Guy W. J. Moss

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.

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.

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.

SK3 is an important component of K+ channels mediating the afterhyperpolarization in cultured rat SCG neurones

1 Our aim was to identify the small‐conductance Ca2+‐activated K+ channel(s) (SK) underlying the apamin‐sensitive afterhyperpolarization (AHP) in rat superior cervical ganglion (SCG) neurones. 2 Degenerate oligonucleotide primers designed to the putative calmodulin‐binding domain conserved in all mammalian SK channel sequences were employed to detect SK DNA in a cDNA library from rat SCG. Only a single band, corresponding to a fragment of the rSK3 gene, was amplified. 3 Northern blot analysis employing a PCR‐generated rSK3 fragment showed the presence of mRNA coding for SK3 in SCG as well in other rat peripheral tissues including adrenal gland and liver. 4 The same rSK3 fragment enabled the isolation of a full‐length rSK3 cDNA from the library. Its sequence was closely similar to, but not identical with, that of the previously reported rSK3 gene. 5 Expression of the rSK3 gene in mammalian cell lines (CHO, HEK cells) caused the appearance of a K+ conductance with SK channel properties. 6 The application of selective SK blocking agents (including apamin, scyllatoxin and newer non‐peptidic compounds) showed these homomeric SK3 channels to have essentially the same pharmacological characteristics as the SCG afterhyperpolarization, but to differ from those of homomeric SK1 and SK2 channels. 7 Immunohistochemistry using a rSK3 antipeptide antibody revealed the presence of SK3 protein in the cell bodies and processes of cultured SCG neurones. 8 Taken together, these results identify SK3 as a major component of the SK channels responsible for the afterhyperpolarization of cultured rat SCG neurones.

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 ~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, ~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.

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

Summary 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 Ca2+ channels. This semiautomated approach allows direct investigation of the distribution and properties of presynaptic ion channels at small central synapses. Video Abstract

Contact-Free Scanning and Imaging with the Scanning Ion Conductance Microscope

Scanning ion conductance microscopy (SICM) offers the ability to obtain very high-resolution topographical images of living cells. One of the great advantages of SICM lies in its ability to perform contact-free scanning. However, it is not yet clear when the requirements for this scan mode are met. We have used finite element modeling (FEM) to examine the conditions for contact-free scanning. Our findings provide a framework for understanding the contact-free mode of SICM and also extend previous findings with regard to SICM resolution. Finally, we demonstrate the importance of our findings for accurate biological imaging.

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.

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

•  Cystic fibrosis (CF) is a common genetic disease caused by loss‐of‐function mutations in the cystic fibrosis transmembrane conductance regulator gene, which encodes a channel protein, selective for anions. •  In the lungs, the site of the most severe symptoms, CF causes abnormal electrolyte transport in epithelial cells which line the airways. •  Airway epithelial ion transport can be assessed by measuring the trans‐epithelial potential difference (Vt) which shows characteristic changes in CF individuals. We developed a biophysical model of ion transport in human nasal epithelia, in order to investigate quantitatively which transport parameters underlie these observed bioelectric changes. •  We found that loss of apical Cl− permeability alone is insufficient to explain the bioelectric properties of CF epithelia. An increase of apical Na+ permeability must also occur. •  This insight has important implications for our understanding of the physiology of CF disease, and hence for potential therapies aimed at correcting the CF ion transport defect.