The patch-clamp technique and more recently the high throughput patch-clamp technique have contributed to major advances in the characterization of ion channels. However, the whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that increasing current amplitude profoundly impacts the accuracy of the biophysical analyses of macroscopic ion currents under study. Using mathematical kinetic models of a cardiac voltage-gated sodium channel and a cardiac voltage-gated potassium channel, we demonstrated how large current amplitude and series resistance artefacts induce an undetected alteration in the actual membrane potential and affect the characterization of voltage-dependent activation and inactivation processes. We also computed how dose-response curves are hindered by high current amplitudes. This is of high interest since stable cell lines frequently demonstrating high current amplitudes are used for safety pharmacology using the high throughput patch-clamp technique. It is therefore critical to set experimental limits for current amplitude recordings to prevent inaccuracy in the characterization of channel properties or drug activity, such limits being different from one channel type to another. Based on the predictions generated by the kinetic models, we draw simple guidelines for good practice of whole-cell voltage-clamp recordings.

Long Q-T syndrome (LQTS) is an ion channel disease of the heart featuring single gene inheritance. It is characterized by prolonged QT interval, abnormal T wave, torsade de points (TdP) on electrocardiogram, with recurrent syncope, convulsion and even sudden death. Although the overall prevalence of LQTS is not high, the disease has attracted attention of cardiologists for its high incidence of sudden cardiac death. The compilation of this guideline has referred to the consensus of basic and clinical research, guidelines of other countries, and summarized the clinical manifestations, molecular basis, diagnostic criteria, treatment and prognosis, and genetic counseling of LQTS, with an aim to standardize its clinical diagnosis and treatment.

Measurement of axonal excitability provides an in vivo indication of the properties of the nerve membrane and of the ion channels expressed on these axons. Axonal excitability techniques have been utilised to investigate the pathophysiological mechanisms underlying neurological diseases. This document presents guidelines derived for such studies, based on a consensus of international experts, and highlights the potential difficulties when interpreting abnormalities in diseased axons. The present manuscript provides a state-of-the-art review of the findings of axonal excitability studies and their interpretation, in addition to suggesting guidelines for the optimal performance of excitability studies.

Intensive scientific research devoted in the recent years to understand the molecular mechanisms or neurodegeneration in spinocerebellar ataxias (SCAs) are identifying new pathways and targets providing new insights and a better understanding of the molecular pathogenesis in these diseases. In this consensus manuscript, the authors discuss their current views on the identified molecular processes causing or modulating the neurodegenerative phenotype in spinocerebellar ataxias with the common opinion of translating the new knowledge acquired into candidate targets for therapy. The following topics are discussed: transcription dysregulation, protein aggregation, autophagy, ion channels, the role of mitochondria, RNA toxicity, modulators of neurodegeneration and current therapeutic approaches. Overall point of consensus includes the common vision of neurodegeneration in SCAs as a multifactorial, progressive and reversible process, at least in early stages. Specific points of consensus include the role of the dysregulation of protein folding, transcription, bioenergetics, calcium handling and eventual cell death with apoptotic features of neurons during SCA disease progression. Unresolved questions include how the dysregulation of these pathways triggers the onset of symptoms and mediates disease progression since this understanding may allow effective treatments of SCAs within the window of reversibility to prevent early neuronal damage. Common opinions also include the need for clinical detection of early neuronal dysfunction, for more basic research to decipher the early neurodegenerative process in SCAs in order to give rise to new concepts for treatment strategies and for the translation of the results to preclinical studies and, thereafter, in clinical practice.

Developing an understanding of the mechanism of voltage-gated ion channels in molecular terms requires knowledge of the structure of the active and resting conformations. Although the active-state conformation is known from x-ray structures, an atomic resolution structure of a voltage-dependent ion channel in the resting state is not currently available. This has motivated various efforts at using computational modeling methods and molecular dynamics (MD) simulations to provide the missing information. A comparison of recent computational results reveals an emerging consensus on voltage-dependent gating from computational modeling and MD simulations. This progress is highlighted in the broad context of preexisting work about voltage-gated channels.

Mutations that inhibit Kv11.1 ion channel activity contribute to abnormalities of cardiac repolarization that can lead to long QT2 (LQT2) cardiac arrhythmias and sudden death. However, for most of these mutations, nothing is known about the molecular mechanism linking Kv11.1 malfunction to cardiac death. We have previously demonstrated that disease-related mutations that create consensus sites for kinases on ion channels can dramatically change ion channel activity. Here, we show that a LQT2-associated mutation can inhibit Kv11.1 ion channel activity by perturbing a consensus site for the Ser/Thr protein kinase C α (PKCα). We first reveal by mass spectrometry analysis that Ser890 of the Kv11.1 ion channel is phosphorylated. Then, we demonstrate by a phospho-detection immunoassay combined with genetic manipulation that PKCα phosphorylates Ser890. Furthermore, we show that Ser890 phosphorylation is associated with an increase in Kv11.1 membrane density with alteration of recovery from inactivation. In addition, a newly discovered and as yet uncharacterized LQT2-associated nonsynonymous single nucleotide polymorphism 2660 G→A within the human ether-á-go-go-related gene 1 coding sequence, which replaces arginine 887 with a histidine residue (R887H), strongly inhibits PKCα-dependent phosphorylation of residue Ser890 on Kv11.1, and ultimately inhibits surface expression and current density. Taken together, our data provide a functional link between this channel mutation and LQT2.

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Transcription factors (TFs) are proteins which bind to specific DNA sequences and thus participate in the regulation of the initiation of transcription. We report in this communication our observations that several of these proteins interact with lipid membranes and form ion-permeable channels. For each of the TFs that we studied, the single channel conductance was distinctively different, i.e. each TF had its own electrical signature. More importantly, we show for the first time that addition of cognate double-stranded DNA sequences leads to a specific response: an increase in the conductance of the TF-containing membrane. Strikingly, the effect of cognate DNA was observed when it was added to the trans-side of the membrane (opposite to where the TF was added), strongly suggesting that the TFs span the membrane and that the DNA-binding domain is trans-accessible. Alterations in the primary structure of the TF factors in their basic and DNA-binding regions change the characteristics of the conductance of the protein-containing membranes as well as the response to DNA addition, reinforcing the notion that the changes we measure are due to specific interactions.

Perfused rat liver is a well-established model for studies on hepatic metabolism. The different perfusion systems, the technical requirements and the surgical preparation steps are described. A main advantage of liver perfusion is the maintenance of liver architecture-rendering it a feasible model for the study of interactions between parenchymal and non-parenchymal cells. Furthermore, steady-state conditions allow the calculation of metabolic flux rates and the reversibility of agonist-induced effects can be studied within the same preparation. As the polarity of the cells is maintained, sinusoidal uptake, metabolism and biliary excretion of substances can be studied. Some applications of liver perfusion and special techniques are described, the advantages specified and the limitations of this model discussed. Due to recent developments in monitoring of liver hemodynamics, extracellular ion concentrations and changes of liver cell volume, liver perfusion is one of the best-controlled experimental systems in the study of hepatic physiology.