Monovalent Cations Contribute to T-type Calcium Channel (Ca<Subscript>V</Subscript>3.1 and Ca<Subscript>V</Subscript>3.2) Selectivity

Low voltage-activated (LVA) Ca²⁺ channels regulate chemical signaling by their ability to select for Ca²⁺. Whereas Ca²⁺ is the main permeating species through Ca²⁺ channels, Ca²⁺ permeation may be modified by abundant intra- and extracellular monovalent cations. Therefore, we explored monovalent cation regulation of LVA Ca²⁺ permeation in the cloned T-type Ca²⁺ channels 1G (Ca_V3.1) and 1H (Ca_V3.2). In physiological [Ca²⁺], the reversal potential in symmetrical Li⁺ was 19 mV in 1G and 18 mV in 1H, in symmetrical Cs⁺ the reversal potential was 36 mV in 1G and 37 mV in 1H, and in the bi-ionic condition with Li⁺ in the bath and Cs⁺ in the pipette, the reversal potential was 46 mV in both 1G and 1H. When Cs⁺ was used in the pipette, replacement of external Cs⁺ with Li⁺ (or Na⁺) shifted the reversal potential positive by 5–6 mV and increased the net inward current in 1G. Taken together the data indicate that in physiological [Ca²⁺], external Li⁺ (or Na⁺) permeates more readily than external Cs⁺, resulting in a positive shift of the reversal potential. We conclude that external monovalent cations dictate T-type Ca²⁺ channel selectivity by permeating through the channel. Similar to Li⁺, we previously reported that external [H⁺] can regulate T-type Ca²⁺ channel selectivity. 1Hs selectivity was more sensitive to external pH changes compared to 1G. When Cs⁺ was used in the pipette and Li⁺ was used in the bath external acidification from pH_o 7.4 to 6.0 caused a negative shift of the reversal by 8 mV in 1H. Replacement of internal Cs⁺ with Li⁺ reduced the pH-induced shift of the reversal potential to 2 mV. We conclude that, similar to other external monovalent cations, H⁺ can modify T-type Ca²⁺ channel selectivity. However, in contrast to external monovalent ions that readily permeate, H⁺ regulate T-type Ca²⁺ channel selectivity by increasing the relative permeability of the internal monovalent cation. Present address: B.P. Delisle, Department of Medicine, The University of Wisconsin, Madison, WI 53706, USA     

The mechano-gated K<Subscript>2P</Subscript> channel TREK-1

The versatility of neuronal electrical activity is largely conditioned by the expression of different structural and functional classes of K⁺ channels. More than 80 genes encoding the main K⁺ channel alpha subunits have been identified in the human genome. Alternative splicing, heteromultimeric assembly, post-translational modification and interaction with auxiliary regulatory subunits further increase the molecular and functional diversity of K⁺ channels. Mammalian two-pore domain K⁺ channels (K_2P) make up one class of K⁺ channels along with the inward rectifiers and the voltage- and/or calcium-dependent K⁺ channels. Each K_2P channel subunit is made up of four transmembrane segments and two pore-forming (P) domains, which are arranged in tandem and function as either homo- or heterodimeric channels. This novel structural arrangement is associated with unusual gating properties including “background” or “leak” K⁺ channel activity, in which the channels show constitutive activity at rest. In this review article, we will focus on the lipid-sensitive mechano-gated K_2P channel TREK-1 and will emphasize on the polymodal function of this “unconventional” K⁺ channel. EBSA Satellite meeting: Ion channels, Leeds, July 2007.     

Modulation of synaptic potentials and cell excitability by dendritic K<Subscript>IR</Subscript> and K<Subscript>As</Subscript> channels in nucleus accumbens medium spiny neurons: A computational study

The nucleus accumbens (NAc), a critical structure of the brain reward circuit, is implicated in normal goal-directed behaviour and learning as well as pathological conditions like schizophrenia and addiction. Its major cellular substrates, the medium spiny (MS) neurons, possess a wide variety of dendritic active conductances that may modulate the excitatory post synaptic potentials (EPSPs) and cell excitability. We examine this issue using a biophysically detailed 189-compartment stylized model of the NAc MS neuron, incorporating all the known active conductances. We find that, of all the active channels, inward rectifying K⁺ (K_IR) channels play the primary role in modulating the resting membrane potential (RMP) and EPSPs in the down-state of the neuron. Reduction in the conductance of K_IR channels evokes facilitatory effects on EPSPs accompanied by rises in local input resistance and membrane time constant. At depolarized membrane potentials closer to up-state levels, the slowly inactivating A-type potassium channel (K_As) conductance also plays a strong role in determining synaptic potential parameters and cell excitability. We discuss the implications of our results for the regulation of accumbal MS neuron biophysics and synaptic integration by intrinsic factors and extrinsic agents such as dopamine.     

Determinant Role of Membrane Helices in K<Subscript>ATP</Subscript> Channel Gating

The ATP-sensitive K⁺ (K_ATP) channels couple chemical signals to cellular activity, in which the control of channel opening and closure (i.e., channel gating) is crucial. Transmembrane helices play an important role in channel gating. Here we report that the gating of Kir6.2, the core subunit of pancreatic and cardiac K_ATP channels, can be switched by manipulating the interaction between two residues located in transmembrane domains (TM) 1 and 2 of the channel protein. The Kir6.2 channel is gated by ATP and proton, which inhibit and activate the channel, respectively. The channel gating involves two residues, namely, Thr71 and Cys166, located at the interface of the TM1 and TM2. Creation of electrostatic attraction between these sites reverses the channel gating, which makes the ATP an activator and proton an inhibitor of the channel. Electrostatic repulsion with two acidic residues retains or even enhances the wild-type channel gating. A similar switch of the pH-dependent channel gating was observed in the Kir2.1 channel, which is normally pH- insensitive. Thus, the manner in which the TM1 and TM2 helices interact appears to determine whether the channels are open or closed following ligand binding.^*These authors contributed equally to this work.     

Characteristics of Omega-conotoxin GVI A and MVIIC Binding to Ca<Subscript>v</Subscript> 2.1 and Ca<Subscript>v</Subscript> 2.2 Channels Captured by Anti-Ca<Superscript>2+</Superscript> Channel Peptide Antibodies

A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of ¹²⁵I-omega-conotoxin (ω-CTX) GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Ca_v2.1 and Ca_v2.2 channels). The results for the NBM were as follows. (1) The ED₅₀ values for specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels were about 68 and 60 pM, respectively, and very similar to those (87 and 35 pM, respectively) to crude membranes from chick brain. (2) The specific ¹²⁵I-ω-CTX GVIA (100 pM) binding was inhibited by ω-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 μM each), but not by ω-agaconotoxin (Aga) IVA, calciseptine, ω-CTX SVIB, ω-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 μM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC₅₀ value of about 100 μg protein/ml). (3) The specific ¹²⁵I-ω-CTX MVIIC (60 pM) binding was inhibited by ω-CTX MVIIC, ω-CTX GVIA, ω-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 μM, each), but not by ω-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 μM each) or 100 μg protein/ml CaM. These results suggested that the characteristics of the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels in the NBM were very similar to those to crude membranes from chick brain, although the IC₅₀ values for CaM and free Ca²⁺ of CaM were about 33- and 5000-fold higher, respectively, than those for the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to crude membranes.     

Thiamine Deficiency Increases Ca<Superscript>2+</Superscript> Current and Ca<Subscript>V</Subscript>1.2 L-type Ca<Superscript>2+</Superscript> Channel Levels in Cerebellum Granular Neurons

Thiamine (vitamin B1) is co-factor for three pivotal enzymes for glycolytic metabolism: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase. Thiamine deficiency leads to neurodegeneration of several brain regions, especially the cerebellum. In addition, several neurodegenerative diseases are associated with impairments of glycolytic metabolism, including Alzheimer’s disease. Therefore, understanding the link between dysfunction of the glycolytic pathway and neuronal death will be an important step to comprehend the mechanism and progression of neuronal degeneration as well as the development of new treatment for neurodegenerative states. Here, using an in vitro model to study the effects of thiamine deficiency on cerebellum granule neurons, we show an increase in Ca²⁺ current density and Ca_V1.2 expression. These results indicate a link between alterations in glycolytic metabolism and changes to Ca²⁺ dynamics, two factors that have been implicated in neurodegeneration.     

A Target Repurposing Approach Identifies N-myristoyltransferase as a New Candidate Drug Target in Filarial Nematodes

Myristoylation is a lipid modification involving the addition of a 14-carbon unsaturated fatty acid, myristic acid, to the N-terminal glycine of a subset of proteins, a modification that promotes their binding to cell membranes for varied biological functions. The process is catalyzed by myristoyl-CoA:protein N-myristoyltransferase (NMT), an enzyme which has been validated as a drug target in human cancers, and for infectious diseases caused by fungi, viruses and protozoan parasites. We purified Caenorhabditis elegans and Brugia malayi NMTs as active recombinant proteins and carried out kinetic analyses with their essential fatty acid donor, myristoyl-CoA and peptide substrates. Biochemical and structural analyses both revealed that the nematode enzymes are canonical NMTs, sharing a high degree of conservation with protozoan NMT enzymes. Inhibitory compounds that target NMT in protozoan species inhibited the nematode NMTs with IC₅₀ values of 2.5–10 nM, and were active against B. malayi microfilariae and adult worms at 12.5 µM and 50 µM respectively, and C. elegans (25 µM) in culture. RNA interference and gene deletion in C. elegans further showed that NMT is essential for nematode viability. The effects observed are likely due to disruption of the function of several downstream target proteins. Potential substrates of NMT in B. malayi are predicted using bioinformatic analysis. Our genetic and chemical studies highlight the importance of myristoylation in the synthesis of functional proteins in nematodes and have shown for the first time that NMT is required for viability in parasitic nematodes. These results suggest that targeting NMT could be a valid approach for the development of chemotherapeutic agents against nematode diseases including filariasis.     

In vitro fluorescence assay to study the folding of K<Subscript>v</Subscript> ion channels

A fluorescence assay to check the folding of potassium K_v channels expressed in vitro has been developed. For this aim, the fluorescently labeled channel blocker, recombinant agitoxin of yellow scorpion was employed. The level of expression of various K_v channels in vitro has been tested. It has been demonstrated that K_v2 channels form clusters on the cell surface, which are not associated with actin filaments. On the other hand, K_v10 channels form larger clusters, which are associated with actin, indicating the principal differences in the organization of cytoplasmic domains of K_v2 and K_v10 channels.     

K<Subscript>ATP</Subscript> channel polymorphism is associated with left ventricular size in hypertensive individuals: a large-scale community-based study

ATP-sensitive K⁺ (K_ATP) channel mutations have been identified in individuals with dilated cardiomyopathy and overt heart failure. Here, a common E23K functional polymorphism in the Kir6.2 channel pore versus cardiac phenotype was studied in a cross-sectional community-based cohort (n = 2,031). The KK genotype was associated with greater left ventricular size among subjects with increased stress load due to hypertension. These findings implicate Kir6.2 K23 as a risk factor for adverse subclinical myocardial remodeling, and underscore the significance of cardiac K_ATP channels within the population.     

The influence of Sc doping on structural,electronic and optical properties of Be<Subscript>12</Subscript>O<Subscript>12</Subscript>, Mg<Subscript>12</Subscript>O<Subscript>12</Subscript> and Ca<Subscript>12</Subscript>O<Subscript>12</Subscript> nanocages: a DFT study

Density functional theory (DFT) calculations were used to study the effect of scandium doping on the structural, energetic, electronic, linear and nonlinear optical (NLO) properties of Be₁₂O₁₂, Mg₁₂O₁₂ and Ca₁₂O₁₂ nanoclusters. Scandium (Sc) doping on nanoclusters leads to narrowing of their E _g, which enhances their conductance greatly. Also, the polarizability (α) and first hyperpolarizability (β₀) of nanoclusters were dramatically increased as Be, Mg or Ca atoms are substituted with a Sc atom. Among all clusters, α and β₀ values for Sc-doped Ca₁₂O₁₂ were the largest. Consequently, the effect of the doping atom, as well as of cluster size, on electronic and optical properties was explored. Time dependent (TD)-DFT calculations were also carried out to confirm the β₀ values; the results show that the higher value of first hyperpolarizability belongs to Sc-doped Ca₁₂O₁₂, which has the smallest transition energy (ΔEgn). The results obtained show that these clusters can be candidates for using in electronic devices and NLO materials in industry.     

Mitochondrial K<Subscript>ATP</Subscript> channels-derived reactive oxygen species activate pro-survival pathway in pravastatin-induced cardioprotection

Reactive oxygen species (ROS) are important intracellular signaling molecules and are implicated in cardioprotective pathways including ischemic preconditioning. Statins have been shown to have cardioprotective effects against ischemia/reperfusion injury, however, the precise mechanisms remain to be elucidated. We hypothesized that ROS-mediated signaling cascade may be involved in pravastatin-induced cardioprotection. Cultured rat cardiomyocytes were exposed to H₂O₂ for 30 min to induce cell injury. Pravastatin significantly suppressed H₂O₂-induced cell death evaluated by propidium iodide staining and the MTT assay. Incubation with pravastatin activated catalase, and prevented a ROS burst induced by H₂O₂, which preserved mitochondrial membrane potential. Protective effects were induced very rapidly within 10 min, which was concordant with the up-regulation of phosphorylated ERK1/2. L-NAME, 5HD, N-acetylcysteine (NAC) and staurosporine inhibited ERK1/2 phosphorylation and also reduced pravastatin-induced cardioprotection, suggesting NO, mitochondrial K_ATP (mitoK_ATP) channels, ROS and PKC should be involved in the cardioprotective signaling. We also demonstrated that pravastatin moderately up-regulated ROS generation in a 5HD-inhibitable manner. In isolated perfused rat heart experiments, pravastatin administered 10 min prior to no-flow global ischemia significantly improved left ventricular functional recovery, and also reduced infarct size, which were attenuated by the treatment with NAC, 5HD, L-NAME or staurosporine. Administration of pravastatin from the beginning of reperfusion also conferred cardioprotection. Pravastatin protected the cardiomyocytes against oxidative stress by preventing the ROS burst and preserving mitochondrial function. Moderately up-regulated ROS production by mitoK_ATP channels opening is involved in the pro-survival signaling cascade activated by pravastatin.     

Ca<Subscript>v</Subscript>1.3 and BK Channels for Timing and Regulating Cell Firing

L-type Ca²⁺ channels (LTCCs, Ca_v1) open readily during membrane depolarization and allow Ca²⁺ to enter the cell. In this way, LTCCs regulate cell excitability and trigger a variety of Ca²⁺-dependent physiological processes such as: excitation–contraction coupling in muscle cells, gene expression, synaptic plasticity, neuronal differentiation, hormone secretion, and pacemaker activity in heart, neurons, and endocrine cells. Among the two major isoforms of LTCCs expressed in excitable tissues (Ca_v1.2 and Ca_v1.3), Ca_v1.3 appears suitable for supporting a pacemaker current in spontaneously firing cells. It has steep voltage dependence and low threshold of activation and inactivates slowly. Using Ca_v1.3^−/− KO mice and membrane current recording techniques such as the dynamic and the action potential clamp, it has been possible to resolve the time course of Ca_v1.3 pacemaker currents that regulate the spontaneous firing of dopaminergic neurons and adrenal chromaffin cells. In several cell types, Ca_v1.3 is selectively coupled to BK channels within membrane nanodomains and controls both the firing frequency and the action potential repolarization phase. Here we review the most critical aspects of Ca_v1.3 channel gating and its coupling to large conductance BK channels recently discovered in spontaneously firing neurons and neuroendocrine cells with the aim of furnishing a converging view of the role that these two channel types play in the regulation of cell excitability.     

Cross-talks between Ca<Superscript>2+</Superscript>/CaM and H<Subscript>2</Subscript>O<Subscript>2</Subscript> in abscisic acid-induced antioxidant defense in leaves of maize plants exposed to water stress

Here we examined whether Ca²⁺/Calmodulin (CaM) is involved in abscisic acid (ABA)-induced antioxidant defense and the possible relationship between CaM and H₂O₂ in ABA signaling in leaves of maize (Zea mays L.) plants exposed to water stress. An ABA-deficient mutant vp5 and its wild type were used for the experimentation. We found that water stress enhanced significantly the contents of CaM and H₂O₂, and the activities of chloroplastic and cytosolic superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), and the gene expressions of the CaM1, cAPX, GR1 and SOD4 in leaves of wild-type maize. However, the increases mentioned above were almost arrested in vp5 plants and in the wild-type plants pretreated with ABA biosynthesis inhibitor tungstate (T), suggesting that ABA is required for water stress-induced H₂O₂ production, the enhancement of CaM content and antioxidant defense. Besides, we showed that the up-regulation of water stress-induced antioxidant defense was almost completely blocked by pretreatment with Ca²⁺ inhibitors, CaM antagonists and reactive oxygen (ROS) manipulators. Moreover, the analysis of time course of CaM and H₂O₂ production under water stress showed that the increase in CaM content preceded that of H₂O₂. These results suggested that Ca²⁺/CaM and H₂O₂ were involved in the ABA-induced antioxidant defense under water stress, and the increases of Ca²⁺/CaM contents triggered H₂O₂ production, which inversely affected the contents of CaM. Thus, a cross-talk between Ca²⁺/CaM and H₂O₂ may play a pivotal role in the ABA signaling.     

Ca<Superscript>2+</Superscript> and CaM are Involved in NO- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Adventitious Root Development in Marigold

Our previous results have demonstrated that both nitric oxide (NO) and hydrogen peroxide (H₂O₂) are involved in the promotion of adventitious root development in marigold (Tagetes erecta L.). However, not much is known about the intricate molecular network of adventitious root development triggered by NO and H₂O₂. In this study, the involvement of calcium (Ca²⁺) and calmodulin (CaM) in NO- and H₂O₂-induced adventitious rooting in marigold was investigated. Exogenous Ca²⁺ was capable of promoting adventitious rooting, with a maximal biological response at 50 μM CaCl₂. Ca²⁺ chelators and CaM antagonists prevented NO- and H₂O₂-induced adventitious rooting, indicating that both endogenous Ca²⁺ and CaM may play crucial roles in the adventitious rooting induced by NO and H₂O₂. NO and H₂O₂ treatments increased the endogenous content of Ca²⁺ and CaM, suggesting that NO and H₂O₂ enhanced adventitious rooting by stimulating the endogenous Ca²⁺ and CaM levels. Moreover, treatment with Ca²⁺ enhanced the endogenous levels of NO and H₂O₂. Additionally, Ca²⁺ might be involved as an upstream signaling molecule for CaM during NO- and H₂O₂-induced rooting. Altogether, the results suggest that both Ca²⁺ and CaM are two downstream signaling molecules in adventitious rooting induced by NO and H₂O₂.     

Activation of cloned BK<Subscript>Ca</Subscript> channels in nitric oxide-induced apoptosis of HEK293 cells

The large conductance Ca²⁺-activated K⁺ (BK_Ca) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK_Ca has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK_Ca channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the α-subunit of BK_Ca channel, hSloα, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloα cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloα cells exhibited functional features similar to native BK_Ca channel in VSMCs. Exposuring of HEK- hSloα cells to S-nitroso-N-acetyl-penicillamine increased the hSloα channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloα channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 μM NS1619, the specific agonist of BK_Ca, independently increased hSloα currents and induced apoptosis. These results indicated that activation of cloned BK_Ca channel was involved in NO-induced apoptosis of HEK293 cells.     

Swelling-Induced Ca<Superscript>2+</Superscript> Influx and K<Superscript>+</Superscript> Efflux in American Alligator Erythrocytes

The American alligator can hibernate during winter, which may lead to osmotic imbalance because of reduced kidney function and lack of food consumption during this period. Accordingly, we hypothesized that their red blood cells would have a well-developed regulatory volume decrease (RVD) to cope with the homeostatic challenges associated with torpor. Osmotic fragility was determined optically, mean cell volume was measured by electronic sizing, and changes in intracellular Ca²⁺ concentration were visualized using fluorescence microscopy and fluo-4-AM. Osmotic fragility increased and the ability to regulate volume was inhibited when extracellular Na⁺ was replaced with K⁺, or when cells were exposed to the K⁺ channel inhibitor quinine, indicating a requirement of K⁺ efflux for RVD. Addition of the ionophore gramicidin to the extracellular medium decreased osmotic fragility and also potentiated volume recovery, even in the presence of quinine. In addition, hypotonic shock (0.5× Ringer) caused an increase in cytosolic Ca²⁺, which resulted from Ca²⁺ influx because it was not observed when extracellular Ca²⁺ was chelated with EGTA (ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid). Furthermore, cells loaded with BAPTA-AM (1,2-bis(2-aminophenoxymethyl)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl) ester) or exposed to a low Ca²⁺-EGTA hypotonic Ringer had a greater osmotic fragility and also failed to recover from cell swelling, indicating that extracellular Ca²⁺ was needed for RVD. Gramicidin reversed the inhibitory effect of low extracellular Ca²⁺. Finally, and surprisingly, the Ca²⁺ ionophore A23187 increased osmotic fragility and inhibited volume recovery. Taken together, our results show that cell swelling activated a K⁺ permeable pathway via a Ca²⁺-dependent mechanism, and this process mediated K⁺ loss during RVD.     

Preload-induced changes in isometric tension and [Ca<Superscript>2+</Superscript>]<Subscript>i</Subscript> in rat myocardium

Preload-induced changes of active tension and [Ca²⁺]i are “dissociated” in mammalian myocardium. This study aimed to describe the distinct effects of preload at low and physiological [Ca²⁺]_o. Rat RV papillary muscles were studied in isometric conditions at 25‡C and 0.33 Hz at 1 mM (hypo-Ca group) and 2.5 mM [Ca²⁺]_o (normal-Ca group). [Ca²⁺]_i was monitored with fura-2/AM. Increase of preload caused a rise of active tension in hypo-Ca and normal-Ca groups whereas peak fluorescence rose significantly only at low [Ca²⁺]_o. End-diastolic tension, end-diastolic level of fluorescence, time-to-peak tension, but not time-to-peak of Ca²⁺ transient, progressively increased with preload. Mechanical relaxation decelerated with preload while Ca²⁺ transient decay time decreased in the initial phase and increased in the late phase, resulting in a prominent “bump” configuration. The “bump” was assessed as a ratio of its area to the fluorescence trace area. It was a new finding that the preload-induced rise of this ratio was twice as large in hypo-Ca. Our results indicate that preload-induced changes in active tension and [Ca²⁺]_i are “dissociated” in rat myocardium, with relatively higher expression at low [Ca²⁺]_o. Ca-dependence of Ca-TnC association/dissociation kinetics is thought to be a main contributor to these preload-induced effects.