Biomedical subjects

Vera A Golovina

Publications and source records attributed to Vera A Golovina.

6 recordsLinked to original sources

PubMed2006

Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor.

Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent "junctional" sarcoplasmic/endoplasmic reticulum (jS/ER) constitute specialized Ca(2+) signaling complexes in many cell types. We examined the possibility that some Ca(2+) signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The Ca(2+) sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the alpha1 or alpha2 isoform of the Na(+) pump catalytic (alpha) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that alpha2(f)GCaMP2, like native Na(+) pumps with alpha2-subunits, sorted to PM domains that colocalized with subjacent S/ER; alpha1(f)GCaMP2, like Na(+) pumps with alpha1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca(2+) signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca(2+) channel-mediated Ca(2+) entry after S/ER unloading with cyclopiazonic acid (in Ca(2+)-free medium). When cytosolic Ca(2+) diffusion was markedly restricted with EGTA, however, only alpha2(f)GCaMP2 detected the local, store-operated Ca(2+) channel-mediated Ca(2+) entry signal. Thus, alpha1 Na(+) pumps are apparently excluded from the PM microdomains occupied by alpha2 Na(2+) pumps. The jS/ER and adjacent PM may communicate by Ca(2+) signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca(2+) signals in other subPM microdomains and signals associated with other organelles.

Adenosine Triphosphate↗

PubMed2005

TRPC1 functions as a store-operated Ca2+ channel in intestinal epithelial cells and regulates early mucosal restitution after wounding.

An increase in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) results from Ca(2+) release from intracellular stores and extracellular Ca(2+) influx through Ca(2+)-permeable ion channels and is crucial for initiating intestinal epithelial restitution to reseal superficial wounds after mucosal injury. Capacitative Ca(2+) entry (CCE) induced by Ca(2+) store depletion represents a major Ca(2+) influx mechanism, but the exact molecular components constituting this process remain elusive. This study determined whether canonical transient receptor potential (TRPC)1 served as a candidate protein for Ca(2+)-permeable channels mediating CCE in intestinal epithelial cells and played an important role in early epithelial restitution. Normal intestinal epithelial cells (the IEC-6 cell line) expressed TRPC1 and TPRC5 and displayed typical records of whole cell store-operated Ca(2+) currents and CCE generated by Ca(2+) influx after depletion of intracellular stores. Induced TRPC1 expression by stable transfection with the TRPC1 gene increased CCE and enhanced cell migration during restitution. Differentiated IEC-Cdx2L1 cells induced by forced expression of the Cdx2 gene highly expressed endogenous TRPC1 and TRPC5 and exhibited increased CCE and cell migration. Inhibition of TRPC1 expression by small interfering RNA specially targeting TRPC1 not only reduced CCE but also inhibited cell migration after wounding. These findings strongly suggest that TRPC1 functions as store-operated Ca(2+) channels and plays a critical role in intestinal epithelial restitution by regulating CCE and intracellular [Ca(2+)](cyt).

Animals↗

PubMed2005

Visualization of localized store-operated calcium entry in mouse astrocytes. Close proximity to the endoplasmic reticulum.

Unloading of endoplasmic reticulum (ER) Ca(2+) stores activates influx of extracellular Ca(2+) through 'store-operated' Ca(2+) channels (SOCs) in the plasma membrane (PM) of most cells, including astrocytes. A key unresolved issue concerning SOC function is their spatial relationship to ER Ca(2+) stores. Here, using high resolution imaging with the membrane-associated Ca(2+) indicator, FFP-18, it is shown that store-operated Ca(2+) entry (SOCE) in primary cultured mouse cortical astrocytes occurs at plasma membrane-ER junctions. In the absence of extracellular Ca(2+), depletion of ER Ca(2+) stores using cyclopiazonic acid, an ER Ca(2+)-ATPase inhibitor, and caffeine transiently increases the sub-plasma-membrane Ca(2+) concentration ([Ca(2+)](SPM)) within a restricted space between the plasma membrane and adjacent ER. Restoration of extracellular Ca(2+) causes localized Ca(2+) influx that first increases [Ca(2+)](SPM) in the same restricted regions and then, with a delay, in ER-free regions. Antisense knockdown of the TRPC1 gene, proposed to encode endogenous SOCs, markedly reduces SOCE measured with Fura-2. High resolution immunocytochemistry with anti-TRPC1 antibody reveals that these TRPC-encoded SOCs are confined to the PM microdomains adjacent to the underlying 'junctional' ER. Thus, Ca(2+) entry through TRPC-encoded SOCs is closely linked, not only functionally, but also structurally, to the ER Ca(2+) stores.

Animals↗

PubMed2002

Na+ pump alpha 2-subunit expression modulates Ca2+ signaling.

The role of the Na(+) pump alpha(2)-subunit in Ca(2+) signaling was examined in primary cultured astrocytes from wild-type (alpha(2)+/+ = WT) mouse fetuses and those with a null mutation in one [alpha(2)+/- = heterozygote (Het)] or both [alpha(2)-/- = knockout (KO)] alpha(2) genes. Na(+) pump catalytic (alpha) subunit expression was measured by immunoblot; cytosol [Na(+)] ([Na(+)](cyt)) and [Ca(2+)] ([Ca(2+)](cyt)) were measured with sodium-binding benzofuran isophthalate and fura 2 by using digital imaging. Astrocytes express Na(+) pumps with both alpha(1)- ( approximately 80% of total alpha) and alpha(2)- ( approximately 20% of total alpha) subunits. Het astrocytes express approximately 50% of normal alpha(2); those from KO express none. Expression of alpha(1) is normal in both Het and KO cells. Resting [Na(+)](cyt) = 6.5 mM in WT, 6.8 mM in Het (P > 0.05 vs. WT), and 8.0 mM in KO cells (P < 0.001); 500 nM ouabain (inhibits only alpha(2)) equalized [Na(+)](cyt) at 8 mM in all three cell types. Resting [Ca(2+)](cyt) = 132 nM in WT, 162 nM in Het, and 196 nM in KO cells (both P < 0.001 vs. WT). Cyclopiazonic acid (CPA), which inhibits endoplasmic reticulum (ER) Ca(2+) pumps and unloads the ER, induces transient (in Ca(2+)-free media) or sustained (in Ca(2+)-replete media) elevation of [Ca(2+)](cyt). These Ca(2+) responses to 10 microM CPA were augmented in Het as well as KO cells. When CPA was applied in Ca(2+)-free media, the reintroduction of Ca(2+) induced significantly larger transient rises in [Ca(2+)](cyt) (due to Ca(2+) entry through store-operated channels) in Het and KO cells than in WT cells. These results correlate with published evidence that alpha(2) Na(+) pumps and Na(+)/Ca(2+) exchangers are confined to plasma membrane microdomains that overlie the ER. The data suggest that selective reduction of alpha(2) Na(+) pump activity can elevate local [Na(+)] and, via Na(+)/Ca(2+) exchange, [Ca(2+)] in the tiny volume of cytosol between the plasma membrane and ER. This, in turn, augments adjacent ER Ca(2+) stores and thereby amplifies Ca(2+) signaling without elevating bulk [Na(+)](cyt).

Animals↗

PubMed2002

Activation of K(+) channels and increased migration of differentiated intestinal epithelial cells after wounding.

Early mucosal restitution occurs by epithelial cell migration to reseal superficial wounds after injury. Differentiated intestinal epithelial cells induced by forced expression of the Cdx2 gene migrate over the wounded edge much faster than undifferentiated parental cells in an in vitro model. This study determined whether these differentiated intestinal epithelial cells exhibit increased migration by altering voltage-gated K(+) (Kv) channel expression and cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)). Stable Cdx2-transfected IEC-6 cells (IEC-Cdx2L1) with highly differentiated phenotype expressed higher basal levels of Kv1.1 and Kv1.5 mRNAs and proteins than parental IEC-6 cells. Neither IEC-Cdx2L1 cells nor parental IEC-6 cells expressed voltage-dependent Ca(2+) channels. The increased expression of Kv channels in differentiated IEC-Cdx2L1 cells was associated with an increase in whole cell K(+) currents, membrane hyperpolarization, and a rise in [Ca(2+)](cyt). The migration rates in differentiated IEC-Cdx2L1 cells were about four times those of parental IEC-6 cells. Inhibition of Kv channel expression by polyamine depletion decreased [Ca(2+)](cyt), reduced myosin stress fibers, and inhibited cell migration. Elevation of [Ca(2+)](cyt) by ionomycin promoted myosin II stress fiber formation and increased cell migration. These results suggest that increased migration of differentiated intestinal epithelial cells is mediated, at least partially, by increasing Kv channel activity and Ca(2+) influx during restitution.

Animals↗

PubMed2002

Organization of Ca2+ stores in vascular smooth muscle: functional implications.

Much evidence suggests that caffeine/ryanodine (Caf/Ry)-releasable and inositol-1,4,5-trisphosphate (InsP3)-releasable Ca2+ stores in the sarcoplasmic reticulum (SR) of smooth muscles are at least partially distinct. We directly visualized SR stores in primary-cultured rat mesenteric artery myocytes with high-resolution digital imaging and the low-affinity Ca2, indicator, Furaptra (Kd = 75.6 microM). The SR appears to be a continuous tubular network. Nevertheless, SR Ca2+ stores are organized into small, separate, functionally independent compartments. Cyclopiazonic acid (CPA; inhibits SR (Ca2+ pump) and Caf (or Ry) release Ca2+ from different, spatially distinct compartments. Similar heterogeneity is seen with serotonin (acts via InsP3), which unloads only the CPA-sensitive compartments. Some of the SR ('junctional' SR; jSR) lies within 12-15 nm of the plasmalemma (PL). The jSR, the overlying PL microdomains, and the intervening, tiny volume of cytosol form junctional complexes ('PLasmERosomes'). Na+ pumps with high-ouabain-affinity alpha2 or alpha3 subunits, Na+/Ca2+ exchangers, and store-operated channels are confined to these PL microdomains, whereas Na+ pumps with low-ouabain-affinity alpha1 subunits and plasma membrane Ca2+ pumps are uniformly distributed. As a result of this organization, low-dose ouabain can selectively modulate Na+ and Ca2+ concentrations in the PLasmERosomes and jSR Ca2+ stores, and can thereby regulate Ca2+ signalling.

Adenosine Triphosphatases↗