Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes.

The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown. Using single-molecule subunit counting, we determined that the number of KCNE2 subunits in complex with the pore-forming subunits of human HCN channels differs with each HCN isoform and is dynamic with respect to concentration. These interactions can be altered by KCNE2 gene-variants with functional implications. The results provide an additional consideration necessary to understand heart rhythm, pain, and epileptic disorders.

Isoform dependent regulation of human HCN channels by cholesterol.

Cholesterol has been shown to regulate numerous ion channels. HCN channels represent the molecular correlate of If or Ih in sinoatrial node (SAN) and neuronal cells. Previous studies have implicated a role for cholesterol in the regulation of rabbit HCN4 channels with effects on pacing in the rabbit SAN. Using electrophysiological and biochemical approaches, we examined the effect of cholesterol modulation on human HCN1, HCN2 and HCN4 isoforms. Patch-clamp experiments uncovered isoform specific differences in the effect of cholesterol on gating kinetics upon depletion by MßCD or mevastatin or enrichment using MßCD/cholesterol. Most dramatically cholesterol had isoform specific effects on mode-shifting, which has been suggested to play a key role in stabilizing firing rate and preventing arrhythmic firing in SAN cells and neurons. Mode-shifting in HCN1 channels was insensitive to cholesterol manipulation, while HCN2 and HCN4 were strongly affected. Trafficking of each isoform to the plasma membrane was also affected by cholesterol modulation differentially between isoforms, however, each isoform remained localized in lipid raft domains after cholesterol depletion. These effects may contribute to the side effects of cholesterol reducing therapies including disrupted heart rhythm and neuropathic pain, as well as the susceptibility of sinus dysfunction in patients with elevated cholesterol.

Identification of a cholesterol-binding pocket in inward rectifier K(+) (Kir) channels.

Cholesterol is the major sterol component of all mammalian plasma membranes. Recent studies have shown that cholesterol inhibits both bacterial (KirBac1.1 and KirBac3.1) and eukaryotic (Kir2.1) inward rectifier K(+) (Kir) channels. Lipid-sterol interactions are not enantioselective, and the enantiomer of cholesterol (ent-cholesterol) does not inhibit Kir channel activity, suggesting that inhibition results from direct enantiospecific binding to the channel, and not indirect effects of changes to the bilayer. Furthermore, conservation of the effect of cholesterol among prokaryotic and eukaryotic Kir channels suggests an evolutionary conserved cholesterol-binding pocket, which we aimed to identify. Computational experiments were performed by docking cholesterol to the atomic structures of Kir2.2 (PDB: 3SPI) and KirBac1.1 (PDB: 2WLL) using Autodock 4.2. Poses were assessed to ensure biologically relevant orientation and then clustered according to location and orientation. The stability of cholesterol in each of these poses was then confirmed by molecular dynamics simulations. Finally, mutation of key residues (S95H and I171L) in this putative binding pocket found within the transmembrane domain of Kir2.1 channels were shown to lead to a loss of inhibition by cholesterol. Together, these data provide support for this location as a biologically relevant pocket.

Phosphoinositide regulation of inward rectifier potassium (Kir) channels.

Inward rectifier potassium (Kir) channels are integral membrane proteins charged with a key role in establishing the resting membrane potential of excitable cells through selective control of the permeation of K(+) ions across cell membranes. In conjunction with secondary anionic phospholipids, members of this family are directly regulated by phosphoinositides (PIPs) in the absence of other proteins or downstream signaling pathways. Different Kir isoforms display distinct specificities for the activating PIPs but all eukaryotic Kir channels are activated by PI(4,5)P2. On the other hand, the bacterial KirBac1.1 channel is inhibited by PIPs. Recent crystal structures of eukaryotic Kir channels in apo and lipid bound forms reveal one specific binding site per subunit, formed at the interface of N- and C-terminal domains, just beyond the transmembrane segments and clearly involving some of the key residues previously identified as controlling PI(4,5)P2 sensitivity. Computational, biochemical, and biophysical approaches have attempted to address the energetic determinants of PIP binding and selectivity among Kir channel isoforms, as well as the conformational changes that trigger channel gating. Here we review our current understanding of the molecular determinants of PIP regulation of Kir channel activity, including in context with other lipid modulators, and provide further discussion on the key questions that remain to be answered.

Activity of exporters of Escherichia coli in Corynebacterium glutamicum, and their use to increase L-threonine production.

L-Threonine is an important biotechnological product and Corynebacterium glutamicum is able to synthesize and accumulate this amino acid to high intracellular levels. We here use four exporters of Escherichia coli and show that three of them operate in C. glutamicum, with RhtA and RhtC being the most effective. Whereas RhtA was unspecific, resulting in L-homoserine together with L-threonine excretion, this was not the case with RhtC. Expression of rhtC reduced the intracellular L-threonine concentration from 140 to 11 mM and resulted in maximal excretion rates of 11.2 nmol min(-1) mg(-1) as compared to 2.3 nmol min(-1) mg(-1) obtained without rhtC expression. In combination with an ilvA mutation generated and introduced into the chromosome, an accumulation of up to 54 mM L-threonine was achieved as compared to 21 mM obtained with the ancestor strain. This shows that expression of rhtC is the pivotal point for industrial relevant L-threonine production with C. glutamicum, and might encourage in general the use of heterologous exporters in the field of white biotechnology to make full use of biosynthesis pathways.

Antibiotic-impregnated PMMA hip spacers: Current status.

The infection rate after primary hip arthroplasty lies at 1-2%. In the past few years, a two-stage protocol with the implantation of an antibiotic-loaded spacer has become a popular procedure in the treatment of infected hip joint arthroplasties. In this review, we pay special attention to the elution characteristics of the spacers, their mechanical stability and the clinical response. We conclude that hip spacers are an effective method in the treatment of hip joint infections, with success rates of over 90%.

Rupture of the anterior tibial tendon: three clinical cases, anatomical study, and literature review.

BACKGROUND: We report three cases of anterior tibial tendon ruptures and the results of an anatomical study in regard to the tendon's insertion site and a literature review. METHODS: Three patients were referred to our hospital with anterior tibial tendon ruptures. In the anatomical study, 53 feet were dissected, looking in particular for variants of the bony insertion of the tendon. RESULTS: Two patients had surgical treatment (one primary repair and one semimembranosus tendon graft) and one conservative treatment. After a mean followup of 14 weeks all patients had satisfactory outcomes. In the anatomical study, we noted three different insertion sites: in 36 feet the tendon inserted into the medial side of the cuneiform and the base of the first metatarsal bone and in 13 feet only into the medial side of the cuneiform bone. In the remaining four feet the tendon inserted into the cuneiform and the first metatarsal bone, but an additional tendon was noted taking its origin from the anterior tibial tendon near its insertion into the medial cuneiform and attaching to the proximal part of the first metatarsal. CONCLUSIONS: According to literature, surgical repair is the treatment of choice for acute ruptures and for patients with high activity levels. For chronic ruptures and patients with low demands, conservative management may lead to an equally good outcome. Knowledge of the anatomy in this region may be helpful for diagnosis and for the interpretation of intraoperative findings and choosing the most appropriate surgical procedure.

Postoperative myalgia after succinylcholine: no evidence for an inflammatory origin.

UNLABELLED: A common side effect associated with succinylcholine is postoperative myalgia. The pathogenesis of this myalgia is still unclear; inflammation has been suggested but without convincing evidence. We designed the present study to investigate whether an inflammatory reaction contributes to this myalgia. The incidence and severity of succinylcholine-associated myalgia was determined in 64 patients pretreated with saline or dexamethasone before succinylcholine (n = 32 for each). Incidence and severity of myalgia did not differ significantly between the two groups: 15 patients in the dexamethasone group complained of myalgia compared with 18 patients in the saline group, and severe myalgia was reported by five patients and three patients, respectively (not significant). At 48 h after surgery, 12 patients in both groups still suffered from myalgia (not significant). In addition, interleukin-6 (IL-6) as an early marker of inflammation was assessed in a subgroup of 10 patients pretreated with saline. We found an increase of IL-6 for only three patients, but only one patient reported myalgia; no relationship between myalgia and the increase of IL-6 was found. In conclusion, there is no evidence for an inflammatory origin of succinylcholine-associated myalgia. IMPLICATIONS: Administration of dexamethasone before succinylcholine was not effective in decreasing the incidence or the severity of succinylcholine-induced postoperative myalgia. Furthermore, there was no significant relationship between postoperative myalgia and time course of interleukin-6 concentrations, a marker of inflammation. Pretreatment with dexamethasone is not justified to prevent postoperative myalgia after succinylcholine.