Zebrafish cardiac repolarization does not functionally depend on the expression of the hERG1b-like transcript.

Zebrafish provide a translational model of human cardiac function. Their similar cardiac electrophysiology enables screening of human cardiac repolarization disorders, drug arrhythmogenicity, and novel antiarrhythmic therapeutics. However, while zebrafish cardiac repolarization is driven by delayed rectifier potassium channel current (IKr), the relative role of alternate channel transcripts is uncertain. While human ether-a-go-go-related-gene-1a (hERG1a) is the dominant transcript in humans, expression of the functionally distinct alternate transcript, hERG1b, modifies the electrophysiological and pharmacologic IKr phenotype. Studies of zebrafish IKr are frequently translated without consideration for the presence and impact of hERG1b in humans. Here, we performed phylogenetic analyses of all available KCNH genes from Actinopterygii (ray-finned fishes). Our findings confirmed zebrafish cardiac zkcnh6a as the paralog of human hERG1a (hKCNH2a), but also revealed evidence of a hERG1b (hKCNH2b)-like N-terminally truncated gene, zkcnh6b, in zebrafish. zkcnh6b is a teleost-specific variant that resulted from the 3R genome duplication. qRT-PCR showed dominant expression of zkcnh6a in zebrafish atrial and ventricular tissue, with low levels of zkcnh6b. Functional evaluation of zkcnh6b in a heterologous system showed no discernable function under the conditions tested, and no influence on zkcnh6a function during the zebrafish ventricular action potential. Our findings provide the first descriptions of the zkcnh6b gene, and show that, unlike in humans, zebrafish cardiac repolarization does not rely upon co-assembly of zERG1a/zERG1b. Given that hERG1b modifies IKr function and drug binding in humans, our findings highlight the need for consideration when translating hERG variant effects and toxicological screens in zebrafish, which lack a functional hERG1b-equivalent gene.

SUMO1 modification of PKD2 channels regulates arterial contractility.

PKD2 (polycystin-2, TRPP1) channels are expressed in a wide variety of cell types and can regulate functions, including cell division and contraction. Whether posttranslational modification of PKD2 modifies channel properties is unclear. Similarly uncertain are signaling mechanisms that regulate PKD2 channels in arterial smooth muscle cells (myocytes). Here, by studying inducible, cell-specific Pkd2 knockout mice, we discovered that PKD2 channels are modified by SUMO1 (small ubiquitin-like modifier 1) protein in myocytes of resistance-size arteries. At physiological intravascular pressures, PKD2 exists in approximately equal proportions as either nonsumoylated (PKD2) or triple SUMO1-modifed (SUMO-PKD2) proteins. SUMO-PKD2 recycles, whereas unmodified PKD2 is surface-resident. Intravascular pressure activates voltage-dependent Ca2+ influx that stimulates the return of internalized SUMO-PKD2 channels to the plasma membrane. In contrast, a reduction in intravascular pressure, membrane hyperpolarization, or inhibition of Ca2+ influx leads to lysosomal degradation of internalized SUMO-PKD2 protein, which reduces surface channel abundance. Through this sumoylation-dependent mechanism, intravascular pressure regulates the surface density of SUMO-PKD2-mediated Na+ currents (INa) in myocytes to control arterial contractility. We also demonstrate that intravascular pressure activates SUMO-PKD2, not PKD2, channels, as desumoylation leads to loss of INa activation in myocytes and vasodilation. In summary, this study reveals that PKD2 channels undergo posttranslational modification by SUMO1, which enables physiological regulation of their surface abundance and pressure-mediated activation in myocytes and thus control of arterial contractility.

TMEM16A channel upregulation in arterial smooth muscle cells produces vasoconstriction during diabetes.

The pathological involvement of anion channels in vascular dysfunction that occurs during type 2 diabetes (T2D) is unclear. Here, we tested the hypothesis that TMEM16A, a calcium-activated chloride (Cl-) channel, contributes to modifications in arterial contractility during T2D. Our data indicate that T2D increased TMEM16A mRNA in arterial smooth muscle cells and total and surface TMEM16A protein in resistance-size cerebral and hindlimb arteries of mice. To examine vascular cell types in which TMEM16A protein increased and the functional consequences of TMEM16A upregulation during T2D, we generated tamoxifen-inducible, smooth muscle cell-specific TMEM16A knockout (TMEM16A smKO) mice. T2D increased both TMEM16A protein and Cl- current density in arterial smooth muscle cells of control (TMEM16Afl/fl) mice. In contrast, T2D did not alter arterial TMEM16A protein or Cl- current density in smooth muscle cells of TMEM16A smKO mice. Intravascular pressure stimulated greater vasoconstriction (myogenic tone) in the arteries of T2D TMEM16Afl/fl mice than in the arteries of nondiabetic TMEM16Afl/fl mice. This elevation in myogenic tone in response to T2D was abolished in the arteries of T2D TMEM16A smKO mice. T2D also reduced Akt2 protein and activity in the arteries of T2D mice. siRNA-mediated knockdown of Akt2, but not Akt1, increased arterial TMEM16A protein in nondiabetic mice. In summary, data indicate that T2D is associated with an increase in TMEM16A expression and currents in arterial smooth muscle cells that produces vasoconstriction. Data also suggest that a reduction in Akt2 function drives these pathological alterations during T2D.NEW & NOTEWORTHY We investigated the involvement of TMEM16A channels in vascular dysfunction during type 2 diabetes (T2D). TMEM16A message, protein, and currents were higher in smooth muscle cells of resistance-size arteries during T2D. Pressure stimulated greater vasoconstriction in the arteries of T2D mice that was abolished in the arteries of TMEM16A smKO mice. Akt2 protein and activity were both lower in T2D arteries, and Akt2 knockdown elevated TMEM16A protein. We propose that a decrease in Akt2 function stimulates TMEM16A expression in arterial smooth muscle cells, leading to vasoconstriction during T2D.

The cardiac L-type calcium channel alpha subunit is a target for direct redox modification during oxidative stress-the role of cysteine residues in the alpha interacting domain.

Cardiovascular disease is the leading cause of death in the Western world. The incidence of cardiovascular disease is predicted to further rise with the increase in obesity and diabetes and with the aging population. Even though the survival rate from ischaemic heart disease has improved over the past 30 years, many patients progress to a chronic pathological condition, known as cardiac hypertrophy that is associated with an increase in morbidity and mortality. Reactive oxygen species (ROS) and calcium play an essential role in mediating cardiac hypertrophy. The L-type calcium channel is the main route for calcium influx into cardiac myocytes. There is now good evidence for a direct role for the L-type calcium channel in the development of cardiac hypertrophy. Cysteines on the channel are targets for redox modification and glutathionylation of the channel can modulate the function of the channel protein leading to the onset of pathology. The cysteine responsible for modification of L-type calcium channel function has now been identified. Detailed understanding of the role of cysteines as possible targets during oxidative stress may assist in designing therapy to prevent the development of hypertrophy and heart failure.

Author Correction: Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Arterial smooth muscle cell PKD2 (TRPP1) channels regulate systemic blood pressure.

Systemic blood pressure is determined, in part, by arterial smooth muscle cells (myocytes). Several Transient Receptor Potential (TRP) channels are proposed to be expressed in arterial myocytes, but it is unclear if these proteins control physiological blood pressure and contribute to hypertension in vivo. We generated the first inducible, smooth muscle-specific knockout mice for a TRP channel, namely for PKD2 (TRPP1), to investigate arterial myocyte and blood pressure regulation by this protein. Using this model, we show that intravascular pressure and α1-adrenoceptors activate PKD2 channels in arterial myocytes of different systemic organs. PKD2 channel activation in arterial myocytes leads to an inward Na+ current, membrane depolarization and vasoconstriction. Inducible, smooth muscle cell-specific PKD2 knockout lowers both physiological blood pressure and hypertension and prevents pathological arterial remodeling during hypertension. Thus, arterial myocyte PKD2 controls systemic blood pressure and targeting this TRP channel reduces high blood pressure.

Membrane depolarization activates BK channels through ROCK-mediated ß1 subunit surface trafficking to limit vasoconstriction.

Membrane depolarization of smooth muscle cells (myocytes) in the small arteries that regulate regional organ blood flow leads to vasoconstriction. Membrane depolarization also activates large-conductance calcium (Ca2+)-activated potassium (BK) channels, which limits Ca2+ channel activity that promotes vasoconstriction, thus leading to vasodilation. We showed that in human and rat arterial myocytes, membrane depolarization rapidly increased the cell surface abundance of auxiliary BK ß1 subunits but not that of the pore-forming BKα channels. Membrane depolarization stimulated voltage-dependent Ca2+ channels, leading to Ca2+ influx and the activation of Rho kinase (ROCK) 1 and 2. ROCK1/2-mediated activation of Rab11A promoted the delivery of ß1 subunits to the plasma membrane by Rab11A-positive recycling endosomes. These additional ß1 subunits associated with BKα channels already at the plasma membrane, leading to an increase in apparent Ca2+ sensitivity and activation of the channels in pressurized arterial myocytes and vasodilation. Thus, membrane depolarization activates BK channels through stimulation of ROCK- and Rab11A-dependent trafficking of ß1 subunits to the surface of arterial myocytes.

Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel.

The "Fight or Flight" response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the ß-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca2+ channel (Cav1.2) is absolutely required. Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca2+ channel pore forming α1 subunit using a novel approach. We used a cell free system where we could assess direct effects of PKA on human purified channel protein function reconstituted in proteoliposomes. In addition to assessing open probability of channel protein we used semi-quantitative fluorescent phosphoprotein detection and MS/MS mass spectrometry analysis to demonstrate the PKA specificity of the site. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and the channel protein with C terminus truncated at aa1504. A protein kinase A anchoring protein (AKAP) was not required. We find the novel PKA phosphorylation site at Ser1458 is in close proximity to the Repeat IV S6 region and induces a conformational change in the channel protein that is necessary and sufficient for increased calcium influx through the channel.

Evidence for redox sensing by a human cardiac calcium channel.

Ion channels are critical to life and respond rapidly to stimuli to evoke physiological responses. Calcium influx into heart muscle occurs through the ion conducting α1C subunit (Cav1.2) of the L-type Ca(2+) channel. Glutathionylation of Cav1.2 results in increased calcium influx and is evident in ischemic human heart. However controversy exists as to whether direct modification of Cav1.2 is responsible for altered function. We directly assessed the function of purified human Cav1.2 in proteoliposomes. Truncation of the C terminus and mutation of cysteines in the N terminal region and cytoplasmic loop III-IV linker did not alter the effects of thiol modifying agents on open probability of the channel. However mutation of cysteines in cytoplasmic loop I-II linker altered open probability and protein folding assessed by thermal shift assay. We find that C543 confers sensitivity of Cav1.2 to oxidative stress and is sufficient to modify channel function and posttranslational folding. Our data provide direct evidence for the calcium channel as a redox sensor that facilitates rapid physiological responses.