in stimulus intensity or repetition rate. These shifts included significant decreases in latency with increasing intensity for peaks I-IV and increases in latency with increases in repetition rate for peaks III, V, and VI. Inter-peak intervals were similar to those observed in human. Furthermore, ketamine anesthesia significantly delayed the latencies of most peaks (except I, V, and VII). Some differences between monkey and human BAEPs were evident in the relative amplitude of specific peaks. For example, peak V is typically most prominent in human, while this was true for peak III in monkey. The similarities between unanesthetized monkey and human inter-peak intervals suggest that the times required for impulses to reach particular brain-stem areas are conserved across primate species that vary in brain size. This supports the hypothesis that comparably numbered BAEP peaks in monkey and human index homologous processes. The data also suggest that the differences between animal and human BAEPs commonly reported may result from the use of anesthetics. In summary, unanesthetized monkey BAEPs resemble human BAEPs in morphology, number of peaks, polarity, latency variability, inter-peak intervals, slow potentials superimposed on the high-frequency peaks, and variations in morphology, amplitude, and resolution of peaks as a function of recording site. Thus, unanesthetized monkey BAEPs may be an excellent model for investigating the neural substrates of human BAEP or for determining species differences in acoustic processing among primates.

Anesthesia↗

PubMed1988

Endogenous event-related potentials in monkey: the role of task relevance, stimulus probability, and behavioral response.

Monkeys were trained in auditory discrimination tasks resembling human paradigms in which long-latency endogenous components, such as P300, are typically recorded. Morphological, topographical, and functional properties of the monkey event-related potentials (ERPs) were analyzed to determine similarities and differences with human ERPs reported in the literature. ERPs were recorded from epidural electrodes in monkeys trained to produce operant responses. In a conditional discrimination (CD) task, tone pips (2 kHz or 6 kHz, 40 msec duration, and 60 dB above nHL) were presented every 4-8 sec. Target tones presented during 'time-in' (TI) were rewarded when followed by a response in the correct post-stimulus interval (400-3000 msec). In contrast, tones presented during 'time-out' (TO) were not rewarded. Under both conditions, tones elicited an initial frontally dominant triphasic complex (P56-N92-P157). Additionally, TI target tones followed by a response elicited a large negativity (N358) having maximal amplitude over mid-frontal regions and followed by a parietally distributed positivity (P658). The scalp distribution and covariation with task requirements of N358 resemble those reported for the human 'O' wave. ERPs were also recorded in an auditory oddball paradigm in which tone pips (2 kHz and 6 kHz, 40 msec duration, and 60 dB above nHL) were presented in random order every second. Monkeys trained in the CD paradigm, along with additional subjects, were trained to make delayed responses following target tones embedded in a background of different-pitch tones. Tone probabilities were varied in different sessions from 90-10, 70-30, to 50-50 to assess the effects of probability. Background and target tones elicited a triphasic complex (P52-N110-P159) similar in latency and distribution to that recorded in the CD task. Additionally, target tones in this paradigm elicited a long-latency positive component (LPC) that exhibited an inverse relationship with stimulus probability. LPC had an onset latency of approximately 150-200 msec, a duration of approximately 300 msec, and multiple peaks (P244 and P376). These data indicate the importance of stimulus context in eliciting long-latency endogenous activity. It further suggests that strong analogies exist between monkey and human potentials recorded under similar paradigms. The effects of task relevance, stimulus probability, and the act of producing behavioral responses are similar to the effects of these variables on analogous human potentials.(ABSTRACT TRUNCATED AT 400 WORDS)

Acoustic Stimulation↗