Faculty Bibliography

The immature and mature heart differ from each other in terms of excitability, action potential properties, contractility, and relaxation. This includes upregulation of repolarizing K(+) currents, an enhanced inward rectifier K(+) (Kir) current, and changes in Ca(2+), Na(+), and Cl(-) currents. At the molecular level, the developmental regulation of ion channels is scantily described. Using a large-scale real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay, we performed a comprehensive analysis of ion channel transcript expression during perinatal development in the embryonic (embryonic day 17.5), neonatal (postnatal days 1-2), and adult Swiss-Webster mouse hearts. These data are compared with publicly available microarray data sets (Cardiogenomics project). Developmental mRNA expression for several transcripts was consistent with the published literature. For example, transcripts such as Kir2.1, Kir3.1, Nav1.5, Cav1.2, etc. were upregulated after birth, whereas others [e.g., Ca(2+)-activated K(+) (KCa)2.3 and minK] were downregulated. Cl(-) channel transcripts were expressed at higher levels in immature heart, particularly those that are activated by intracellular Ca(2+). Defining alterations in the ion channel transcriptome during perinatal development will lead to a much improved understanding of the electrophysiological alterations occurring in the heart after birth. Our study may have important repercussions in understanding the mechanisms and consequences of electrophysiological alterations in infants and may pave the way for better understanding of clinically relevant events such as congenital abnormalities, cardiomyopathies, heart failure, arrhythmias, cardiac drug therapy, and the sudden infant death syndrome

K(ATP) channels are involved in regulating coronary function, but the contribution of endothelial K(ATP) channels remains largely uncharacterized. We generated a transgenic mouse model to specifically target endothelial K(ATP) channels by expressing a dominant negative Kir6.1 subunit only in the endothelium. These animals had no obvious overt phenotype and no early mortality. Histologically, the coronary endothelium in these animals was preserved. There was no evidence of increased susceptibility to ergonovine-induced coronary vasospasm. However, isolated hearts from these animals had a substantially elevated basal coronary perfusion pressure. The K(ATP) channel openers, adenosine and levcromakalim, decreased the perfusion pressure whereas the K(ATP) channel blocker glibenclamide failed to produce a vasoconstrictive response. The inducible endothelial nitric oxide pathway was intact, as evidenced by vasodilation caused by bradykinin. In contrast, basal endothelin-1 release was significantly elevated in the coronary effluent from these hearts. Treatment of mice with bosentan (endothelin-1 receptor antagonist) normalized the coronary perfusion pressure, demonstrating that the elevated endothelin-1 release was sufficient to account for the increased coronary perfusion pressure. Pharmacological blockade of K(ATP) channels led to elevated endothelin-1 levels in the coronary effluent of isolated mouse and rat hearts as well as enhanced endothelin-1 secretion from isolated human coronary endothelial cells. These data are consistent with a role for endothelial K(ATP) channels to control the coronary blood flow by modulating the release of the vasoconstrictor, endothelin-1

Sorcin is a Ca2+ binding protein implicated in the regulation of intracellular Ca2+ cycling and cardiac excitation-contraction coupling. Structural and human genetic studies suggest that a naturally occurring sequence variant encoding L112-sorcin disrupts an E-F hand Ca2+ binding domain and may be responsible for a heritable form of hypertension and hypertrophic heart disease. We generated transgenic mice overexpressing L112-sorcin in the heart and characterized the effects on Ca2+ regulation and cardiac function both in vivo and in dissociated cardiomyocytes. Hearts of sorcin(F112L) transgenic mice were mildly dilated but ventricular function was preserved and systemic blood pressure was normal. Sorcin(F112L) myocytes were smaller than control cells and displayed complex alterations in Ca2+ regulation and contractility, including a slowed inactivation of L-type Ca2+ current, enhanced Ca2+ spark width, duration, and frequency, and increased Na+-Ca2+ exchange activity. In contrast, mice with cardiac-specific overexpression of wild-type sorcin displayed directionally opposite effects on L-type Ca2+ channel function and Ca2+ spark behavior. These data further define the role of sorcin in cardiac excitation-contraction coupling and highlight its negative regulation of SR calcium release. Our results also suggest that additional factors may be responsible for the development of cardiac hypertrophy and hypertension in humans expressing the L112-sorcin sequence variant.

The macroscopic volume-regulated anion current (VRAC) is regulated by both intracellular and extracellular ATP, which has important implications in signaling and regulation of cellular excitability. The outwardly rectifying Cl(-) channel (ORCC) is a major contributor to the VRAC. This study investigated the effects of intracellular and extracellular ATP on the ORCCs expressed in the human cardiovascular system. With inside-out single-channel patch-clamp techniques, ORCCs were recorded from myocytes isolated from human atrium and septal ventricle and from primary cells originating from human coronary artery endothelium and human coronary artery smooth muscle. ORCCs from all of these tissues had similar biophysical properties, i.e., they were outwardly rectifying in symmetrical Cl(-) solutions, exhibited a slope conductance of approximately 90-100 pS at positive potentials and approximately 22 pS at negative potentials, and had a high open probability that was independent of voltage or time. The presence of ATP at the cytosolic face of the membrane increased the number of patches that contained functional ORCC but had no effect on gating. In contrast, 'extracellular' ATP (in pipette solution) had no effect on the proportion of patches in which ORCC was detected but strongly reduced the open probability by increasing the closed dwell time. The potency order for nucleotides to affect gating was ATPgammaS > ATP = UTP > ADP > AMP, which suggests that a negatively charged phosphate group is involved in ORCC block. Our findings are consistent with a role of ORCC in the human cardiovasculature (atrium, ventricle, and coronary arteries). Regulation of ORCC by extracellular ATP suggests that this channel may have an important role in maintaining electrical activity and membrane potential under conditions in which extracellular ATP levels are elevated, such as with ATP release from nerve endings or during pathophysiological conditions

Glycolysis is an evolutionary conserved metabolic pathway that provides small amounts of energy in the form of ATP when compared to other pathways such as oxidative phosphorylation or fatty acid oxidation. The ATP levels inside metabolically active cells are not constant and the local ATP level will depend on the site of production as well as the respective rates of ATP production, diffusion and consumption. Membrane ion transporters (pumps, exchangers and channels) are located at sites distal to the major sources of ATP formation (the mitochondria). We review evidence that the glycolytic complex is associated with membranes; both at the plasmalemma and with membranes of the endo/sarcoplasmic reticular network. We examine the evidence for the concept that many of the ion transporters are regulated preferentially by the glycolytic process. These include the Na(+)/K(+)-ATPase, the H(+)-ATPase, various types of Ca(2+)-ATPases, the Na(+)/H(+) exchanger, the ATP-sensitive K(+) channel, cation channels, Na(+) channels, Ca(2+) channels and other channels involved in intracellular Ca(2+) homeostasis. Regulation of these pumps, exchangers and ion channels by the glycolytic process has important consequences in a variety of physiological and pathophysiological processes, and a better understanding of this mode of regulation may have important consequences for developing future strategies in combating disease and developing novel therapeutic approaches

Monitoring Editor: Guido Guidotti KCa3.1 is an intermediate conductance Ca(2+)-activated K channels that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca2+ influx, cell volume and chloride secretion. We recently found that the KCa3.1 channel also specifically requires PI(3)P for channel activity and is inhibited by myotubularin related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2 or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for their biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxyterminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data when taken together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca2+ gating of KCa3.1

The Epac family of cAMP-regulated guanine nucleotide exchange factors (cAMPGEFs, also known as Epac1 and Epac2) mediate stimulatory actions of the second messenger cAMP on insulin secretion from pancreatic beta cells. Because Epac2 is reported to interact in vitro with the isolated nucleotide-binding fold-1 (NBF-1) of the beta cell sulfonylurea receptor-1 (SUR1), we hypothesized that cAMP might act via Epac1 and/or Epac2 to inhibit beta cell ATP-sensitive K+ channels (KATP channels; a hetero-octomer of SUR1 and Kir6.2). If so, Epac-mediated inhibition of KATP channels might explain prior reports that cAMP-elevating agents promote beta cell depolarization, Ca2+ influx, and insulin secretion. Here we report that Epac-selective cAMP analogs (2'-O-Me- cAMP; 8-pCPT-2'-O-Me-cAMP; 8-pMeOPT-2'-O-Me-cAMP), but not a cGMP analog (2'-O-Me-cGMP), inhibit the function of KATP channels in human beta cells and rat INS-1 insulin-secreting cells. Inhibition of KATP channels is also observed when cAMP, itself, is administered intracellularly, whereas no such effect is observed upon administration N6-Bnz-cAMP, a cAMP analog that activates protein kinase A (PKA) but not Epac. The inhibitory actions of Epac-selective cAMP analogs at KATP channels are mimicked by a cAMP agonist (Sp-8-Br-cAMPS), but not a cAMP antagonist (Rp-8-Br-cAMPS), and are abrogated following transfection of INS-1 cells with a dominant- negative Epac1 that fails to bind cAMP. Because both Epac1 and Epac2 co-immunoprecipitate with full-length SUR1 in HEK cell lysates, such findings delineate a novel mechanism of second messenger signal transduction in which cAMP acts via Epac to modulate ion channel function, an effect measurable as the inhibition of KATP channel activity in pancreatic beta cells

The Ca2+ -activated K+ channel KCa3.1 is required for Ca2+ influx and the subsequent activation of B and T cells. Inhibitors of KCa3.1 are in development to treat autoimmune diseases and transplant rejection, underscoring the importance in understanding how these channels are regulated. We show that nucleoside diphosphate kinase B (NDPK-B), a mammalian histidine kinase, functions downstream of PI(3)P to activate KCa3.1. NDPK-B directly binds and activates KCa3.1 by phosphorylating histidine 358 in the carboxyl terminus of KCa3.1. Endogenous NDPK-B is also critical for KCa3.1 channel activity and the subsequent activation of CD4 T cells. These findings provide one of the best examples whereby histidine phosphorylation regulates a biological process in mammals, and provide an example whereby a channel is regulated by histidine phosphorylation. The critical role for NDPK-B in the reactivation of CD4 T cells indicates that understanding NDPK-B regulation should uncover novel pathways required for T cell activation