Control of ion channel expression for patch clamp recordings using an inducible expression system in mammalian cell lines

BACKGROUND: Many molecular studies of ion channel function rely on the ability to obtain high quality voltage clamp recordings using the patch clamp technique. For a variety of channel types studied in mammalian cell heterologous expression systems, the lack of experimenter control over expression levels severely hinders the ability to obtain a high percentage of cells with an expression level suitable for high quality recordings. Moreover, it has been nearly impossible to obtain expression levels in mammalian cells well suited for single channel recordings. We describe here the use of an inducible promoter system in a stably transfected mammalian cell line that produces nearly 100% success in obtaining ion channel expression levels suitable for either whole cell or single ion channel studies. RESULTS: We used a tetracycline-regulated expression system to control K(+ )channel expression in a CHO (Chinese hamster ovary) cell line. Current magnitudes within a reasonably narrow range could be easily and reliably obtained for either macroscopic or single channel recordings. Macroscopic currents of 1 – 2 nA could be obtained in nearly 100% of cells tested. The desired expression level could be obtained within just 2 to 3 hours, and remained stable at room temperature. Very low expression levels of transfected channels could also be obtained, which resulted in a >70% success rate in the ability to record single channel currents from a patch. Moreover, at these low expression levels, it appeared that endogenous channels produced little or no contamination. CONCLUSION: This approach to controlling ion channel expression is relatively simple, greatly enhances the speed and efficiency with which high quality macroscopic current data can be collected, and makes it possible to easily and reliably record single channel currents in a mammalian cell heterologous expression system. Whereas we demonstrate the ability of this system to control expression levels of voltage-gated K(+ )channels, it should be applicable to all other channel types that express well in mammalian expression systems

The External TEA Binding Site and C-Type Inactivation in Voltage-Gated Potassium Channels

AbstractThe location of the tetraethylammonium (TEA) binding site in the outer vestibule of K+ channels, and the mechanism by which external TEA slows C-type inactivation, have been considered well-understood. The prevailing model has been that TEA is coordinated by four amino acid side chains at the position equivalent to Shaker T449, and that TEA prevents a constriction that underlies inactivation via a foot-in-the-door mechanism at this same position. However, a growing body of evidence has suggested that this picture may not be entirely correct. In this study, we reexamined these two issues, using both the Kv2.1 and Shaker potassium channels. In contrast to results previously obtained with Shaker, substitution of the tyrosine at Kv2.1 position 380 (equivalent to Shaker 449) with a threonine or cysteine had a relatively minor effect on TEA potency. In both Kv2.1 and Shaker, modification of cysteines at position 380/449 by 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET) proceeded at identical rates in the absence and presence of TEA. Additional experiments in Shaker demonstrated that TEA bound well to C-type inactivated channels, but did not interfere with MTSET modification of C449 in inactivated channels. Together, these findings rule out the possibility that TEA binding involves an intimate interaction with the four side chains at the position equivalent to Shaker 449. Moreover, these results argue against the model whereby TEA slows inactivation via a foot-in-the-door mechanism at position 449, and also argue against the hypothesis that the position 449 side chains move toward the center of the conduction pathway during inactivation. Occupancy by TEA completely prevented MTSET modification of a cysteine in the outer-vestibule turret (Kv2.1 position 356/Shaker position 425), which has been shown to interfere with both TEA binding and the interaction of K+ with an external binding site. Together, these data suggest that TEA is stabilized in a more external position in the outer vestibule, and does not bind via direct coordination with any specific outer-vestibule residues

Control of Single Channel Conductance in the Outer Vestibule of the Kv2.1 Potassium Channel

Current magnitude in Kv2.1 potassium channels is modulated by external [K+]. In contrast to behavior expected from the change in electrochemical driving force, outward current through Kv2.1 channels becomes larger when extracellular [K+] is increased within the physiological range. The mechanism that underlies this unusual property involves the opening of Kv2.1 channels into one of two different outer vestibule conformations, which are defined by their sensitivity to TEA. Channels that open into a TEA-sensitive conformation generate larger macroscopic currents, whereas channels that open into a TEA-insensitive conformation generate smaller macroscopic currents. At higher [K+], more channels open into the TEA-sensitive conformation. In this manuscript, we examined the mechanism by which the conformational change produced a change in current magnitude. We started by testing the simplest hypothesis: that each pharmacologically defined channel conformation produces a different single channel conductance, one smaller and one larger, and that the [K+]-dependent change in current magnitude reflects the [K+]-dependent change in the percentage of channels that open into each of the two conformations. Using single channel and macroscopic recordings, as well as hidden Markov modeling, we were able to quantitatively account for [K+]-dependent regulation of macroscopic current with this model. Combined with previously published work, these results support a model whereby an outer vestibule lysine interferes with K+ flux through the channel, and that the [K+]-dependent change in orientation of this lysine alters single channel conductance by changing the level of this interference. Moreover, these results provide an experimental example of single channel conductance being modulated at the outer end of the conduction pathway by a mechanism that involves channel activation into open states with different outer vestibule conformations

synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses

Comparison across 12 countries on knowledge, attitude, and behavior scores about medication errors in Intensive Care Units : an international study

OBJECTIVE: The aim of the study was to explore the degree of agreement of intensive care unit nurses working on a set of medication error preventive strategies and to examine possible predictors of nurses’ knowledge, attitude and behavior. MATERIALS AND METHODS: Observational, international, and cross-sectional study. Iran, Malta, Spain, Pakistan, Nepal, Qatar, Ecuador, Australia, Finland, Italy, Egypt, and Jordan were the countries included in this survey. To collect data, the Knowledge, Attitude and Behavior in Medication Errors questionnaire was used. A descriptive statistical analysis was performed for the socio-demographic characteristics of the sample and three multiple logistic regressions were performed. RESULTS: The international sample consists of 1383 nurses, of whom 478 (34.6%) were men and 900 (65.1%) were women and their mean age was 35.61 years with a range of 19-61. Descriptive statistics conducted on the international sample show a medium to high degree of agreement among participants concerning some preventive strategies of medication error. In addition, the results of the present study show a strong relationship between positive nurses’ attitudes and correct behaviors and/or adequate knowledge, as well as between adequate knowledge and correct behaviors (p< 0.01). CONCLUSIONS: Further studies are needed to explore the issue of medication error concerning nurses’ cultural backgrounds, as well as to assess similarities and disparities among international nurses.peer-reviewe

Effect of External pH on Activation of the Kv1.5 Potassium Channel

We studied the mechanism by which external acidification from pH 7.3 to 6.8 reduced current magnitude in the Kv1.5 potassium channel. At physiological external [K(+)], a shift in the voltage-dependence of activation was entirely responsible for the acidification-induced decrease in Kv1.5 current magnitude (pK = 7.15). Elevation of external [Ca(2+)] or [Mg(2+)] identically shifted activation curves to the right and identically shifted the pH-sensitivity of the activation curves to more acidic values. Similar observations were made with the Kv2.1 K(+) channel, except that the pK for the activation shift was out of the physiological range. These data are consistent with a mechanism by which acidification shifted activation via modification of a local surface potential. Elimination of eight positive charges within the outer vestibule of the conduction pathway had no effect on the voltage-dependence of activation at pH 7.3 or higher, which suggested that sites exposed to the conduction pathway within the outer vestibule did not directly contribute to the relevant local surface potential. However, mutations at position 487 (within the conduction pathway) displaced the pK of the pH-sensitive shift in activation, such that the sensitivity of Kv1.5 current to physiologically relevant changes in pH was reduced or eliminated. These results suggest that, among voltage-gated K(+) channels, activation in Kv1.5 is uniquely sensitive to physiologically relevant changes in pH because the pK for the sites that contribute to the local surface potential effect is near pH 7. Moreover, the pK for the activation shift depends not only on the nature of the sites involved but also on structural orientation conferred, in part, by at least one residue within the conduction pathway

Natural Bizbenzoquinoline Derivatives Protect Zebrafish Lateral Line Sensory Hair Cells from Aminoglycoside Toxicity

Moderate to severe hearing loss affects 360 million people worldwide and most often results from damage to sensory hair cells. Hair cell damage can result from aging, genetic mutations, excess noise exposure, and certain medications including aminoglycoside antibiotics. Aminoglycosides are effective at treating infections associated with cystic fibrosis and other life-threatening conditions such as sepsis, but cause hearing loss in 20–30% of patients. It is therefore imperative to develop new therapies to combat hearing loss and allow safe use of these potent antibiotics. We approach this drug discovery question using the larval zebrafish lateral line because zebrafish hair cells are structurally and functionally similar to mammalian inner ear hair cells and respond similarly to toxins. We screened a library of 502 natural compounds in order to identify novel hair cell protectants. Our screen identified four bisbenzylisoquinoline derivatives: berbamine, E6 berbamine, hernandezine, and isotetrandrine, each of which robustly protected hair cells from aminoglycoside-induced damage. Using fluorescence microscopy and electrophysiology, we demonstrated that the natural compounds confer protection by reducing antibiotic uptake into hair cells and showed that hair cells remain functional during and after incubation in E6 berbamine. We also determined that these natural compounds do not reduce antibiotic efficacy. Together, these natural compounds represent a novel source of possible otoprotective drugs that may offer therapeutic options for patients receiving aminoglycoside treatment