Biomedical subjects

Yuchun Gu

Publications and source records attributed to Yuchun Gu.

7 recordsLinked to original sources

PubMed2005

Mechanisms of light adaptation in Drosophila photoreceptors.

Phototransduction in Drosophila is mediated by a phospholipase C (PLC) cascade culminating in activation of transient receptor potential (TRP) channels. Ca(2+) influx via these channels is required for light adaptation, but although several molecular targets of Ca(2+)-dependent feedback have been identified, their contribution to adaptation is unclear. By manipulating cytosolic Ca(2+) via the Na(+)/Ca(2+) exchange equilibrium, we found that Ca(2+) inhibited the light-induced current (LIC) over a range corresponding to steady-state light-adapted Ca(2+) levels (0.1-10 microM Ca(2+)) and accurately mimicked light adaptation. However, PLC activity monitored with genetically targeted PIP(2)-sensitive ion channels (Kir2.1) was first inhibited by much higher (>/= approximately 50 microM) Ca(2+) levels, which occur only transiently in vivo. Ca(2+)-dependent inhibition of PLC, but not the LIC, was impaired in mutants (inaC) of protein kinase C (PKC). The results indicate that light adaptation is primarily mediated downstream of PLC and independently of PKC by Ca(2+)-dependent inhibition of TRP channels. This is interpreted as a strategy to prevent inhibition of PLC by global steady-state light-adapted Ca(2+) levels, whereas rapid inhibition of PLC by local Ca(2+) transients is required to terminate the response and ensures that PIP(2) reserves are not depleted during stimulation.

Adaptation, Ocular↗

PubMed2005

Light activation, adaptation, and cell survival functions of the Na+/Ca2+ exchanger CalX.

In sensory neurons, Ca(2+) entry is crucial for both activation and subsequent attenuation of signaling. Influx of Ca(2+) is counterbalanced by Ca(2+) extrusion, and Na(+)/Ca(2+) exchange is the primary mode for rapid Ca(2+) removal during and after sensory stimulation. However, the consequences on sensory signaling resulting from mutations in Na(+)/Ca(2+) exchangers have not been described. Here, we report that mutations in the Drosophila Na(+)/Ca(2+) exchanger calx have a profound effect on activity-dependent survival of photoreceptor cells. Loss of CalX activity resulted in a transient response to light, a dramatic decrease in signal amplification, and unusually rapid adaptation. Conversely, overexpression of CalX had reciprocal effects and greatly suppressed the retinal degeneration caused by constitutive activity of the TRP channel. These results illustrate the critical role of Ca(2+) for proper signaling and provide genetic evidence that Ca(2+) overload is responsible for a form of retinal degeneration resulting from defects in the TRP channel.

Adaptation, Ocular↗

PubMed2005

Group III metabotropic glutamate receptor activation inhibits Ca2+ influx and nitric oxide synthase activity in bone marrow stromal cells.

Nitric oxide (NO) is pivotal to bone physiology. In the central nervous system constitutive, Ca(2+)-calmodulin regulated NO synthase activity and glutamate signalling are intimately linked. Since L-glutamate signalling occurs in bone and is implicated in bone regulation, we have investigated the effect of L-glutamate on NO synthase in bone-derived cells. Treatment of marrow stromal cells with L-glutamate reduced basal NO synthase activity by 40%. Imaging showed that L-glutamate caused a rapid, usually localised and slowly-reversible fall in [Ca(2+)](i). This effect was resistant to disruption of intracellular Ca(2+) stores but sensitive to extracellular La(3+) or omission of extracellular Ca(2+), demonstrating that glutamate acts by inhibition of membrane Ca(2+) influx. The only previous description of such an effect of L-glutamate is via activation of the group III receptor, mGluR6, in the retina. Using Western blotting and RT-PCR we detected mGluR6 protein and transcripts in marrow stromal cells. The effects of L-glutamate on NOS activity and [Ca(2+)](i) in marrow stromal cells were abolished by a group III mGluR inhibitor, (S)-2-amino-2-methyl-4-phosphonobutyric acid. Recording of membrane potential showed that, similarly to the effects of retinal mGluR6 activation, L-glutamate induced membrane hyperpolarisation (-16 +/- 2 mV), which was also sensitive to group III mGluR inhibition. L-glutamate had no effect on cAMP levels. We conclude that activation of a group III mGluR in bone marrow stromal cells inhibits a Ca(2+)-permeable plasma membrane channel, reducing [Ca(2+)](i) and suppressing generation of NO. These observations directly link bone L-glutamate signalling to processes central to bone growth and regulation.

Animals↗

PubMed2004

In vivo light-induced and basal phospholipase C activity in Drosophila photoreceptors measured with genetically targeted phosphatidylinositol 4,5-bisphosphate-sensitive ion channels (Kir2.1).

The phosphatidylinositol 4,5-bisphosphate (PIP(2))-sensitive inward rectifier channel Kir2.1 was expressed in Drosophila photoreceptors and used to monitor in vivo PIP(2) levels. Since the wild-type (WT) Kir2.1 channel appeared to be saturated by the prevailing PIP(2) concentration, we made a single amino acid substitution (R228Q), which reduced the effective affinity for PIP(2) and yielded channels generating currents proportional to the PIP(2) levels relevant for phototransduction. To isolate Kir2.1 currents, recordings were made from mutants lacking both classes of light-sensitive transient receptor potential channels (TRP and TRPL). Light resulted in the effective depletion of PIP(2) by phospholipase C (PLC) in approximately three or four microvilli per absorbed photon at rates exceeding approximately 150% of total microvillar phosphoinositides per second. PIP(2) was resynthesized with a half-time of approximately 50 s. When PIP(2) resynthesis was prevented by depriving the cell of ATP, the Kir current spontaneously decayed at maximal rates representing a loss of approximately 40% loss of total PIP(2) per minute. This loss was attributed primarily to basal PLC activity, because it was greatly decreased in norpA mutants lacking PLC. We tried to confirm this by using the PLC inhibitor U73122; however, this was found to act as a novel inhibitor of the Kir2.1 channel. PIP(2) levels were reduced approximately 5-fold in the diacylglycerol kinase mutant (rdgA), but basal PLC activity was still pronounced, consistent with the suggestion that raised diacylglycerol levels are responsible for the constitutive TRP channel activity characteristic of this mutant.

Animals↗

PubMed2002

High-resolution scanning patch-clamp: new insights into cell function.

Cell specialization is often governed by the spatial distribution of ion channels and receptors on the cell surface. So far, little is known about functional ion channel localization. This is due to a lack of satisfactory methods for investigating ion channels in an intact cell and simultaneously determining the channels' positions accurately. We have developed a novel high-resolution scanning patch-clamp technique that enables the study of ion channels, not only in small cells, such as sperm, but in submicrometer cellular structures, such as epithelial microvilli, fine neuronal dendrites, and, particularly, T-tubule openings of cardiac myocytes. In cardiac myocytes, as in most excitable cells, action potential propagation depends essentially on the properties of ion channels that are functionally and spatially coupled. We found that the L-type calcium and chloride channels are distributed and colocalized in the region of T-tubule openings, but not in other regions of the myocyte. In addition, chloride channels were found in narrowly defined regions of Z-grooves. This finding suggests a new synergism between these types of channels that may be relevant for action potential propagation along the T-tubule system and excitation-contraction coupling.

Animals↗

PubMed2002

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.

Animals↗

PubMed2001

Identification and localization of T-type voltage-operated calcium channel subunits in human male germ cells. Expression of multiple isoforms.

Low voltage activated, voltage-operated Ca(2+) channels are expressed in rodent male germ cells and are believed to be pivotal in induction of the acrosome reaction in mouse spermatozoa. However, in humans, very little is known about expression of voltage-operated Ca(2+) channels in male germ cells or their function. We have used reverse transcription-polymerase chain reaction, in situ hybridization, and patch clamp recording to investigate the expression of low voltage activated voltage-operated Ca(2+) channels in human male germ cells. We report that full-length transcripts for both alpha(1G) and alpha(1H) low voltage activated channel subunits are expressed in human testis. Multiple isoforms of alpha(1G) are present in the testis and at least two isoforms of alpha(1H), including a splice variant not previously described in the human. Transcripts for all the isoforms of both alpha(1G) and alpha(1H) were detected by reverse transcription-polymerase chain reaction on mRNA isolated from human spermatogenic cells. In situ hybridization for alpha(1G) and alpha(1H) localized transcripts both in germ cells and in other cell types in the testis. Within the seminiferous tubules, alpha(1H) was detected primarily in germ cells. Using the whole cell patch clamp technique, we detected T-type voltage-operated Ca(2+) channel currents in isolated human male germ cells, although the current amplitude and frequency of occurrence were low in comparison to the occurrence of T-currents in murine male germ cells. We conclude that low voltage activated voltage-operated Ca(2+) channels are expressed in cells of the human male germ line.

Animals↗