Targeting the IKs Channel PKA Phosphorylation Axis to Restore Its Function in High-Risk LQT1 Variants.

BACKGROUND: The KCNQ1+KCNE1 (IKs) potassium channel plays a crucial role in cardiac adaptation to stress, in which ß-adrenergic stimulation phosphorylates the IKs channel through the cyclic adenosine monophosphate (cAMP)/PKA (protein kinase A) pathway. Phosphorylation increases the channel current and accelerates repolarization to adapt to an increased heart rate. Variants in KCNQ1 can cause long-QT syndrome type 1 (LQT1), and those with defective cAMP effects predispose patients to the highest risk of cardiac arrest and sudden death. However, the molecular connection between IKs channel phosphorylation and channel function, as well as why high-risk LQT1 mutations lose cAMP sensitivity, remain unclear. METHODS: Regular patch clamp and voltage clamp fluorometry techniques were utilized to record pore opening and voltage sensor movement of wild-type and mutant KCNQ1/IKs channels. The clinical phenotypic penetrance of each LQT1 mutation was analyzed as a metric for assessing their clinical risk. The patient-specific-induced pluripotent stem-cell model was used to test mechanistic findings in physiological conditions. RESULTS: By systematically elucidating mechanisms of a series of LQT1 variants that lack cAMP sensitivity, we identified molecular determinants of IKs channel regulation by phosphorylation. These key residues are distributed across the N-terminus of KCNQ1 extending to the central pore region of IKs. We refer to this pattern as the IKs channel PKA phosphorylation axis. Next, by examining LQT1 variants from clinical databases containing 10 579 LQT1 carriers, we found that the distribution of the most high-penetrance LQT1 variants extends across the IKs channel PKA phosphorylation axis, demonstrating its clinical relevance. Furthermore, we found that a small molecule, ML277, which binds at the center of the phosphorylation axis, rescues the defective cAMP effects of multiple high-risk LQT1 variants. This finding was then tested in high-risk patient-specific induced pluripotent stem cell-derived cardiomyocytes, where ML277 remarkably alleviates the beating abnormalities. CONCLUSIONS: Our findings not only elucidate the molecular mechanism of PKA-dependent IKs channel phosphorylation but also provide an effective antiarrhythmic strategy for patients with high-risk LQT1 variants.

The Transient Receptor Potential Vanilloid 2 (TRPV2) Channel Facilitates Virus Infection Through the Ca2+ -LRMDA Axis in Myeloid Cells.

The transient receptor potential vanilloid 2 (TRPV2) channel is a nonselective cation channel that has been implicated in multiple sensory processes in the nervous system. Here, it is shown that TRPV2 in myeloid cells facilitates virus penetration by promoting the tension and mobility of cell membrane through the Ca2+ -LRMDA axis. Knockout of TRPV2 in myeloid cells or inhibition of TRPV2 channel activity suppresses viral infection and protects mice from herpes simplex virus 1 (HSV-1) and vesicular stomatitis virus (VSV) infection. Reconstitution of TRPV2 but not the Ca2+ -impermeable mutant TRPV2E572Q into LyZ2-Cre;Trpv2fl/fl bone marrow-derived dendritic cells (BMDCs) restores viral infection. Mechanistically, knockout of TRPV2 in myeloid cells inhibits the tension and mobility of cell membrane and the penetration of viruses, which is restored by reconstitution of TRPV2 but not TRPV2E572Q . In addition, knockout of TRPV2 leads to downregulation of Lrmda in BMDCs and BMDMs, and knockdown of Lrmda significantly downregulates the mobility and tension of cell membrane and inhibits viral infections in Trpv2fl/fl but not LyZ2-Cre;Trpv2fl/fl BMDCs. Consistently, complement of LRMDA into LyZ2-Cre;Trpv2fl/fl BMDCs partially restores the tension and mobility of cell membrane and promotes viral penetration and infection. These findings characterize a previously unknown function of myeloid TRPV2 in facilitating viral infection though the Ca2+ -LRMDA axis.

Tyrosine phosphorylation tunes chemical and thermal sensitivity of TRPV2 ion channel.

Transient receptor potential vanilloid 2 (TRPV2) is a multimodal ion channel implicated in diverse physiopathological processes. Its important involvement in immune responses has been suggested such as in the macrophages' phagocytosis process. However, the endogenous signaling cascades controlling the gating of TRPV2 remain to be understood. Here, we report that enhancing tyrosine phosphorylation remarkably alters the chemical and thermal sensitivities of TRPV2 endogenously expressed in rat bone marrow-derived macrophages and dorsal root ganglia (DRG) neurons. We identify that the protein tyrosine kinase JAK1 mediates TRPV2 phosphorylation at the molecular sites Tyr(335), Tyr(471), and Tyr(525). JAK1 phosphorylation is required for maintaining TRPV2 activity and the phagocytic ability of macrophages. We further show that TRPV2 phosphorylation is dynamically balanced by protein tyrosine phosphatase non-receptor type 1 (PTPN1). PTPN1 inhibition increases TRPV2 phosphorylation, further reducing the activation temperature threshold. Our data thus unveil an intrinsic mechanism where the phosphorylation/dephosphorylation dynamic balance sets the basal chemical and thermal sensitivity of TRPV2. Targeting this pathway will aid therapeutic interventions in physiopathological contexts.

All the cells in our body have a membrane that separates their interior from the outside environment. However, studded across this barrier are numerous ion channels which allow the cell to sense and react to changes in its surroundings. This includes the ion channel TRPV2, which opens in response to mechanical pressure, certain chemical signals, or rising temperature levels. Many types of cell express TRPV2, including cells in the nervous system, muscle, and the immune system. However, despite being extensively studied, it is still not clear how TRPV2 opens and closes upon encountering high temperatures. In particular, previous work suggested that TRPV2 only responds when a cell's surroundings reach around 52°C, which is a much higher temperature than cells inside our body normally encounter, even during a fever. To help resolve this mystery, Mo, Pang et al. studied TRPV2 in neurons responsible for sending sensory information and in immune cells called macrophages which had been extracted from rodents and grown in the laboratory. They found that when the cells were bathed in solutions containing magnesium ions, their TRPV2 channels were more sensitive to a number of different cues, including temperature. Further biochemical experiments showed that magnesium ions do not directly affect TRPV2, but increase the activity of another protein called JAK1. The magnesium ions caused JAK1 to attach specialized structures called phosphorylation tags to TRPV2. This modification (known as phosphorylation) made the channel more sensitive, allowing it to open in response to temperatures as low as 40°C. Mo, Pang et al. found that inhibiting JAK1 reduced the activity of TRPV2. Conversely, inhibiting the enzyme that removes the phosphorylation tags, called PTPN1, increased the channel's activity. They also discovered that when JAK1 was blocked, macrophages were less able to 'eat up' bacteria, which is one of their main roles in the immune system. Taken together these experiments advance our understanding of how TRPV2 becomes active. The balance between the phosphorylation by JAK1 and the dephosphorylation by PTPN1 controls the temperature at which TRPV2 opens. Since TRPV2 contributes to several biological functions, including the development of the nervous system, the maintenance of heart muscles, and inflammation, these findings will be important to scientists in a broad range of fields.

Isolation and genomic characterization of lymphocytic choriomeningitis virus in ticks from northeastern China.

Lymphocytic choriomeningitis virus (LCMV) is a rodent-borne arenavirus that is considered a neglected cause of neurologic diseases in humans. In this study, we described genomic characterization of newly isolated LCMVs in Haemaphysalis longicornis, Dermacentor nuttalli, Dermacentor silvarum and Ixodes persulcatus in Jilin Province, northeastern China. The complete sequences of the small (S) and large (L) segments of LCMVs in ticks contained 3,375 and 7,235-7,241 nucleotides, respectively. Sequence comparison showed 82.1%-86.0% identity of S segment with other lineage I strains at the nucleotide level and 91.2%-97.5% at the deduced amino acid level, while a lower identity was observed in the L segment at both nucleotide (75.4%-82.2%) and amino acid (82.4%-93.4%) levels. Phylogenetic analysis grouped the tick LCMVs together with the lineage I strains, but in an isolated cluster with a high bootstrap value. Bayesian analysis indicated that the molecular evolutionary rate was estimated to be 3.3 × 10-4 substitutions/site/year for the S segment and 6.3 × 10-4 substitutions/site/year for the L segment, and the time to most recent common ancestor was 1980 and 1970 years ago, respectively, showing that tick LCMVs were predicted to originate between 1970s and 1980s. A long evolutionary history and high prevalence of LCMV in H. longicornis were found compared to other tick species. This study represented the first report on isolation of LCMV in China, showing that LCMV is circulating among ticks in Jilin Province, but the role of ticks in the epidemiology of LCMV remains to be explored.