Primary extra-cranial meningioma following total hip replacement.

A 61-year-old man presented with pain at the left hip and decreased mobility 10 years after total hip replacement. Imaging demonstrated a large destructive expansile mass adjacent to the prosthesis. Histological analysis confirmed the presence of an extra-cranial meningioma. Primary tumours after total hip replacement are rare and include soft tissue sarcomas, bone sarcomas and lymphomas. To our knowledge, no previous cases of primary extracranial meningioma have been identified. The imaging features, histology, pathogenesis and differential diagnosis are discussed.

The S631A mutation causes a mechanistic switch in the block of hERG channels by CnErg1.

We have studied the interaction of CnErg1, a member of the gamma-KTX subfamily of scorpion toxins with the inactivation-deficient S631A hERG channel. In the background of this mutation, we observed a mechanistic switch from turret block, characteristic of the action of gamma-KTXs on Kv11-type channels, to pore plugging, characteristic of alpha-KTX block of Kv1-type channels. We suggest this reflects destabilization of the outer pore (turret region) of hERG allowing access of the toxin molecule to directly plug the conduction pathway.

Mechanism of block of the hERG K+ channel by the scorpion toxin CnErg1.

The scorpion toxin CnErg1 binds to human ether-a-go-go related gene (hERG) K(+) channels with a 1:1 stoichiometry and high affinity. However, in contrast to other scorpion toxin-ion channel interactions, the inhibition of macroscopic hERG currents by high concentrations of CnErg1 is incomplete. In this study, we have probed the molecular basis for this incomplete inhibition. High concentrations of CnErg1 had only modest effects on hERG gating that could not account for the incomplete block. Furthermore, the residual current in the presence of 1 microM CnErg1 had normal single channel conductance. Analysis of the kinetics of CnErg1 interaction with hERG indicated that CnErg1 binding is not diffusion-limited. A bimolecular binding scheme that incorporates an initial encounter complex and permits normal ion conduction was able to completely reproduce both the kinetics and steady-state level of CnErg1-hERG binding. This scheme provides a simple kinetic explanation for incomplete block; that is, relatively fast backward compared to forward rate constants for the interconversion of the toxin-channel encounter complex and the blocked toxin-channel complex. We have also examined the temperature-dependence of CnErg1 binding to hERG. The dissociation constant, K(d), for CnErg1 increases from 7.3 nM at 22 degrees C to 64 nM at 37 degrees C (i.e., the affinity decreases as temperature increases) and the proportion of binding events that lead to channel blockade decreases from 70% to 40% over the same temperature range. These temperature-dependent effects on CnErg1 binding correlate with a temperature-dependent decrease in the stability of the putative CnErg1 binding site, the amphipathic alpha-helix in the outer pore domain of hERG, assayed using circular dichroism spectropolarimetry. Collectively, our data provides a plausible kinetic explanation for incomplete blockade of hERG by CnErg1 that is consistent with the proposed highly dynamic conformation of the outer pore domain of hERG.

Effect of S5P alpha-helix charge mutants on inactivation of hERG K+ channels.

The ether-à-go-go (EAG) family of voltage-gated K(+) channels contains three subfamilies, EAG, ether-à-go-go related (ERG) and ether-à-go-go like (ELK). The human ether-à-go-go related gene (hERG) K(+) channel has been of significant interest because loss of function in the hERG channel is associated with a markedly increased risk of cardiac arrhythmias. The hERG channel has unusual kinetics with slow activation and deactivation but very rapid and voltage-dependent inactivation. The outer pore region of the hERG K(+) channel is predicted to be different from that of other members of the voltage-gated K(+) channel family. HERG has a much longer linker between the fifth transmembrane domain (SS) and the pore helix (S5P linker) compared to other families of voltage-gated K(+) channels (43 amino acids compared to 14-23 amino acids). Further, the S5P linker contains an amphipathic alpha-helix that in hERG channels probably interacts with the mouth of the pore to modulate inactivation. The human EAG and rat ELK2 channels (hEAG and rELK2) show reduced or no inactivation in comparison to hERG channels, yet both channels are predicted to contain a similarly long S5P linker to that of hERG. In this study, we have constructed a series of chimaeric channels consisting of the S1-S6 of hERG but with the S5P alpha-helical region of either hEAG or rELK2, and one consisting of the S1-S6 of rELK2 but with the S5P alpha-helical region of hERG to investigate the role of the S5P linker in inactivation. Our studies show that charged residues on the alpha-helix of the S5P linker contribute significantly to the differences in inactivation characteristics of the EAG family channels. Further, individually mutating each of the hydrophilic residues on the S5P alpha-helix of hERG to a charged residue had significant effects on the voltage dependence of inactivation and the two residues with the greatest affect when mutated to a lysine, N588 and Q592, both lie on the same face of the S5P alpha -helix. We suggest that inactivation of hERG involves the interaction of this face of the S5P alpha-helix with a charged residue on the remainder of the outer pore domain of the channel.

A potassium ion channel is involved in cytokine production by activated human macrophages.

Macrophages play an important role in immune and inflammatory responses, largely through secretion of bioactive molecule such as cytokines. While calcium is known to be an important regulator of this process, less is known about the role of other ions and the ion channels that regulate them. We have previously implicated an outwardly rectifying potassium channel (Kor) in this process and for this reason we have investigated the role of potassium (K+) and K+ channels in the regulation of tumour necrosis factor-alpha (TNF-alpha)and interleukin (IL)-8 production by activated human culture-derived macrophages. The effect of blockade of Kor is to inhibit phorbol myristate acetate (PMA)-induced cytokine production by translational or post-translational mechanisms, an effect that is duplicated by increasing extracellular K+. By contrast, the effects of K+ on LPS-stimulated cells are far more complex and are probably mediated through the change of osmolality and occur largely at the mRNA level. This data directly implicates K+, and its regulation through Kor, in early events following PMA stimulation of these cells.

Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution.

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.

Therapeutic drug monitoring: antiarrhythmic drugs.

Antiarrhythmic agents are traditionally classified according to Vaughan Williams into four classes of action. Class I antiarrhythmic agents include most of the drugs traditionally thought of as antiarrhythmics, and have as a common action, blockade of the fast-inward sodium channel on myocardium. These agents have a very significant toxicity, and while they are being used less, therapeutic drug monitoring (TDM) does significantly increase the safety with which they can be administered. Class II agents are antisympathetic drugs, particularly the b-adrenoceptor blockers. These are generally safe agents which do not normally require TDM. Class III antiarrhythmic agents include sotalol and amiodarone. TDM can be useful in the case of amiodarone to monitor compliance and toxicity but is generally of little value for sotalol. Class IV antiarrhythmic drugs are the calcium channel blockers verapamil and diltiazem. These are normally monitored by haemodynamic effects, rather than using TDM. Other agents which do not fall neatly into the Vaughan Williams classification include digoxin and perhexiline. TDM is very useful for monitoring the administration (and particularly the safety) of both of these agents.

The diagnostic accuracy of mammography in the evaluation of male breast disease.

BACKGROUND: The role of mammography in the evaluation of male patients presenting with breast disease is controversial. This controversy is a function of the lack of specific data concerning the diagnostic accuracy of mammography when used in this clinical setting. The purpose of this study was to define the diagnostic accuracy of mammography in the evaluation of male breast disease. METHODS: One hundred and four prebiopsy mammograms from 100 patients with tissue diagnoses were read blindly by two independent radiologists, and placed into one of five predetermined categories: definitely malignant, possibly malignant, gynecomastia, benign mass, and normal. Radiologic/pathologic correlation was performed and the sensitivity (Sn), specificity (Sp), positive (Ppv) and negative predictive value (Npv), and accuracy (Ac) for each of the mammographic diagnostic category determined. RESULTS: The pathologic diagnoses were 12 cancers, including 1 patient with bilateral breast cancer, 70 cases of gynecomastia, 16 benign masses, and 6 normals. The accuracy data for the mammographic diagnostic categories are as follows: malignant (combined definitely and possibly malignant), Sn 92%, Sp 90%, Ppv 55%, Npv 99%, Ac 90%; and overall benignity (combined gynecomastia, benign mass, and normal), Sn 90%, Sp 92%, Ppv 99%, Npv 55%, Ac 90%. Six cancers (50%) coexisted with gynecomastia. CONCLUSIONS: Mammography can accurately distinguish between malignant and benign male breast disease. Although not a replacement for clinical examination, its routine use could substantially reduce the need for biopsy in patients whose mammograms and clinical examination suggest benign disease.

HERG K+ channels: friend and foe.

The K+ channel encoded by the human ether-à-go-go related gene (HERG) is one of many ion channels that are crucial for normal action potential repolarization in cardiac myocytes. HERG encodes the pore-forming subunit of the rapid component of the delayed rectifier K+ channel, I(K(Vr)). HERG K+ channels are of considerable pharmaceutical interest as possible therapeutic targets for anti-arrhythmic agents and as the molecular target responsible for the cardiac toxicity of a wide range of pharmaceutical agents. Recent studies of the molecular basis of the promiscuity of HERG K+ channel drug binding has not only started to shed light on this tricky pharmaceutical problem but has also provided further insights into the structure and function of HERG K+ channels.

Comparative effects of azimilide and ambasilide on the human ether-a-go-go-related gene (HERG) potassium channel.

OBJECTIVE: To evaluate the effects of azimilide and ambasilide on the biophysical properties of the human-ether-a-go-go-related (HERG) channel. METHODS: HERG was stably transfected into Chinese hamster ovary (CHO-K1) cells and currents were measured using a whole cell, voltage-clamp technique. RESULTS: Azimilide had a 'dual effect', inhibiting current at voltage steps above -40 mV and augmenting current at -40 and -50 mV. Tail current inhibition following a step to +30 mV did not vary with temperature (IC(50) 610 nM at 22 degrees C and 560 nM at 37 degrees C). The agonist effect at -50 mV was concentration-dependent and correlated with a hyperpolarizing shift in the V(1/2) of activation (r=0.98, P<0.05). Time constants of inactivation were faster and there was a -10 mV shift in the V(1/2) of steady state inactivation suggestive of open and inactivated state binding. By comparison, ambasilide inhibited HERG channels with lower potency (IC(50) 3.6 microM), in a voltage- and time-dependent but frequency-independent manner (0.03-1 Hz). Ambasilide had no effect on activation or inactivation gating but prolonged both fast and slow components of deactivation consistent with unbinding from the open state. The net effect of both drugs was similar during a voltage ramp which simulated a cardiac action potential. CONCLUSIONS: Inhibition of HERG channels by azimilide and ambasilide exhibits a similar time and voltage-dependence. While both exhibit affinity for the open state, azimilide also binds to inactivated channels.

Inhibition of HERG potassium channels by the antimalarial agent halofantrine.

Halofantrine is a widely used antimalarial agent which has been associated with prolongation of the 'QT interval' of the electrocardiogram (ECG), torsades de pointes and sudden death. Whilst QT prolongation is consistent with halofantrine-induced increases in cardiac ventricular action potential duration, the cellular mechanism for these observations has not been previously reported. The delayed rectifier potassium channel, I(Kr), is a primary site of action of drugs causing QT prolongation and is encoded by the human-ether-a-go-go-related gene (HERG). We examined the effects of halofantrine on HERG potassium channels stably expressed in Chinese hamster ovary (CHO-K1) cells. Halofantrine blocked HERG tail currents elicited on repolarization to -60 mV from +30 mV with an IC(50) of 196.9 nM. The therapeutic plasma concentration range for halofantrine is 1.67-2.98 microM. Channel inhibition by halofantrine exhibited time-, voltage- and use-dependence. Halofantrine did not alter the time course of channel activation or deactivation, but inactivation was accelerated and there was a 20 mV hyperpolarizing shift in the mid-activation potential of steady-state inactivation. Block was enhanced by pulses that render channels inactivated, and channel blockade increased with increasing duration of depolarizing pulses. We conclude that HERG channel inhibition by halofantrine is the likely underlying cellular mechanism for QT prolongation. Our data suggest preferential binding of halofantrine to the open and inactivated channel states.

Functional characterization of the NCC27 nuclear protein in stable transfected CHO-K1 cells.

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO-K1 and dominantly localized to the nucleus and nuclear membrane. When CHO-K1 cells are transfected with NCC27-expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane-resident channel in its own right. In CHO-K1 cells transfected with epitope-tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.

The nuclear chloride ion channel NCC27 is involved in regulation of the cell cycle.

NCC27 is a nuclear chloride ion channel, identified in the PMA-activated U937 human monocyte cell line. NCC27 mRNA is expressed in virtually all cells and tissues and the gene encoding NCC27 is also highly conserved. Because of these factors, we have examined the hypothesis that NCC27 is involved in cell cycle regulation. Electrophysiological studies in Chinese hamster ovary (CHO-K1) cells indicated that NCC27 chloride conductance varied according to the stage of the cell cycle, being expressed only on the plasma membrane of cells in G2/M phase. We also demonstrate that Cl- ion channel blockers known to block NCC27 led to arrest of CHO-K1 cells in the G2/M stage of the cell cycle, the same stage at which this ion channel is selectively expressed on the plasma membrane. These data strongly support the hypothesis that NCC27 is involved, in some as yet undetermined manner, in regulation of the cell cycle.

Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride: affinity for open and inactivated states.

1 Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high affinity blockade of IKr (encoded by HERG). 2 In a chronic transfection model using CHO-K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC50 16. 4 nM at 20-22 degrees C and 23.6 nM at 37 degrees C). 3 Channel inhibition exhibited time-, voltage- and frequency-dependence. In an envelope of tails test, channel blockade increased from 27+/-8% after a 120 ms depolarizing step to 50+/-4% after a 1.0 s step. These findings suggested affinity for open and/or inactivated channel states. 4 Inactivation was significantly accelerated by cisapride in a concentration-dependent manner and there was a small (-7 mV) shift in the voltage dependence of steady state inactivation. 5 Channel blockade by cisapride was modulated by [K+]o, with a 26% reduction in the potency of channel blockade when [K+]o was increased from 1 to 10 mM. 6 In conclusion, HERG channel inhibition by cisapride exhibits features consistent with open and inactivated state binding and is sensitive to external potassium concentration. These features may have significant clinical implications with regard to the mechanism and treatment of cisapride-induced proarrhythmia.

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.

1. The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. 2. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. 3. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. 4. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8 +/- 0.2 and 5.3 +/- 0.4 ms for activation and 20.0 +/- 1.2 and 96.6+/-6.7 ms for inactivation at +45 mV at 23 degrees C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation= -6.7+/-2.5 mV, V1,2 of steady state inactivation= -51.3+/-0.2 mV at 23 degrees C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37 degrees C) compared to room temperature (23 degrees C). 5. The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 micromol L(-1). Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.