Abstract-Local, rhythmic, subsarcolemmal Ca 2ϩ releases (LCRs) from the sarcoplasmic reticulum (SR) during diastolic depolarization in sinoatrial nodal cells (SANC) occur even in the basal state and activate an inward Na ϩ -Ca 2ϩ exchanger current that affects spontaneous beating. Why SANC can generate spontaneous LCRs under basal conditions, whereas ventricular cells cannot, has not previously been explained. Here we show that a high basal cAMP level of isolated rabbit SANC and its attendant increase in protein kinase A (PKA)-dependent phosphorylation are obligatory for the occurrence of spontaneous, basal LCRs and for spontaneous beating. Gradations in basal PKA activity, indexed by gradations in phospholamban phosphorylation effected by a specific PKA inhibitory peptide were highly correlated with concomitant gradations in LCR spatiotemporal synchronization and phase, as well as beating rate. Higher levels of basal PKA inhibition abolish LCRs and spontaneous beating ceases. Stimulation of ␤-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca 2ϩ cycling and beating rate is also present in vivo, as the regulation of beating rate by local ␤-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca 2ϩ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca 2ϩ balance and spontaneous SR Ca 2ϩ cycling, ie, phospholamban and L-type Ca 2ϩ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na ϩ -Ca 2ϩ exchanger current and is crucial for both basal and reserve cardiac pacemaker function. R ecent studies have demonstrated that in sinoatrial (SA) nodal cells (SANC) generate local, rhythmic, subsarcolemmal Ca 2ϩ releases (LCRs) under basal conditions, ie, even in the absence of experimental Ca 2ϩ loading or stimulation of ␤-adrenergic receptors (␤-ARs). [1][2][3] In rabbit SANC, spontaneous, rhythmic LCRs occur during the late diastolic depolarization and activate Na ϩ -Ca 2ϩ exchanger (NCX) to generate an inward current that accelerates the depolarization rate, and, thus, LCRs are involved in control of spontaneous beating rate of SANC. 1 The mechanisms that permit SANC, but not ventricular myocytes, to generate rhythmic LCRs under basal conditions, however, have not been delineated.Spontaneous SR Ca 2ϩ release is facilitated by factors that increase the rate at which the SR can pump Ca 2ϩ , foremost among which are elevated cell Ca 2ϩ or elevated cAMP and its attendant protein kinase A (PKA)-dependent protein phosphorylation that results from intense ␤-AR stimulation. Whereas the cytosolic Ca 2ϩ concentration does not appreciably differ in rabbit ventricular cells and SANC, 2,4 the cAMP level of the intact SA node is high, 5 and it has been suspected that the basal cAMP level within SANC is elevated. 6,7 The SA node, however, is highly innervated, and neither the basal cAMP le...

Abstract-Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase A-dependent local subsarcolemmal ryanodine receptor Ca 2ϩ releases (LCRs). LCRs activated an inward Na ϩ /Ca 2ϩ exchange current that increases the terminal diastolic depolarization rate and, therefore, the spontaneous SANC beating rate. Basal cAMP in SANCs is elevated, suggesting that cAMP degradation by phosphodiesterases (PDEs) may be low. Surprisingly, total suppression of PDE activity with a broad-spectrum PDE inhibitor, 3Ј-isobutylmethylxanthine (IBMX), produced a 9-fold increase in the cAMP level, doubled cAMP-mediated, protein kinase A-dependent phospholamban phosphorylation, and increased SANC firing rate by Ϸ55%, indicating a high basal activity of PDEs in SANCs. A comparison of specific PDE1 to -5 inhibitors revealed that the specific PDE3 inhibitor, milrinone, accelerated spontaneous firing by Ϸ47% (effects of others were minor) and increased amplitude of L-type Ca 2ϩ current (I Ca,L ) by Ϸ46%, indicating that PDE3 was the major constitutively active PDE in the basal state. PDE-dependent control of the spontaneous SANC firing was critically dependent on subsarcolemmal LCRs, ie, PDE inhibition increased LCR amplitude and size and decreased LCR period, leading to earlier and augmented LCR Ca 2ϩ release, Na ϩ /Ca 2ϩ exchange current, and an increase in the firing rate. When ryanodine receptors were disabled by ryanodine, neither IBMX nor milrinone was able to amplify LCRs, accelerate diastolic depolarization rate, or increase the SANC firing rate, despite preserved PDE inhibition-induced augmentation of I Ca,L amplitude. Thus, basal constitutive PDE activation provides a novel and powerful mechanism to decrease cAMP, limit cAMP-mediated, protein kinase A-dependent increase of diastolic ryanodine receptor Ca 2ϩ release, and restrict the spontaneous SANC beating rate. Key Words: sinoatrial node Ⅲ phosphodiesterase Ⅲ ryanodine receptors Ⅲ local Ca 2ϩ release T he sinoatrial (SA) node is the primary physiological pacemaker of the heart. The pacemaker action potential (AP) is initiated within the SA node center and then propagates to the atria and ventricle to initiate contraction. 1,2 Our recent studies have demonstrated that spontaneous firing of SA node pacemaker cells (SANCs) is controlled by local subsarcolemmal Ca 2ϩ releases (LCRs) from ryanodine receptors (RyR) that occur during the second half of spontaneous diastolic depolarization (DD), just prior to the AP upstroke. 3 LCRs activate inward Na ϩ /Ca 2ϩ exchange (NCX) current that accelerates the rate of DD, leading to an earlier occurrence of the subsequent spontaneous AP, ie, to an increase in the beating rate. 3 Although LCRs do not require membrane depolarization, 4 they are critically dependent on levels of cAMP and cAMP-mediated, protein kinase (PKA)-dependent phosphorylation, both of which are markedly higher in SANCs than in atrial or ventricular myocytes, because of constitutive activation of adenylyl cyclases (ACs). 5 T...

releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.Numerous studies over the past decade have indicated that intracellular Ca 2ϩ release is a key feature of normal cardiac pacemaker cell automaticity (1). More recently it has been demonstrated that the basal level of global cAMP in rabbit sinoatrial nodal cells (SANC) 3 exceeds that in ventricular myocytes (2).The high basal cAMP in SANC mediates robust basal protein kinase A (PKA)-dependent phosphorylation of specific surface membrane ion channels and Ca 2ϩ cycling proteins, which regulates the periodicity and amplitude of spontaneous, sarcoplasmic reticulum generated, local Ca 2ϩ releases in the absence of cell Ca 2ϩ overload (2). Local Ca 2ϩ releases emanate from ryanodine receptors of sarcoplasmic reticulum that lies beneath the sarcolemma (10 -15 nm), near the Na/Ca exchanger (NCX) proteins (3). Local Ca 2ϩ releases occur mainly during the late part of the spontaneous diastolic depolarization and activate an inward NCX current (4 -7). This imparts an exponential character to the late diastolic depolarization (5,8,9), facilitating the achievement of the threshold for opening of L-type Ca 2ϩ channels, which generate the rapid upstroke of the subsequent action potential (AP). Thus, cAMP-mediated, PKA-dependent phosphorylation of surface membrane ion channels and SR Ca 2ϩ cycling proteins control the SANC basal spontaneous rhythmic firing (2).The mechanisms that underlie a high basal cAMP in SANC are unknown. The failure of ␤ 1 or ␤ 2 adrenergic receptor (␤-AR) inverse agonists to alter the spontaneous, basal SANC firing rate indicates that high levels of cAMP are not due to constitutively active ␤-ARs (2). Although a reduction in phosphodiesterase (PDE) activity could, in part, account for elevated cAMP levels in SANC, recent evidence suggests that basal PDE activity of SANC is not reduced, but rather, appears to be elevated (10). Moreover, inhibition of basal adenylyl cyclase (AC) activity in SANC substantially reduces cAMP and cAMP-mediated, PKA-dependent phosphorylation of phospholamban (2) suggesting a high constitutive (basal) level of AC activity. Whereas there is some evidence to indicate that SANC harbor Ca 2ϩ -activated AC isoforms (11, 12), direct evidence for Ca 2ϩ activation of AC activity, and the specific cell microdomains in which this may occur, are lacking. Using multiple approaches we show that both Ca 2ϩ -regulated ACs reside in lipid microdomains and that Ca 2ϩ activation of AC activity occurs within these domains.

Heart rate and contractility are sensitive to stretch. To better understand the origin of these effects, we have studied the effect of mechanical stimuli on a model system of tissue-cultured heart cells. Gently prodding cells with a pipette produced a Ca2+ influx that often led to waves of calcium-induced calcium release (CICR) spreading from the site of stimulation. Ca2+ release could also be produced by pulling on neighboring cells. The response was blocked by removing extracellular Ca2+ or by adding 20 microM Gd3+ to normal saline. The mechanical sensitivity probably arose from stretch-activated ion channels (SACs) based on several lines of evidence. Chick heart cells contain nonselective cation SACs that pass Ca2+ as well as Na+ and K+. Both the SACs and the fluorescence response are blocked by 20 microM Gd3+. Removal of Ca2+ from the extracellular medium blocked the fluorescent response. Cultures without SACs (grown in the absence of embryo extract) had no mechanically induced fluxes. These data contradict the recent claim that SAC activity is a patch-clamp artifact (C.E. Morris and R. Horn, Science Wash. DC 256: 1246-1249, 1991). The SACs had a density of approximately 1/micron 2 and were expected to pass less than 20 fA of Ca2+ current under physiological conditions. The change in intracellular concentration of Ca2+ ([Ca2+]i) resulting from activation of SACs may be too small to induce CICR unless the channels pass current into a restricted space (N. LeBlanc and J.R. Hume, Science Wash. DC 248: 372, 1990).(ABSTRACT TRUNCATED AT 250 WORDS)

SummaryWe have identified and characterized a protein of enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7 that shares homology with antigen 43 and AIDA-I of E. coli. The gene encoding this protein consists of a 2850 bp open reading frame and was named cah for calcium binding antigen 43 homologue. The prototype EHEC strain EDL933 possesses identical duplicate copies of cah ( cah 1 and cah 2 ), which showed 100% identity at the nucleotide level. We showed that E. coli K-12 containing the recombinant cah gene produced two proteins, an ≈ ≈ ≈ ≈ 80 kDa outer membrane protein and a 43.0 kDa heatextractable protein. The Cah protein contains a predicted 52-amino-acid extended signal sequence found in several autotransporter proteins, and Nterminal sequencing data indicated that the 43.0 kDa passenger protein was derived from cleavage of the signal sequence from alanine at position 53. Phenotypes such as autoaggregation and change in bacterial shape were observed when a recombinant plasmid containing the cah gene was introduced into a laboratory E. coli strain, and these phenotypes were eliminated upon mutation of the cah gene. The passenger domain contains six domains found in calcium-binding proteins, and the recombinant Cah passenger protein bound 45 Ca 2 + . In E. coli O157:H7, Cah is a heat-extractable protein, the expression of which is induced in minimal essential media and under divalent ion-depleting conditions; it also participates in the formation of biofilms. Our results provide insight into the expression, secretion and preliminary features of the calcium-binding Cah autotransporter protein of EHEC O157:H7.

The sodium-calcium exchanger, NCX1, is a ubiquitously expressed membrane protein essential in calcium homeostasis for many cells including those in mammalian heart and brain. The function of NCX1 depends on subcellular ("local") factors, the phosphorylation state of NCX1, and the subcellular location of NCX1 within the cell. Here we investigate the molecular organization of NCX1 within the cardiac myocyte. We show that NCX1 is dynamically phosphorylated by protein kinase A (PKA)-dependent phosphorylation in vitro. We also provide evidence that the regulation of this phosphorylation is attributed to the existence of an NCX1 macromolecular complex. Specifically, we show that the macromolecular complex includes both the catalytic and regulatory subunits of PKA. However, only the RI regulatory subunit is found in this macromolecular complex, not RII. Other critical regulatory enzymes are also associated with NCX1, including protein kinase C (PKC) and two serine/ threonine protein phosphatases, PP1 and PP2A. Importantly, the protein kinase A-anchoring protein, mAKAP, is found and its presence in the macromolecular complex suggests that these regulatory enzymes are coordinately positioned to regulate NCX1 as has been found in diverse cells for a number of channel proteins. Dual immunocytochemical staining showed the colocalization of NCX1 protein with mAKAP and PKA-RI proteins in cardiomyocytes. Finally, leucine/isoleucine zipper motifs have been identified as possible sites of interaction. Our finding of an NCX1 macromolecular complex in heart suggests how NCX1 regulation is achieved in heart and other cells. The existence of the NCX1 macromolecular complex may also provide an explanation for recent controversial findings.The Na ϩ /Ca 2ϩ exchanger, NCX1, is an integral membrane protein that is expressed in many tissues and is involved in cellular Ca 2ϩ homeostasis (1, 2). The expression level of NCX1 is modulated during development (3, 4) and under pathological conditions (5-9). The Na ϩ /Ca 2ϩ exchanger activity has been shown to be affected by the ions that it transports (Na ϩ and Ca 2ϩ ) (10 -13), by protons (14,15), by phosphatidylinositol 4,5-bisphosphate in the membrane (16), and by exogenous agents including intracellular application of an inhibitor peptide (XIP) (17). However, regulation of NCX1 by PKA 1 has remained controversial (18 -23).Early studies suggested that ATP-dependent regulation of NCX1 occurred in squid axons (24,25) and in cardiac sarcolemmal vesicles (26), but these studies did not distinguish between direct ATP binding and ATP-dependent phosphorylation. Several studies were designed to resolve this problem. Hilgemann and colleagues (11,12,27,28) investigated the question by measuring NCX1 currents in giant excised patches. The work used two preparations, NCX1-expressing Xenopus oocytes (27) or cardiac myocytes expressing native NCX1 (11,12,28). These investigations found no functional change in cardiac NCX1 activity following application of PKA or protein kinase C (PKC) catalytic subunits to...

With use of single-channel patch-clamp recording, we found five distinct types of stretch-activated ion channels (SACs) in tissue-cultured embryonic chick cardiac myocytes. With 140 mM K+ saline in the pipette, four channels had linear conductances of approximately equal to 25, 50, 100, and 200 pS and other channel was an inward rectifier of approximately equal to 25 pS at 0 mV membrane potential. The 100- and 200-pS channels were K+ selective, whereas the others passed alkali cations and Ca2+. From reversal potentials, the permeability ratio of K+/Na+, PK/PNa, was 3-7 for nonselective channels and 7-16 for K(+)-selective channels. Channel density was approximately equal to 0.3/microns2 for linear conductances and approximately equal to 0.1/microns2 for inward rectifier. Open-channel noise was a function of pipette filling solution with root-mean-square (RMS) noise increasing in the order K+ < isosmotic sucrose (plus trace ions) < Na+, probably reflecting short-lived block by extracellular ions. All channels were blocked by 20 microM Gd3+. The 25-pS linear channel was also blocked by 12.5 microM tetrodotoxin and 10 microM diltiazem, but the others were insensitive at these concentrations. Extracellular Cs+ and tetraethylammonium chloride did not block any channels. We saw no SAC activity in cells grown without embryo extract (EE), which demonstrates that channel expression, or some necessary cofactor, is under control of growth factors. Basic fibroblast growth factor (FGF) could replace EE in supporting channel expression. The presence of SACs capable of generating inward currents might explain how stretch increases automaticity in the heart. Because some SACs were permeable to Ca2+, they could contribute to the Starling curve and perhaps to initiating stretch-induced hypertrophy.

Unique ATP-inhibitable K ؉ channels (K ATP ) in the kidney determine the rate of urinary K ؉ excretion and play an essential role in extracellular K ؉ balance. Here, we demonstrate that functionally similar low sulfonylurea affinity K ATP channels are formed by two heterologous molecules, products of Kir1.1a and cystic fibrosis transmembrane conductance regulator (CFTR) genes. Co-injection of CFTR and Kir1.1a cRNA into Xenopus oocytes lead to the expression of K ؉ selective channels that retained the high open probability behavior of Kir1.1a but acquired sulfonylurea sensitivity and ATP-dependent gating properties. Similar to the K ATP channels in the kidney but different from K ATP channels in excitable tissues, the Kir1.1a/CFTR channel was inhibited by glibenclamide with micromolar affinity. Since the expression of Kir1.1a and CFTR overlap at sites in the kidney where the low sulfonylurea affinity K ATP are expressed, our study offers evidence that these native K ATP channels are comprised of Kir1.1a and CFTR. The implication that Kir subunits can interact with ABC proteins beyond the subfamily of sulfonylurea receptors provides an intriguing explanation for functional diversity in K ATP channels.