Tomoe Y. Nakamura

A role for frequenin, a Ca2+-binding protein, as a regulator of Kv4 K+-currents

Frequenin, a Ca2+-binding protein, has previously been implicated in the regulation of neurotransmission, possibly by affecting ion channel function. Here, we provide direct evidence that frequenin is a potent and specific modulator of Kv4 channels, the principal molecular components of subthreshold activating A-type K+ currents. Frequenin increases Kv4.2 current amplitudes (partly by enhancing surface expression of Kv4.2 proteins) and it slows the inactivation time course in a Ca2+-dependent manner. It also accelerates recovery from inactivation. Closely related Ca2+-binding proteins, such as neurocalcin and visinin-like protein (VILIP)-1 have no such effects. Specificity for Kv4 currents is suggested because frequenin does not modulate Kv1.4 or Kv3.4 currents. Frequenin has negligible effects on Kv4.1 current inactivation time course. By using chimeras made from Kv4.2 and Kv4.1 subunits, we determined that the differential effects of frequenin are mediated by means of the Kv4 N terminus. Immunohistochemical analysis demonstrates that frequenin and Kv4.2 channel proteins are coexpressed in similar neuronal populations and have overlapping subcellular localizations in brain. Coimmunoprecipitation experiments demonstrate that a physical interaction occurs between these two proteins in brain membranes. Together, our data provide strong support for the concept that frequenin may be an important Ca2+-sensitive regulatory component of native A-type K+ currents.

Immunolocalization of KATP channel subunits in mouse and rat cardiac myocytes and the coronary vasculature

BackgroundElectrophysiological data suggest that cardiac KATP channels consist of Kir6.2 and SUR2A subunits, but the distribution of these (and other KATP channel subunits) is poorly defined. We examined the localization of each of the KATP channel subunits in the mouse and rat heart.ResultsImmunohistochemistry of cardiac cryosections demonstrate Kir6.1 protein to be expressed in ventricular myocytes, as well as in the smooth muscle and endothelial cells of coronary resistance vessels. Endothelial capillaries also stained positive for Kir6.1 protein. Kir6.2 protein expression was found predominantly in ventricular myocytes and also in endothelial cells, but not in smooth muscle cells. SUR1 subunits are strongly expressed at the sarcolemmal surface of ventricular myocytes (but not in the coronary vasculature), whereas SUR2 protein was found to be localized predominantly in cardiac myocytes and coronary vessels (mostly in smaller vessels). Immunocytochemistry of isolated ventricular myocytes shows co-localization of Kir6.2 and SUR2 proteins in a striated sarcomeric pattern, suggesting t-tubular expression of these proteins. Both Kir6.1 and SUR1 subunits were found to express strongly at the sarcolemma. The role(s) of these subunits in cardiomyocytes remain to be defined and may require a reassessment of the molecular nature of ventricular KATP channels.ConclusionsCollectively, our data demonstrate unique cellular and subcellular KATP channel subunit expression patterns in the heart. These results suggest distinct roles for KATP channel subunits in diverse cardiac structures.

Perilipin 5, a Lipid Droplet-binding Protein, Protects Heart from Oxidative Burden by Sequestering Fatty Acid from Excessive Oxidation

Background: Perilipin family proteins are important in determining the properties of lipid droplets (LDs). Results: Perilipin 5-deficient mice lack detectable LDs, exhibit enhanced fatty acid oxidation, and suffer increased ROS production in the heart. Conclusion: Perilipin 5 protects the heart from oxidative burden by sequestering fatty acid from excessive oxidation. Significance: These findings may help to increase understanding of the functions of non-adipose LDs. Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart.

Activation of Na+/H+ Exchanger 1 Is Sufficient to Generate Ca2+ Signals That Induce Cardiac Hypertrophy and Heart Failure

Activation of the sarcolemmal Na+/H+ exchanger (NHE)1 is increasingly documented as a process involved in cardiac hypertrophy and heart failure. However, whether NHE1 activation alone is sufficient to induce such remodeling remains unknown. We generated transgenic mice that overexpress a human NHE1 with high activity in hearts. The hearts of these mice developed cardiac hypertrophy, contractile dysfunction, and heart failure. In isolated transgenic myocytes, intracellular pH was elevated in Hepes buffer but not in physiological bicarbonate buffer, yet intracellular Na+ concentrations were higher under both conditions. In addition, both diastolic and systolic Ca2+ levels were increased as a consequence of Na+-induced Ca2+ overload; this was accompanied by enhanced sarcoplasmic reticulum Ca2+ loading via Ca2+/calmodulin-dependent protein kinase (CaMK)II-dependent phosphorylation of phospholamban. Negative force–frequency dependence was observed with preservation of high Ca2+, suggesting a decrease in myofibril Ca2+ sensitivity. Furthermore, the Ca2+-dependent prohypertrophic molecules calcineurin and CaMKII were highly activated in transgenic hearts. These effects observed in vivo and in vitro were largely prevented by the NHE1 inhibitor cariporide. Interestingly, overexpression of NHE1 in neonatal rat ventricular myocytes induced cariporide-sensitive nuclear translocation of NFAT (nuclear factor of activated T cells) and nuclear export of histone deacetylase 4, suggesting that increased Na+/H+ exchange activity can alter hypertrophy-associated gene expression. However, in transgenic myocytes, contrary to exclusive translocation of histone deacetylase 4, NFAT only partially translocated to nucleus, possibly because of marked activation of p38, a negative regulator of NFAT signaling. We conclude that activation of NHE1 is sufficient to initiate cardiac hypertrophy and heart failure mainly through activation of CaMKII–histone deacetylase pathway.

Unique topology of the internal repeats in the cardiac Na+/Ca2+ exchanger.

Hydropathy analysis predicts 11 transmembrane helices in the cardiac Na+/Ca2+ exchanger. Using cysteine susceptibility analysis and epitope tagging, we here studied the membrane topology of the exchanger, in particular of the highly conserved internal α‐1 and α‐2 repeats. Unexpectedly, we found that the connecting loop in the α‐1 repeat forms a re‐entrant membrane loop with both ends facing the extracellular side and one residue (Asn‐125) being accessible from the inside and that the region containing the α‐2 repeat is mostly accessible from the cytoplasm. Together with other data, we propose that the exchanger may consist of nine transmembrane helices.

Modulation of Kv4 channels, key components of rat ventricular transient outward K+ current, by PKC

Current evidence suggests that members of the Kv4 subfamily may encode native cardiac transient outward current ( I to). Antisense hybrid-arrest with oligonucleotides targeted to Kv4 mRNAs specifically inhibited rat ventricular I to, supporting this hypothesis. To determine whether protein kinase C (PKC) affects I to by an action on these molecular components, we compared the effects of PKC activation on Kv4.2 and Kv4.3 currents expressed in Xenopus oocytes and rat ventricular I to. Phorbol 12-myristate 13-acetate (PMA) suppressed both Kv4.2 and Kv4.3 currents as well as native I to, but not after preincubation with PKC inhibitors (e.g., chelerythrine). An inactive stereoisomer of PMA had no effect. Phenylephrine or carbachol inhibited Kv4 currents only when coexpressed, respectively, with α1C-adrenergic or M1 muscarinic receptors (this inhibition was also prevented by chelerythrine). The voltage dependence and inactivation kinetics of Kv4.2 were unchanged by PKC, but small effects on the rates of inactivation and recovery from inactivation of native I to were observed. Thus Kv4.2 and Kv4.3 proteins are important subunits of native rat ventricular I to, and PKC appears to reduce this current by affecting the molecular components of the channels mediating I to.

Inhibition of rat ventricular IK1 with antisense oligonucleotides targeted to Kir2.1 mRNA

The cardiac inward rectifying K+ current ( I K1) is important in maintaining the maximum diastolic potential. We used antisense oligonucleotides to determine the role of Kir2.1 channel proteins in the genesis of native rat ventricular I K1. A combination of two antisense phosphorothioate oligonucleotides inhibited heterologously expressed Kir2.1 currents in Xenopus oocytes, either when coinjected with Kir2.1 cRNA or when applied in the incubation medium. Specificity was demonstrated by the lack of inhibition of Kir2.2 and Kir2.3 currents in oocytes. In rat ventricular myocytes (4-5 days culture), these oligonucleotides caused a significant reduction of whole cell I K1(without reducing the transient outward K+ current or the L-type Ca2+ current). Cell-attached patches demonstrated the occurrence of multiple channel events in control myocytes (8, 14, 21, 35, 43, and 80 pS). The 21-pS channel was specifically knocked down in antisense-treated myocytes (fewer patches contained this channel, and its open frequency was reduced). These results demonstrate that the Kir2.1 gene encodes a specific native 21-pS K+-channel protein and that this channel has an essential role in the genesis of cardiac I K1.

Novel role of neuronal Ca2+ sensor-1 as a survival factor up-regulated in injured neurons

A molecular basis of survival from neuronal injury is essential for the development of therapeutic strategy to remedy neurodegenerative disorders. In this study, we demonstrate that an EF-hand Ca2+-binding protein neuronal Ca2+ sensor-1 (NCS-1), one of the key proteins for various neuronal functions, also acts as an important survival factor. Overexpression of NCS-1 rendered cultured neurons more tolerant to cell death caused by several kinds of stressors, whereas the dominant-negative mutant (E120Q) accelerated it. In addition, NCS-1 proteins increased upon treatment with glial cell line–derived neurotrophic factor (GDNF) and mediated GDNF survival signal in an Akt (but not MAPK)-dependent manner. Furthermore, NCS-1 is significantly up-regulated in response to axotomy-induced injury in the dorsal motor nucleus of the vagus neurons of adult rats in vivo, and adenoviral overexpression of E120Q resulted in a significant loss of surviving neurons, suggesting that NCS-1 is involved in an antiapoptotic mechanism in adult motor neurons. We propose that NCS-1 is a novel survival-promoting factor up-regulated in injured neurons that mediates the GDNF survival signal via the phosphatidylinositol 3-kinase–Akt pathway.

Expression of ATP-sensitive K+ channel subunits during perinatal maturation in the mouse heart.

Prevailing data suggest that sarcolemmal ATP-sensitive (KATP) channels in the adult heart consist of Kir6.2 and SUR2A subunits, but the expression of other KATP channel subunits (including SUR1, SUR2B, and Kir6.1) is poorly defined. The situation is even less clear for the immature heart, which shows a remarkable resistance to hypoxia and metabolic stress. The hypoxia-induced action potential shortening and opening of sarcolemmal KATP channels that occurs in adults is less prominent in the immature heart. This might be due in part to the different biophysical and pharmacological properties of KATP channels of immature and adult KATP channels. Because these properties are largely conferred by subunit composition, it is important to examine the relative expression levels of the various KATP channel subunits during maturation. We therefore used RNAse protection assays, reverse transcription–PCR approaches, and Western blotting to characterize the mRNA and protein expression profiles of KATP channel subunits in fetal, neonatal, and adult mouse heart. Our data indicate that each of the KATP channel subunits (Kir6.1, Kir6.2, SUR1, SUR2A, and SUR2B) is expressed in the mouse heart at all of the developmental time points studied. However, the expression level of each of the subunits is low in the fetal heart and progressively increases with maturation. Each of the subunits seems to be expressed in ventricular myocytes with a subcellular expression pattern matching that found in the adult. Our data suggest that the KATP channel composition may change during maturation, which has important implications for KATP channel function in the developing heart.

Different effects of the Ca2+-binding protein, KChIP1, on two Kv4 subfamily members, Kv4.1 and Kv4.2

The Ca2+‐binding protein, K+ channel‐interacting protein 1 (KChIP1), modulates Kv4 channels. We show here that KChIP1 affects Kv4.1 and Kv4.2 currents differently. KChIP1 slows Kv4.2 inactivation but accelerates the Kv4.1 inactivation time course. Kv4.2 activation is shifted in a hyperpolarizing direction, whereas a depolarizing shift occurs for Kv4.1. On the other hand, KChIP1 increases the current amplitudes and accelerates recovery from inactivation of both currents. An involvement of the Kv4 N‐terminus in these differential effects is demonstrated using chimeras of Kv4.2 and Kv4.1. These results reveal a novel interaction of KChIP1 with these two Kv4 members. This represents a mechanism to further increase the functional diversity of K+ channels.