The current chemical biology tool box for studying ion channels.

Ion channels play important roles in human physiology and their dysfunction is linked to a variety of diseases. This has sparked considerable interest in their molecular function and pharmacology and generated a need to manipulate them with great precision. The use of high-sensitivity electrophysiological methods allows for the implementation of chemical biology manipulations, as even minute protein amounts can be studied. For example, modification of solvent-accessible cysteines is a powerful tool to site-selectively modify proteins through the introduction of charged moieties or those with fluorescent properties. This has been harnessed to study ion conduction pathways and monitor conformational dynamics. In ligand-directed chemistry, a high-affinity ligand is used to modify an ion channel with a chemical probe via a reactive linker. While these approaches are typically limited to extracellular positions, genetic code expansion provides a means to introduce non-canonical amino acids in any position of the protein. This enables the insertion of subtle analogues of naturally occurring side chains or the protein backbone, as well as amino acids with fluorescent, cross-linking or photo-switchable properties. Finally, protein semi-synthesis enables the simultaneous insertion of multiple modifications, including those that would not be tolerated by the ribosomal translation machinery. Collectively, these chemical biology tools have overcome various shortcomings of conventional mutagenesis and vastly expanded the scope of possible modifications and the type of ion channels they can be applied to. Their application in both heterologous and native cell systems will no doubt play an increasingly important role in ion channel research.

The NALCN channel complex is voltage sensitive and directly modulated by extracellular calcium.

The sodium leak channel (NALCN) is essential for survival in mammals: NALCN mutations are life-threatening in humans and knockout is lethal in mice. However, the basic functional and pharmacological properties of NALCN have remained elusive. Here, we found that robust function of NALCN in heterologous systems requires co-expression of UNC79, UNC80, and FAM155A. The resulting NALCN channel complex is constitutively active and conducts monovalent cations but is blocked by physiological concentrations of extracellular divalent cations. Our data support the notion that NALCN is directly responsible for the increased excitability observed in a variety of neurons in reduced extracellular Ca2+. Despite the smaller number of voltage-sensing residues in NALCN, the constitutive activity is modulated by voltage, suggesting that voltage-sensing domains can give rise to a broader range of gating phenotypes than previously anticipated. Our work points toward formerly unknown contributions of NALCN to neuronal excitability and opens avenues for pharmacological targeting.

Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing.

Manipulation of proteins by chemical modification is a powerful way to decipher their function. However, most ribosome-dependent and semi-synthetic methods have limitations in the number and type of modifications that can be introduced, especially in live cells. Here, we present an approach to incorporate single or multiple post-translational modifications or non-canonical amino acids into proteins expressed in eukaryotic cells. We insert synthetic peptides into GFP, NaV1.5 and P2X2 receptors via tandem protein trans-splicing using two orthogonal split intein pairs and validate our approach by investigating protein function. We anticipate the approach will overcome some drawbacks of existing protein enigineering methods.

[Effect of indomethacin on the intracranial pressure]. / Indomethacins virkning på det intrakranielle tryk.

Twenty patients subjected to craniotomy for supratentorial cerebral tumours were anaesthetized with thiopental, fentanyl, nitrous oxide, and isoflurane. A PaCO2 level averaging 4.8 kPa was achieved. The patients were randomized to intravenous indomethacin 50 mg or placebo administrated after exposure of the dura. A significant decrease in intracranial pressure from 6.5 to 1.5 mmHg (medians) was found after indomethacin administration. This decrease was caused by a significant decrease in cerebral blood flow associated with a significant increase in the arterio-venous oxygen difference. Indomethacin did not affect cerebral oxygen uptake, arteriovenous difference in lactate or the lactate/oxygen index, suggesting that indomethacin did not provoke global cerebral ischaemia. In the indomethacin group, dura was sufficiently relaxed in eight of nine patients, and dura was opened without the occurrence of cerebral swelling. In the placebo group, mannitol supplemented with hypocapnia was applied in five patients. These findings suggest that perioperative treatment with indomethacin is an excellent treatment of intracranial hypertension during normocapnic isoflurane anaesthesia for craniotomy.

Effects of perioperative indomethacin on intracranial pressure, cerebral blood flow, and cerebral metabolism in patients subjected to craniotomy for cerebral tumors.

This study was carried out to evaluate the effects of perioperative indomethacin on intracranial pressure (ICP), cerebral blood flow (CBF), and cerebral metabolism. Twenty patients subjected to craniotomy for supratentorial cerebral tumors were anesthetized with thiopental, fentanyl, nitrous oxide, and isoflurane. A PaCO2 level averaging 4.8 kPa (median) was achieved. The patients were randomized to intravenous indomethacin 50 mg or placebo administrated after exposure of the dura. ICP was measured continuously subdurally with a 22-gauge canula connected to a transducer. CBF and the arteriovenous difference of oxygen (AVDO2) were measured twice, before and after indomethacin/placebo administration. A significant decrease in ICP from 6.5 to 1.5 mm Hg (median) was found after indomethacin administration. This decrease was caused by a significant decrease in CBF associated with a significant increase in AVDO2. Indomethacin did not affect the cerebral metabolic rate of oxygen, the arteriovenous difference of lactate, or the lactate/oxygen index, suggesting that indomethacin did not provoke global cerebral ischemia. In the indomethacin group, dura was sufficiently relaxed in eight of nine patients and dura was opened without the occurrence of cerebral swelling. In one patient, mannitol treatment was necessary to prevent dural tightness. In the placebo group, mannitol supplemented with hypocapnia was applied in five patients. These findings suggest that perioperative treatment with indomethacin is an excellent treatment of intracranial hypertension during normocapnic isoflurane anesthesia for craniotomy.

Multiple pulmonary chondrohamartomas in trachea, bronchi and lung parenchyma. Review of the literature.

Multiple pulmonary chondromatous hamartomas (MPCH) are rare, and MPCH with tracheal involvement have not been, to the best of our knowledge, reported before. Furthermore, there is no agreement in the literature about the origin of these tumours. We report a case of MPCH involving trachea, bronchi and lung parenchyma, incidentally found at the autopsy of a 88-year-old woman who died of acute myocardial infarction, and we review the literature about MPCH. We conclude that there appear to be two different types of multiple pulmonary chondromatous hamartomas; those presented in young patients often linked to gastric leiomyoblastomas and catecholamine-producing paragangliomas (Carney syndrome) with high mortality, and those presented in elderly patients, which seems to be compatible with life.

[Calcifying pre-patellar bursitis. Presentation of a case involving both knees]. / Bursitis calcarea praepatellaris. Proesentation af et tilfoelde med involvering af begge knoe.

A case story is presented of bilateral calcific bursitis of the prepatellar bursa in the same patient. The patient (a 52-year old female) complained of tender swellings of the front of both of her knees. X-ray examination showed several calcifications in both prepatellar bursae. The bursae were successfully excised relieving the patient of her symptoms.

[Head injuries among children in the county of Ringkøbing. 1. An analysis of hospital data]. / Kranietraumer blandt børn i Ringkøbing Amt. 1. En analyse of sygehusdata.

The authors analysed hospital data concerning 1,498 patients aged 0-15 years admitted on 1,548 occasions in the hospitals in Ringkøbing County during the period 1.1.1982 to 31.8.1989 after head injuries. The commonest diagnosis was cerebral concussion (91.0%). Thirteen children (0.8%) died in hospital. A significant preponderance of boys was found together with a significant increase in hospital admissions in the individual age groups with increasing age. The number of hospital admissions decreased during the period and the number of admissions per 1,000 of population was below the average for Denmark. A definite seasonal variation was observed with maximums in May and August and minimums in December and February. No significant increase in the number of admissions on weekdays was observed. The average duration of hospitalization was four days (1-196) and 73.8% of the patients were discharged after three days.

[Cranial injuries among children in the county of Ringkøbing. 2. Analysis of accidents]. / Kranietraumer blandt børn i Ringkøbing Amt. 2. En ulykkesanalyse.

Questionnaires were sent concerning all of the patients aged 0-15 years who were admitted to the Ringkøbing County Hospitals during the period 1.1.1982 to 31.8.1989 with head injuries. The object of the questionnaires was to elucidate the circumstances of the accident which resulted in hospitalization. Questionnaires concerning 988 patients (67.5%) were returned. Definite accumulation of the accidents was observed during the period 12.00-18.00 with 55.2% of all the accidents. The majority of the accidents (72.3%) occurred out-of-doors and the majority of these on transport areas. Bicycle accidents were most common, most frequently in children over the age of four years. Where younger children were concerned, accidents in the home were commonest. Accidents in playgrounds and during sports constituted only 13% of the total material. A relatively great proportion of the children (42.9%) were under supervision, usually from their own family, when the accident occurred. On the basis of this investigation, the authors recommend that possible prophylactic measures in the County of Ringkøbing should be directed towards bicycle accidents among the older children and accidents in the homes where smaller children are concerned.