Abdul M. Ruknudin

High Basal Protein Kinase A–Dependent Phosphorylation Drives Rhythmic Internal Ca2+ Store Oscillations and Spontaneous Beating of Cardiac Pacemaker Cells

Local, rhythmic, subsarcolemmal Ca2+ 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+-Ca2+ 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 β-adrenergic receptors extends the range of PKA-dependent control of LCRs and beating rate beyond that in the basal state. The link between SR Ca2+ cycling and beating rate is also present in vivo, as the regulation of beating rate by local β-adrenergic receptor stimulation of the sinoatrial node in intact dogs is markedly blunted when SR Ca2+ cycling is disrupted by ryanodine. Thus, PKA-dependent phosphorylation of proteins that regulate cell Ca2+ balance and spontaneous SR Ca2+ cycling, ie, phospholamban and L-type Ca2+ channels (and likely others not measured in this study), controls the phase and size of LCRs and the resultant Na+-Ca2+ exchanger current and is crucial for both basal and reserve cardiac pacemaker function.

Protein Kinase A Hyperphosphorylation Increases Basal Current but Decreases β-Adrenergic Responsiveness of the Sarcolemmal Na+-Ca2+ Exchanger in Failing Pig Myocytes

Abstract— The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by &bgr;-adrenergic receptor (&bgr;-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni2+-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P <0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P <0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P <0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to &bgr;-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominately attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although &bgr;-AR responsiveness is decreased because of NCX hyperphosphorylation.

Novel subunit composition of a renal epithelial KATP channel.

Unique ATP-inhibitable K+channels (KATP) 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 KATP 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 KATP channels in the kidney but different from KATP 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 KATP are expressed, our study offers evidence that these native KATP 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 KATP channels.

Constitutive Phosphodiesterase Activity Restricts Spontaneous Beating Rate of Cardiac Pacemaker Cells by Suppressing Local Ca2+ Releases

Spontaneous beating of rabbit sinoatrial node cells (SANCs) is controlled by cAMP-mediated, protein kinase A–dependent local subsarcolemmal ryanodine receptor Ca2+ releases (LCRs). LCRs activated an inward Na+/Ca2+ 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, 3′-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 ≈55%, 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 ≈47% (effects of others were minor) and increased amplitude of L-type Ca2+ current (ICa,L) by ≈46%, 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 Ca2+ release, Na+/Ca2+ 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 ICa,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 Ca2+ release, and restrict the spontaneous SANC beating rate.

Characterization of Cah, a calcium‐binding and heat‐extractable autotransporter protein of enterohaemorrhagic Escherichia coli

We 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 (cah1 and cah2), 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 heat‐extractable protein. The Cah protein contains a predicted 52‐amino‐acid extended signal sequence found in several autotransporter proteins, and N‐terminal 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 45Ca2+. 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.

Ca2+-stimulated basal adenylyl cyclase activity localization in membrane lipid microdomains of cardiac sinoatrial nodal pacemaker cells

Spontaneous, rhythmic subsarcolemmal local Ca2+ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca2+ releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca2+ and cAMP/PKA regulatory components of automaticity are coupled via Ca2+ activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca2+-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca2+ over the entire physiological Ca2+ range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca2+ cycling proteins drive sarcoplasmic reticulum Ca2+ 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.

Functional differences between cardiac and renal isoforms of the rat Na+‐Ca2+ exchanger NCX1 expressed in Xenopus oocytes

1 The transcript of the Na+‐Ca2+ exchanger gene NCX1 undergoes alternative splicing to produce tissue‐specific isoforms. The cloned NCX1 isoforms were expressed in Xenopus oocytes and studied using a two‐electrode voltage clamp method to measure Na+‐Ca2+ exchanger activity. 2 The cardiac isoform (NCX1.1) expressed in oocytes was less sensitive to depolarizing voltages and to activation by [Ca2+]i than the renal isoform (NCX1.3). 3 The cardiac isoform of NCX1 is more sensitive to activation by protein kinase A (PKA) than the renal isoform which may be explained by preferential phosphorylation. The cardiac isoform of NCX1 is phosphorylated to a greater extent than the renal isoform. 4 The action of PKA phosphorylation which increases the activity of the cardiac isoform of the Na+‐Ca2+ exchanger in oocytes was confirmed in adult rat ventricular cardiomyocytes by measuring Na+‐dependent Ca2+ flux. 5 We conclude that alternative splicing of NCX1 confers distinct functional characteristics to tissue‐specific isoforms of the Na+‐Ca2+ exchanger.

The Integration of Spontaneous Intracellular Ca2+ Cycling and Surface Membrane Ion Channel Activation Entrains Normal Automaticity in Cells of the Heart's Pacemaker

Abstract:  Although the ensemble of voltage‐ and time‐dependent rhythms of surface membrane ion channels, the membrane “Clock”, is the immediate cause of a sinoatrial nodal cell (SANC) action potential (AP), it does not necessarily follow that this ion channel ensemble is the formal cause of spontaneous, rhythmic APs. SANC also generates intracellular oscillatory spontaneous Ca2+ releases that ignite excitation (SCaRIE) of the surface membrane via Na+/Ca2+ exchanger activation. The idea that a rhythmic intracellular Ca2+ Clock might keep time for normal automaticity of SANC, however, has not been assimilated into mainstream pacemaker dogma. Recent experimental evidence, derived from simultaneous, confocal imaging of submembrane Ca2+ and membrane potential of SANC, and supported by numerical modeling, indicates that normal automaticity of SANC is entrained and stabilized by the tight integration of the SR Ca2+ Clock that generates rhythmic SCaRIE, and the surface membrane Clock that responds to SCaRIE to immediately produce APs of an adequate shape. Thus, tightly controlled, rhythmic SCaRIE does not merely fine tune SANC AP firing, but is the formal cause of the basal and reserve rhythms, insuring pacemaker stability by rhythmically integrating multiple Ca2+‐dependent functions, and effects normal automaticity by rhythmic ignition of the surface membrane Clock.

Sodium/calcium exchanger expression in the mouse and rat olfactory systems

Sodium/calcium (Na+/Ca2+) exchangers are membrane transport systems that regulate Ca2+‐homeostasis in many eukaryotic cells. In olfactory and vomeronasal sensory neurons ligand‐induced olfactory signal transduction is associated with influx and elevation of intracellular Ca2+, [Ca2+]i. While much effort has been devoted to the characterization of Ca2+‐related excitation and adaptation events of olfactory chemosensory neurons (OSNs), much less is known about mechanisms that return [Ca2+]i to the resting state. To identify proteins participating in the poststimulus Ca2+‐clearance of mouse OSNs, we analyzed the expression of three potassium (K+)‐independent (NCX1, 2, 3) and three K+‐dependent (NCKX1, 2, 3) Na+/Ca2+ exchangers. In situ hybridization showed that mRNAs of all six Na+/Ca2+ exchangers coexist in neurons of the olfactory and vomeronasal systems, and that some are already detectable in the embryo. Of these, NCX1 and NCKX1 represent the most and least abundant mRNAs, respectively. Moreover, immunohistochemistry revealed that the NCX1, 2, and 3 proteins are expressed in nearly all neurons of the olfactory epithelium, the vomeronasal organ, the septal organ of Masera, and the Grueneberg ganglion. These three exchanger proteins display different expression profiles in dendrites, knobs, and plasma membranes of OSNs and in sustentacular cells. Furthermore, we show that NCX1 mRNA in rat olfactory mucosa is expressed as 8 alternative splice variants. This is the first comprehensive analysis of Na+/Ca2+ exchanger expression in the mammalian olfactory system. Our results suggest that Ca2+‐extrusion by OSNs utilizes multiple different Na+/Ca2+ exchangers and that different subtypes are targeted to different subcellular compartments. J. Comp. Neurol. 501:944–958, 2007.

Functional Regulation of Alternatively Spliced Na+/Ca2+ Exchanger (NCX1) Isoforms

Abstract: Alternative splicing of RNA transcripts is a general characteristic for NCX genes in mammals, mollusks, and arthropods. Among the family of three NCX genes in mammals, the NCX1 gene contains six exons, namely, A, B, C, D, E, and F, that make up the alternatively spliced region. Studies of the NCX1 gene transcripts suggested that 16 distinct gene products can be produced from the NCX1 gene. The exons A and B are mutually exclusive when expressed. Generally, exon A‐containing transcripts are predominantly found in excitable cells like cardiomyoctes and neurons, whereas exon B‐containing transcripts are mostly found in nonexcitable cells like astrocytes and kidney cells. Other alternatively spliced exons (C‐F) appear to be cassette‐type exons and are found in various combinations. Interestingly, exon D is present in all characterized transcripts. The alternatively spliced isoforms of NCX1 show tissue‐specific expression patterns, suggesting functional adaptation to tissues. To investigate functional differences among alternatively spliced isoforms of NCX1, we expressed an exon A‐containing transcript present in cardiac tissue (NCX1.1) and an exon B‐containing transcript found in the kidney (NCX1.3) in Xenopus oocytes. We demonstrated that the Na+/Ca2+ exchangers expressed by exon A‐ and exon B‐containing transcripts display differences in activation by PKA and by [Ca2+]i. We also observed that these two isoforms show differences in voltage dependence. Suprisingly, the alternatively spliced isoforms of NCX1 display greater functional differences among themselves than the products of different gene loci, NCX1, NCX2, and NCX3.