1. Guinea-pig ventricle was used in the RNase protection assays to determine which á-isoforms of the Na¤-K¤ pumps are present, and ventricular myocytes were used in whole cell patch clamp studies to investigate the actions of á-and â-adrenergic agonists on Na¤-K¤ pump current. 2. RNase protection assays showed that two isoforms of the á-subunit of the Na¤-K¤-ATPase are present in guinea-pig ventricle. The mRNA for the á1-isoform comprises 82% of the total pump message, the rest being the áµ-isoform. 3. We have previously shown that â-adrenergic agonists affect Na¤-K¤ pump current (Ip) through a protein kinase A (PKA)-dependent pathway. We now show that these â-effects are targeted to the á1-isoform of the Na¤-K¤ pumps. 4. We have also previously shown that á-adrenergic agonists increase Ip through a protein kinase C (PKC)-dependent pathway. We now show that these á-isoform effects are targeted to the áµ-isoform of the Na¤-K¤ pumps. 5. These results suggest the effects of adrenergic activation on Na¤-K¤ pump activity in the heart can be regionally specific, depending on which á-isoform of the Na¤-K¤ pump is expressed.8785
The whole‐cell patch clamp was employed to study Na+‐K+ pump current (Ip) in acutely isolated myocytes. α‐Adrenergic receptors were activated with noradrenaline (NA) after blocking β‐adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str). Activation of α‐receptors by NA increased Ip in a concentration‐dependent manner. The K0.5 depended on intracellular calcium ([Ca2+]i), however maximal stimulation did not. At 15 nm[Ca2+]i the K0.5 was 219 nm NA whereas at 1.4 μm [Ca2+]i it was 3 nm. The voltage dependence of Ip was not shifted by NA at either high or low [Ca2+]i. At each voltage, maximal stimulation of Ip was 14‐15 %. Staurosporine (St), an inhibitor of protein kinase C (PKC), eliminated the α‐receptor‐mediated stimulation of Ip at either high or low[Ca2+]i. The stimulation of Ip was independent of changes in intracellular sodium or external potassium concentrations, and did not reflect a change in affinity for Str. Phenylephrine, methoxamine and metaraminol, three selective α1‐adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an α1‐antagonist, but was unaffected by yohimbine, an α2‐antagonist. We conclude noradrenaline activates ventricular α1‐receptors, which are specifically coupled via PKC to increase Na+‐K+ pump current. The sensitivity of the coupling is [Ca2+]i dependent, however the maximal increase in pump current is [Ca2+]i and voltage independent.
The COVID-19 pandemic has swept over the world in the past months, causing significant loss of life and consequences to human health. Although numerous drug and vaccine developments efforts are underway, many questions remain outstanding on the mechanism of SARS-CoV-2 viral association to angiotensin-converting enzyme 2 (ACE2), its main host receptor, and entry in the cell. Structural and biophysical studies indicate some degree of flexibility in the viral extracellular Spike glycoprotein and at the receptor binding domain-receptor interface, suggesting a role in infection. Here, we perform all-atom molecular dynamics simulations of the glycosylated, full-length membrane-bound ACE2 receptor, in both an apo and spike receptor binding domain (RBD) bound state, in order to probe the intrinsic dynamics of the ACE2 receptor in the context of the cell surface. A large degree of fluctuation in the full length structure is observed, indicating hinge bending motions at the linker region connecting the head to the transmembrane helix, while still not disrupting the ACE2 homodimer or ACE2-RBD interfaces. This flexibility translates into an ensemble of ACE2 homodimer conformations that could sterically accommodate binding of the spike trimer to more than one ACE2 homodimer, and suggests a mechanical contribution of the host receptor towards the large spike conformational changes required for cell fusion. This work presents further structural and functional insights into the role of ACE2 in viral infection that can be exploited for the rational design of effective SARS-CoV-2 therapeutics.Statement of SignificanceAs the host receptor of SARS-CoV-2, ACE2 has been the subject of extensive structural and antibody design efforts in aims to curtail COVID-19 spread. Here, we perform molecular dynamics simulations of the homodimer ACE2 full-length structure to study the dynamics of this protein in the context of the cellular membrane. The simulations evidence exceptional plasticity in the protein structure due to flexible hinge motions in the head-transmembrane domain linker region and helix mobility in the membrane, resulting in a varied ensemble of conformations distinct from the experimental structures. Our findings suggest a dynamical contribution of ACE2 to the spike glycoprotein shedding required for infection, and contribute to the question of stoichiometry of the Spike-ACE2 complex.
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