Fast two-dimensional confocal microscopy and the Ca(2+) indicator fluo-4 were used to study excitation-contraction (E-C) coupling in cat atrial myocytes which lack transverse tubules and contain both subsarcolemmal junctional (j-SR) and central nonjunctional (nj-SR) sarcoplasmic reticulum. Action potentials elicited by field stimulation induced transient increases of intracellular Ca(2+) concentration ([Ca(2+)](i)) that were highly inhomogeneous. Increases started at distinct subsarcolemmal release sites spaced approximately 2 microm apart. The amplitude and the latency of Ca(2+) release from these sites varied from beat to beat. Subsarcolemmal release fused to build a peripheral ring of elevated [Ca(2+)](i), which actively propagated to the center of the cells via Ca(2+)-induced Ca(2+) release. Resting myocytes exhibited spontaneous Ca(2+) release events, including Ca(2+) sparks and local (microscopic) or global (macroscopic) [Ca(2+)](i) waves. The microscopic [Ca(2+)](i) waves propagated in a saltatory fashion along the sarcolemma ("coupled" Ca(2+) sparks) revealing the sequential activation of Ca(2+) release sites of the j-SR. Moreover, during global [Ca(2+)](i) waves, Ca(2+) release was evident from individual nj-SR sites. Ca(2+) release sites were arranged in a regular three-dimensional grid as deduced from the functional data and shown by immunostaining of ryanodine receptor Ca(2+) release channels. The longitudinal and transverse distances between individual Ca(2+) release sites were both approximately 2 microm. Furthermore, electron microscopy revealed a continuous sarcotubular network and one peripheral coupling of j-SR with the sarcolemma per sarcomere. The results demonstrate directly that, in cat atrial myocytes, the action potential-induced whole-cell [Ca(2+)](i) transient is the spatio-temporal summation of Ca(2+) release from subsarcolemmal and central sites. First, j-SR sites are activated in a stochastic fashion by the opening of voltage-dependent sarcolemmal Ca(2+) channels. Subsequently, nj-SR sites are activated by Ca(2+)-induced Ca(2+) release propagating from the periphery.

In cardiac myocytes the type-2 inositol 1,4,5-trisphosphate receptor (IP 3 R2) is the predominant isoform expressed. The IP 3 R2 channel is localized to the SR and to the nuclear envelope. We studied IP stores by approximately 60% relative to the maximum depletion produced by the ionophores ionomycin and A23187. The fluo-5N fluorescence decrease was particularly pronounced in the nuclear periphery, suggesting that the nuclear envelope may represent the predominant nuclear Ca 2+ store. The data indicate that IP 3 can elicit Ca 2+ release from cardiac nuclei resulting in localized nuclear Ca 2+ signals.

A full-length rat type 2 inositol 1,4,5-trisphosphate (InsP(3)) receptor cDNA construct was generated and expressed in COS-1 cells. Targeting of the full-length recombinant type 2 receptor protein to the endoplasmic reticulum was confirmed by immunocytochemistry using isoform specific affinity-purified antibodies and InsP(3)R-green fluorescent protein chimeras. The receptor protein was solubilized and incorporated into proteoliposomes for functional characterization. Single-channel recordings from proteoliposomes fused into planar lipid bilayers revealed that the recombinant protein formed InsP(3)- and Ca(2+)-sensitive ion channels. The unitary conductance ( approximately 250 pS; 220/20 mM Cs(+) as charge carrier), gating, InsP(3), and Ca(2+) sensitivities were similar to those previously described for the native type 2 InsP(3)R channel. However, the maximum open probability of the recombinant channel was slightly lower than that of its native counterpart. These data show that our full-length rat type 2 InsP(3)R cDNA construct encodes a protein that forms an ion channel with functional attributes like those of the native type 2 InsP(3)R channel. The possibility of measuring the function of single recombinant type 2 InsP(3)R is a significant step toward the use of molecular tools to define the determinants of isoform-specific InsP(3)R function and regulation.

The three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (InsP 3 R1) has been determined by electron cryomicroscopy and single-particle reconstruction. The receptor was immunoaffinity-purified and formed functional InsP 3 -and heparin-sensitive channels with a unitary conductance similar to native InsP 3 Rs. The channel structure exhibits the expected 4-fold symmetry and comprises two morphologically distinct regions: a large pinwheel and a smaller square. The pinwheel region has four radial curved spokes interconnected by a central core. The InsP 3 -binding core domain has been localized within each spoke of the pinwheel region by fitting its x-ray structure into our reconstruction. A structural mapping of the amino acid sequences to several functional domains is deduced within the structure of the InsP 3 R1 tetramer.The inositol 1,4,5-trisphosphate receptor (InsP 3 R) 1 is a ligand-gated ion channel that modulates Ca 2ϩ release from intracellular stores. The InsP 3 R is widely distributed in metazoans and plays a key role in diverse physiological functions. In mammals, three different InsP 3 R isoforms are expressed; each is encoded by a distinct gene. The three isoforms are homologous and share ϳ70% amino acid identity. Functional channels are composed of four subunits that tetramerize via determinants localized within the transmembrane regions and the cytosolic C terminus (1-3). Individual cell types often express more than one isoform, which may be present as homo-or heterotetrameric populations. The most characterized type 1 InsP 3 R (InsP 3 R1) forms largely homotetramers in cerebellum with M r of 313,000 (2749 residues) for the monomer. Based on biochemical, molecular biological, and electrophysiological studies, each monomer subunit of the InsP 3 R1 is organized into three principal functional regions: an N-terminal InsP 3 -binding region (residues 226 -578), a central (ϳ1,700-residue) modulatory/coupling region, and a channel-forming region near the C terminus. The channel region contains six transmembrane segments interspersed within residues 2276 -2590 that are critical for membrane targeting, oligomerization, and formation of the ion permeation pathway (1-4). Thus, both ends, the large N-terminal region (residues 1-2275) and the C-terminal region (residues 2590 -2749), are exposed to the cytoplasm.Electron micrographs of the negatively stained detergentsolubilized InsP 3 R1 and freeze-fracture deep etching electron microscopy of endoplasmic reticulum have been used to characterize the morphology of the InsP 3 R1 channel (5, 6). But the three-dimensional shape and dimensions of the channel particles were not determined. Two three-dimensional maps of the InsP 3 R1 were reported recently based on single-particle reconstruction from specimens prepared by ice embedding (7) and negative staining (8). There is a poor agreement between these two maps with respect to the overall appearance and dimensions of the channel structure. In this study, we present a 30-Å three-dimensional s...

Astrocytes respond to neuronal activity by propagating Ca(2+) waves elicited through the inositol 1,4,5-trisphosphate pathway. We have previously shown that wave propagation is supported by specialized Ca(2+) release sites, where a number of proteins, including inositol 1,4,5-trisphosphate receptors (IP(3)R), occur together in patches. The specific IP(3)R isoform expressed by astrocytes in situ in rat brain is unknown. In the present report, we use isoform-specific antibodies to localize immunohistochemically the IP(3)R subtype expressed in astrocytes in rat brain sections. Astrocytes were identified using antibodies against the astrocyte-specific markers, S-100 beta, or GFAP. Dual indirect immunohistochemistry showed that astrocytes in all regions of adult rat brain express only IP(3)R2. High-resolution analysis showed that hippocampal astrocytes are endowed with a highly branched network of processes that bear fine hair-like extensions containing punctate patches of IP(3)R2 staining in intimate contact with synapses. Such an organization is reminiscent of signaling microdomains found in cultured glial cells. Similarly, Bergmann glial cell processes in the cerebellum also contained fine hair-like processes containing IP(3)R2 staining. The IP(3)R2-containing fine terminal branches of astrocyte processes in both brain regions were found juxtaposed to presynaptic terminals containing synaptophysin as well as PSD 95-containing postsynaptic densities. Corpus callosum astrocytes had an elongated morphology with IP(3)R2 studded processes extending along fiber tracts. Our data suggest that PLC-mediated Ca(2+) signaling in astrocytes in rat brain occurs predominantly through IP(3)R2 ion channels. Furthermore, the anatomical arrangement of the terminal astrocytic branches containing IP(3)R2 ensheathing synapses is ideal for supporting glial monitoring of neuronal activity.