Effective silencing of ENaC by siRNA delivered with epithelial-targeted nanocomplexes in human cystic fibrosis cells and in mouse lung.

INTRODUCTION: Loss of the cystic fibrosis transmembrane conductance regulator in cystic fibrosis (CF) leads to hyperabsorption of sodium and fluid from the airway due to upregulation of the epithelial sodium channel (ENaC). Thickened mucus and depleted airway surface liquid (ASL) then lead to impaired mucociliary clearance. ENaC regulation is thus a promising target for CF therapy. Our aim was to develop siRNA nanocomplexes that mediate effective silencing of airway epithelial ENaC in vitro and in vivo with functional correction of epithelial ion and fluid transport. METHODS: We investigated translocation of nanocomplexes through mucus and their transfection efficiency in primary CF epithelial cells grown at air-liquid interface (ALI).Short interfering RNA (SiRNA)-mediated silencing was examined by quantitative RT-PCR and western analysis of ENaC. Transepithelial potential (Vt), short circuit current (Isc), ASL depth and ciliary beat frequency (CBF) were measured for functional analysis. Inflammation was analysed by histological analysis of normal mouse lung tissue sections. RESULTS: Nanocomplexes translocated more rapidly than siRNA alone through mucus. Transfections of primary CF epithelial cells with nanocomplexes targeting αENaC siRNA, reduced αENaC and ßENaC mRNA by 30%. Transfections reduced Vt, the amiloride-sensitive Isc and mucus protein concentration while increasing ASL depth and CBF to normal levels. A single dose of siRNA in mouse lung silenced ENaC by approximately 30%, which persisted for at least 7 days. Three doses of siRNA increased silencing to approximately 50%. CONCLUSION: Nanoparticle-mediated delivery of ENaCsiRNA to ALI cultures corrected aspects of the mucociliary defect in human CF cells and offers effective delivery and silencing in vivo.

Increased apical Na+ permeability in cystic fibrosis is supported by a quantitative model of epithelial ion transport.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an anion channel. In the human lung CFTR loss causes abnormal ion transport across airway epithelial cells. As a result CF individuals produce thick mucus, suffer persistent bacterial infections and have a much reduced life expectancy. Trans-epithelial potential difference (Vt) measurements are routinely carried out on nasal epithelia of CF patients in the clinic. CF epithelia exhibit a hyperpolarised basal Vt and a larger Vt change in response to amiloride (a blocker of the epithelial Na(+) channel, ENaC). Are these altered bioelectric properties solely a result of electrical coupling between the ENaC and CFTR currents, or are they due to an increased ENaC permeability associated with CFTR loss? To examine these issues we have developed a quantitative mathematical model of human nasal epithelial ion transport. We find that while the loss of CFTR permeability hyperpolarises Vt and also increases amiloride-sensitive Vt, these effects are too small to account for the magnitude of change observed in CF epithelia. Instead, a parallel increase in ENaC permeability is required to adequately fit observed experimental data. Our study provides quantitative predictions for the complex relationships between ionic permeabilities and nasal Vt, giving insights into the physiology of CF disease that have important implications for CF therapy.

Method for estimating the tip geometry of scanning ion conductance microscope pipets.

Scanning ion conductance microscopy (SICM) offers the ability to perform contact-free, high-resolution imaging of biological cells and tissues at physiological conditions. However, imaging resolution is highly dependent on the geometry of the SICM probe, which is generally not known. Small, high-resolution probes are too fine to image optically and, to date, geometry estimation has usually required electron microscopy (EM). This is time-consuming and prone to failure and cannot provide information about the crucial internal geometry of the probe. Here we demonstrate a new method for determining SICM tip geometry that overcomes the limitations of EM imaging. The method involves fitting an analytical model to current changes during quasi-controlled breakage of the pipet tip. The data can be routinely obtained using the SICM apparatus itself and our method thus opens the way for substantially better quantification in SICM imaging and measurement.

Nanoscale live-cell imaging using hopping probe ion conductance microscopy.

We describe hopping mode scanning ion conductance microscopy that allows noncontact imaging of the complex three-dimensional surfaces of live cells with resolution better than 20 nm. We tested the effectiveness of this technique by imaging networks of cultured rat hippocampal neurons and mechanosensory stereocilia of mouse cochlear hair cells. The technique allowed examination of nanoscale phenomena on the surface of live cells under physiological conditions.

A Nanosensor Toolbox for Rapid, Label-Free Measurement of Airway Surface Liquid and Epithelial Cell Function.

Ciliated lung epithelial cells and the airway surface liquid (ASL) comprise one of the body's most important protective systems. This system is finely tuned, and perturbations to ASL rheology, ASL depth, ASL pH, the transepithelial potential, and the cilia beat frequency are all associated with disease pathology. Further, these apparently distinct properties interact with each other in a complex manner. For example, changes in ASL rheology can result from altered mucin secretion, changes in ASL pH, or changes in ASL depth. Thus, one of the great challenges in trying to understand airway pathology is that the properties of the ASL/epithelial cell system need to be assessed near-simultaneously and without perturbing the sample. Here, we show that nanosensor probes mounted on a scanning ion conductance microscope make this possible for the first time, without any need for labeling. We also demonstrate that ASL from senescence-retarded human bronchial epithelial cells retains its native properties. Our results demonstrate that by using a nanosensor approach, it is possible to pursue faster, more accurate, more coherent, and more informative studies of ASL and airway epithelia in health and disease.

Somatodendritic release of glutamate regulates synaptic inhibition in cerebellar Purkinje cells via autocrine mGluR1 activation.

In the cerebellum, the process of retrograde signaling via presynaptic receptors is important for the induction of short- and long-term changes in inhibitory synaptic transmission at interneuron-Purkinje cell (PC) synapses. Endocannabinoids, by activating presynaptic CB1 receptors, mediate a short-term decrease in inhibitory synaptic efficacy, whereas glutamate, acting on presynaptic NMDA receptors, induces a longer-latency sustained increase in GABA release. We now demonstrate that either low-frequency climbing fiber stimulation or direct somatic depolarization of Purkinje cells results in SNARE-dependent vesicular release of glutamate from the soma and dendrites of PCs. The activity-dependent release of glutamate caused the activation of postsynaptic metabotropic glutamate receptor 1 (mGluR1) on PC somatodendritic membranes, resulting in the cooperative release of endocannabinoids and an mGluR1-mediated slow membrane conductance. The activity of excitatory amino acid transporters regulated the spatial spread of glutamate and thus the extent of PC mGluR1 activation. We propose that activity-dependent somatodendritic glutamate release and autocrine activation of mGluR1 on PCs provides a powerful homeostatic mechanism to dynamically regulate inhibitory synaptic transmission in the cerebellar cortex.

A functional role for small-conductance calcium-activated potassium channels in sensory pathways including nociceptive processes.

We investigated the role of small-conductance calcium-activated potassium (SK) and intermediate-conductance calcium-activated potassium channels in modulating sensory transmission from peripheral afferents into the rat spinal cord. Subunit-specific antibodies reveal high levels of SK3 immunoreactivity in laminas I, II, and III of the spinal cord. Among dorsal root ganglion neurons, both peripherin-positive (C-type) and peripherin-negative (A-type) cells show intense SK3 immunoreactivity. Furthermore, dorsal root-stimulated sensory responses recorded in vitro are inhibited when SK channel activity is increased with 1-ethyl-2-benzimidazolinone (1-EBIO). In vivo electrophysiological recordings show that neuronal responses to naturally evoked nociceptive and nonnociceptive stimuli increase after application of the selective SK channel blocker 8,14-diaza-1,7(1,4)-diquinolinacyclotetradecaphanedium di-trifluoroacetate (UCL 1848), indicating that SK channels are normally active in moderating afferent input. Conversely, neuronal responses evoked by mechanical stimuli are inhibited when SK channel activity is increased with 1-EBIO. These effects are reversed by the subsequent application of UCL 1848. Our data demonstrate that SK channels have an important role in controlling sensory input into the spinal cord.

Nanoscale-targeted patch-clamp recordings of functional presynaptic ion channels.

Direct electrical access to presynaptic ion channels has hitherto been limited to large specialized terminals such as the calyx of Held or hippocampal mossy fiber bouton. The electrophysiology and ion-channel complement of far more abundant small synaptic terminals (≤ 1 µm) remain poorly understood. Here we report a method based on superresolution scanning ion conductance imaging of small synapses in culture at approximately 100-150 nm 3D resolution, which allows presynaptic patch-clamp recordings in all four configurations (cell-attached, inside-out, outside-out, and whole-cell). Using this technique, we report presynaptic recordings of K(+), Na(+), Cl(-), and Ca(2+) channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses.

FMRFamide tagging--how an ionotropic receptor can be used to measure peptide secretion.

In this chapter, we describe a technique, FMRFamide tagging, that in principle can be used to measure the release of any sequenced neuropeptide. The method relies upon the addition of an "electrophysiologically active" tag to the prohormone that encodes the neuropeptide of interest. Secretion of the electrophysiological tag (and thus the peptide of interest) is detected by activation of the ionotropic "tag receptor." Both the tagged prohormone and the tag receptor are expressed in the cell type under investigation. Since the tag and the neuropeptide of interest are on the same prohormone they are co-secreted and thus secretion of the tag reflects the co-secretion of the neuropeptide of interest. This method can be used to detect neuropeptide secretion on a millisecond timescale.

Small conductance Ca2+-activated K+ channels formed by the expression of rat SK1 and SK2 genes in HEK 293 cells.

The rat SK1 gene (rSK1) does not form functional Ca2+-activated potassium channels when expressed alone in mammalian cell lines. Using a selective antibody to the rSK1 subunit and a yellow fluorescent protein (YFP) tag we have discovered that rSK1 expression produces protein that remains largely at intracellular locations. We tested the idea that rSK1 may need an expression partner, rSK2, in order to form functional channels. When rSK1 was co-expressed with rSK2 in HEK 293 cells it increased the current magnitude by 77 +/- 34% (as compared with cells expressing rSK2 alone). Co-expression of rSK1 with rSK2 also changed the channel pharmacology. The sensitivity of SK current to block by apamin was reduced approximately 16-fold from an IC50 of 94 pM (for SK2 alone) to 1.4 nM (for SK2 and SK1 together). The sensitivity to block by UCL 1848 (a potent small molecule blocker of SK channels) was similarly reduced, approximately 26-fold, from an IC50 of 110 pM to 2.9 nM. These data clearly demonstrate that rSK1 and rSK2 subunits interact. The most likely explanation for this is that the subunits are able to form heteromeric assemblies.

The SK3 subunit of small conductance Ca2+-activated K+ channels interacts with both SK1 and SK2 subunits in a heterologous expression system.

The aim of this study was to determine whether functional heteromeric channels can be formed by co-assembly of rat SK3 (rSK3) potassium channel subunits with either SK1 or SK2 subunits. First, to determine whether rSK3 could co-assemble with rSK2 we created rSK3VK (an SK3 mutant insensitive to block by UCL 1848). When rSK3VK was co-expressed with rSK2 the resulting currents had an intermediate sensitivity to UCL 1848 (IC50 of approximately 5 nM compared with 120 pM for rSK2 and >300 nM for rSK3VK), suggesting that rSK3 and rSK2 can form functional heteromeric channels. To detect co-assembly of SK3 with SK1, we initially used a dominant negative construct of the human SK1 subunit (hSK1YP). hSK1YP dramatically reduced the SK3 current, supporting the idea that SK3 and SK1 subunits also interact. To determine whether these assemblies were functional we created rSK3VF, an rSK3 mutant with an enhanced affinity for tetraethylammonium chloride (TEA) (IC50 of 0.3 mM). Co-transfection of rSK3VF and hSK1 produced currents with a sensitivity to TEA not different from that of hSK1 alone (IC50 approximately 15 mM). These results suggest that hSK1 does not produce functional cell-surface assemblies with SK3. Antibody-staining experiments suggested that hSK1 may reduce the number of functional SK3 subunits reaching the cell surface. Additional experiments showed that co-expression of the rat SK1 gene with SK3 also dramatically suppressed SK current. The pharmacology of the residual current was consistent with that of homomeric SK3 assemblies. These results demonstrate interactions that cause changes in protein trafficking, cell surface expression, and channel pharmacology and strongly suggest heteromeric assembly of SK3 with the other SK channel subunits.

Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system.

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.