Josefina Ramos-Franco

Isoform-specific function of single inositol 1,4,5-trisphosphate receptor channels.

The inositol 1,4,5-trisphosphate receptor (InsP3R) family of Ca2+ release channels is central to intracellular Ca2+ signaling in mammalian cells. The InsP3R channels release Ca2+ from intracellular compartments to generate localized Ca2+ transients that govern a myriad of cellular signaling phenomena (Berridge, 1993. Nature. 361:315-325; Joseph, 1996. Cell Signal. 8:1-7; Kume et al., 1997. Science. 278:1940-1943; Berridge, 1997. Nature. 368:759-760). express multiple InsP3R isoforms, but only the function of the single type 1 InsP3R channel is known. Here the single-channel function of single type 2 InsP3R channel is defined for the first time. The type 2 InsP3R forms channels with permeation properties similar to that of the type 1 receptor. The InsP3 regulation and Ca2+ regulation of type 1 and type 2 InsP3R channels are strikingly different. Both InsP3 and Ca2+ are more effective at activating single type 2 InsP3R, indicating that single type 2 channels mobilize substantially more Ca2+ than single type 1 channels in cells. Furthermore, high cytoplasmic Ca2+ concentrations inactivate type 1, but not type 2, InsP3R channels. This indicates that type 2 InsP3R channel is different from the type 1 channel in that its activity will not be inherently self-limiting, because Ca2+ passing through an active type 2 channel cannot feed back and turn the channel off. Thus the InsP3R identity will help define the spatial and temporal nature of local Ca2+ signaling events and may contribute to the segregation of parallel InsP3 signaling cascades in mammalian cells.

Single-Channel Function of Recombinant Type 2 Inositol 1,4,5-Trisphosphate Receptor

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.

Single Channel Function of Recombinant Type-1 Inositol 1,4,5-Trisphosphate Receptor Ligand Binding Domain Splice Variants

In this study we describe the expression and function of the two rat type-1 inositol 1,4,5-trisphosphate receptor (InsP3R) ligand binding domain splice variants (SI+/-/SII+). Receptor protein from COS-1 cells transfected with the type-1 InsP3R expression plasmids (pInsP3R-T1, pInsP3R-T1ALT) or control DNA were incorporated into planar lipid bilayers and the single channel properties of the recombinant receptors were defined. The unitary conductance of the two splice variants were approximately 290 pS with Cs+ as charge carrier and approximately 65 pS with Ca2+ as charge carrier. Both InsP3R expression products consistently behaved like those of the native type-1 receptor isoform isolated from cerebellum in terms of their InsP3, Ca2+, and heparin sensitivity. An InsP3 receptor ligand binding domain truncation lacking the 310 amino-terminal amino acids (pInsP3R-DeltaT1ALT) formed tetrameric complexes but failed to bind InsP3 with high affinity, and did not form functional Ca2+ channels when reconstituted in lipid bilayers. These data suggest that 1) the ligand binding alternative splice site is functionally inert in terms of InsP3 binding and single channel function, and 2) the single channel properties of the expressed recombinant type-1 channel are essentially identical to those of the native channel. This work establishes a foundation from which molecular/biophysical approaches can be used to define the structure-function properties of the InsP3 receptor channel family.

Single ryanodine receptor channel basis of caffeine's action on Ca2+ sparks.

Caffeine (1, 3, 7-trimethylxanthine) is a widely used pharmacological agonist of the cardiac ryanodine receptor (RyR2) Ca(2+) release channel. It is also a well-known stimulant that can produce adverse side effects, including arrhythmias. Here, the action of caffeine on single RyR2 channels in bilayers and Ca(2+) sparks in permeabilized ventricular cardiomyocytes is defined. Single RyR2 caffeine activation depended on the free Ca(2+) level on both sides of the channel. Cytosolic Ca(2+) enhanced RyR2 caffeine affinity, whereas luminal Ca(2+) essentially scaled maximal caffeine activation. Caffeine activated single RyR2 channels in diastolic quasi-cell-like solutions (cytosolic MgATP, pCa 7) with an EC(50) of 9.0 ± 0.4 mM. Low-dose caffeine (0.15 mM) increased Ca(2+) spark frequency ∼75% and single RyR2 opening frequency ∼150%. This implies that not all spontaneous RyR2 openings during diastole are associated with Ca(2+) sparks. Assuming that only the longest openings evoke sparks, our data suggest that a spark may result only when a spontaneous single RyR2 opening lasts >6 ms.

Ryanodine Receptor Luminal Ca2+ Regulation: Swapping Calsequestrin and Channel Isoforms

Sarcoplasmic reticulum (SR) Ca(2+) release in striated muscle is mediated by a multiprotein complex that includes the ryanodine receptor (RyR) Ca(2+) channel and the intra-SR Ca(2+) buffering protein calsequestrin (CSQ). Besides its buffering role, CSQ is thought to regulate RyR channel function. Here, CSQ-dependent luminal Ca(2+) regulation of skeletal (RyR1) and cardiac (RyR2) channels is explored. Skeletal (CSQ1) or cardiac (CSQ2) calsequestrin were systematically added to the luminal side of single RyR1 or RyR2 channels. The luminal Ca(2+) dependence of open probability (Po) over the physiologically relevant range (0.05-1 mM Ca(2+)) was defined for each of the four RyR/CSQ isoform pairings. We found that the luminal Ca(2+) sensitivity of single RyR2 channels was substantial when either CSQ isoform was present. In contrast, no significant luminal Ca(2+) sensitivity of single RyR1 channels was detected in the presence of either CSQ isoform. We conclude that CSQ-dependent luminal Ca(2+) regulation of single RyR2 channels lacks CSQ isoform specificity, and that CSQ-dependent luminal Ca(2+) regulation in skeletal muscle likely plays a relatively minor (if any) role in regulating the RyR1 channel activity, indicating that the chief role of CSQ1 in this tissue is as an intra-SR Ca(2+) buffer.

Role of inositol 1, 4, 5-trisphosphate in the regulation of ventricular Ca2+ signaling in intact mouse heart

Inositol 1,4,5-trisphosphate (InsP(3)R)-mediated Ca(2+) signaling is a major pathway regulating multiple cellular functions in excitable and non-excitable cells. Although InsP(3)-mediated Ca(2+) signaling has been extensively described, its influence on ventricular myocardium activity has not been addressed in contracting hearts at the whole-organ level. In this work, InsP(3)-sensitive intracellular Ca(2+) signals were studied in intact hearts using laser scanning confocal microscopy and pulsed local-field fluorescence microscopy. Intracellular [InsP(3)] was rapidly increased by UV flash photolysis of membrane-permeant caged InsP(3). Our results indicate that the basal [Ca(2+)] increased after the flash photolysis of caged InsP(3) without affecting the action potential (AP)-induced Ca(2+) transients. The amplitude of the basal [Ca(2+)] elevation depended on the intracellular [InsP(3)] reached after the UV flash. Pretreatment with ryanodine failed to abolish the InsP(3)-induced Ca(2+) release (IICR), indicating that this response was not mediated by ryanodine receptors (RyR). Thapsigargin prevented Ca(2+) release from both RyR- and InsP(3)R-containing Ca(2+) stores, suggesting that these pools have similar Ca(2+) reuptake mechanisms. These results were reproduced in acutely isolated cells where photorelease of InsP(3) was able to induce changes in endothelial cells but not in AP-induced transients from cardiomyocytes. Taken together, these results suggest that IICR does not directly regulate cardiac excitation-contraction coupling. To our knowledge, this is the first demonstration of IICR in intact hearts. Consequently, our work provides a reference framework of the spatiotemporal attributes of the IICR under physiological conditions.

Ca2+ sparks and cellular distribution of ryanodine receptors in developing cardiomyocytes from rat

Although abundant ryanodine receptors (RyRs) exist in cardiomyocytes from newborn (NB) rat and despite the maturity of their single-channel properties, the RyR contribution to excitation-contraction (E-C) coupling is minimal. Immature arrangement of RyRs in the Ca(2+) release site of the sarcoplasmic reticulum and/or distant RyRs location from the sarcolemmal Ca(2+) signal could explain this quiescence. Consequently, Ca(2+) sparks and their cellular distribution were studied in NB myocytes and correlated with the formation of dyads and transverse (T) tubules. Ca(2+) sparks were recorded in fluo-4-loaded intact ventricular myocytes acutely dissociated from adult and NB rats (0-9 days old). Sparks were defined/compared in the center and periphery of the cell. Co-immunolocalization of RyRs with dihydropyridine receptors (DHPR) was used to estimate dyad formation, while the development of T tubules was studied using di-8-ANEPPS and diIC12. Our results indicate that in NB cells, Ca(2+) sparks exhibited lower amplitude (1.7+/-0.5 vs. 3.6+/-1.7 F/F(0)), shorter duration (47+/-3.2 vs. 54.1+/-3 ms), and larger width (1.7+/-0.8 vs. 1.2+/-0.4 microm) than in adult. Although no significant changes were observed in the overall frequency, central sparks increased from approximately 60% at 0-1 day to 82% at 7-9 days. While immunolocalization revealed many central release sites at 7-8 days, fluorescence labeling of the plasma membrane showed less abundant internal T tubules. This could imply that although during the first week, release sites emerge forming dyads with DHPR-containing T tubules; some of these T tubules may not be connected to the surface, explaining the RyR quiescence during E-C coupling in NB.

Single channel function of inositol 1,4,5-trisphosphate receptor type-1 and -2 isoform domain-swap chimeras

The InsP3R proteins have three recognized domains, the InsP3-binding, regulatory/coupling, and channel domains (Mignery, G.A., and T.C. Südhof. 1990. EMBO J. 9:3893–3898). The InsP3 binding domain and the channel-forming domain are at opposite ends of the protein. Ligand regulation of the channel must involve communication between these different regions of the protein. This communication likely involves the interceding sequence (i.e., the regulatory/coupling domain). The single channel functional attributes of the full-length recombinant type-1, -2, and -3 InsP3R channels have been defined. Here, two type-1/type-2 InsP3R regulatory/coupling domain chimeras were created and their single channel function defined. One chimera (1-2-1) contained the type-2 regulatory/coupling domain in a type-1 backbone. The other chimera (2-1-2) contained the type-1 regulatory/coupling domain in a type-2 backbone. These chimeric proteins were expressed in COS cells, isolated, and then reconstituted in proteoliposomes. The proteoliposomes were incorporated into artificial planar lipid bilayers and the single-channel function of the chimeras defined. The chimeras had permeation properties like that of wild-type channels. The ligand regulatory properties of the chimeras were altered. The InsP3 and Ca2+ regulation had some unique features but also had features in common with wild-type channels. These results suggest that different independent structural determinants govern InsP3R permeation and ligand regulation. It also suggests that ligand regulation is a multideterminant process that involves several different regions of the protein. This study also demonstrates that a chimera approach can be applied to define InsP3R structure-function.

Platelet-activating factor receptor-dependent activation of the muscarinic K+ current in bullfrog atrial myocytes.

Platelet-activating factor (PAF), a potent signaling lipid implicated as a mediator of pathological responses, has both negative chronotropic and inotropic effects on the heart, although the mechanism(s) involved is not well defined. Because activation of the muscarinic acetylcholine-activated K+ current (IK(ACh)) also produces a negative chronotropic and inotropic response in myocardium, this study examines whether PAF has effects on IK(ACh) in isolated bullfrog atrial myocytes under whole-cell voltage-clamp conditions. We find that 2 microM PAF increases the rate of GTP-gamma-S-mediated IK(ACh) activation (from 0.30 +/- 0.01 min-1 [n = 20] to 0.73 +/- 0.07 min-1 [n = 12], p < 0.005, in the absence of acetylcholine). This effect of 2 microM PAF was blocked by the PAF antagonist CV-3988 (5 microM, 0.33 +/- 0.14 min-1 [n = 12]), suggesting the presence of specific PAF receptors coupled to IK(ACh) activation. Further support for mediation by specific G protein-coupled PAF receptors derives from the inability of PAF to modulate IK(ACh) after maximal activation in the presence of GTP-gamma-S. Eicosatetraynoic acid (ETYA, an inhibitor of 5- and 12-lipoxygenases) did not prevent the PAF-mediated increase in the rate of IK(ACh) activation (10 microM ETYA, 0.28 +/- 0.03 min-1 [n = 7]; 10 microM ETYA plus 2 microM PAF, 0.58 +/- 0.13 min-1 [n = 8]; p < 0.05), suggesting that the observed PAF effect is not mediated by increases in arachidonic acid metabolism.(ABSTRACT TRUNCATED AT 250 WORDS)

Intact Heart Loose Patch Photolysis Reveals Ionic Current Kinetics During Ventricular Action Potentials

RATIONALE Assessing the underlying ionic currents during a triggered action potential (AP) in intact perfused hearts offers the opportunity to link molecular mechanisms with pathophysiological problems in cardiovascular research. The developed loose patch photolysis technique can provide striking new insights into cardiac function at the whole heart level during health and disease. OBJECTIVE To measure transmembrane ionic currents during an AP to determine how and when surface Ca(2+) influx that triggers Ca(2+)-induced Ca(2+) release occurs and how Ca(2+)-activated conductances can contribute to the genesis of AP phase 2. METHODS AND RESULTS Loose patch photolysis allows the measurement of transmembrane ionic currents in intact hearts. During a triggered AP, a voltage-dependent Ca(2+) conductance was fractionally activated (dis-inhibited) by rapidly photo-degrading nifedipine, the Ca(2+) channel blocker. The ionic currents during a mouse ventricular AP showed a fast early component and a slower late component. Pharmacological studies established that the molecular basis underlying the early component was driven by an influx of Ca(2+) through the L-type channel, CaV 1.2. The late component was identified as an Na(+)-Ca(2+) exchanger current mediated by Ca(2+) released from the sarcoplasmic reticulum. CONCLUSIONS The novel loose patch photolysis technique allowed the dissection of transmembrane ionic currents in the intact heart. We were able to determine that during an AP, L-type Ca(2+) current contributes to phase 1, whereas Na(+)-Ca(2+) exchanger contributes to phase 2. In addition, loose patch photolysis revealed that the influx of Ca(2+) through L-type Ca(2+) channels terminates because of voltage-dependent deactivation and not by Ca(2+)-dependent inactivation, as commonly believed.