David C. H. Benton

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.

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.

Enhancement of Hippocampal Pyramidal Cell Excitability by the Novel Selective Slow-Afterhyperpolarization Channel Blocker 3-(Triphenylmethylaminomethyl)pyridine (UCL2077)

The slow afterhyperpolarization (sAHP) in hippocampal neurons has been implicated in learning and memory. However, its precise role in cell excitability and central nervous system function has not been explicitly tested for 2 reasons: 1) there are, at present, no selective inhibitors that effectively reduce the underlying current in vivo or in intact in vitro tissue preparations, and 2) although it is known that a small conductance K+ channel that activates after a rise in [Ca2+]i underlies the sAHP, the exact molecular identity remains unknown. We show that 3-(triphenylmethylaminomethyl)pyridine (UCL2077), a novel compound, suppressed the sAHP present in hippocampal neurons in culture (IC50 = 0.5 μM) and in the slice preparation (IC50 ≈ 10 μM). UCL2077 was selective, having minimal effects on Ca2+ channels, action potentials, input resistance and the medium afterhyperpolarization. UCL2077 also had little effect on heterologously expressed small conductance Ca2+-activated K+ (SK) channels. Moreover, UCL2077 and apamin, a selective SK channel blocker, affected spike firing in hippocampal neurons in different ways. These results provide further evidence that SK channels are unlikely to underlie the sAHP. This study also demonstrates that UCL2077, the most potent, selective sAHP blocker described so far, is a useful pharmacological tool for exploring the role of sAHP channels in the regulation of cell excitability in intact tissue preparations and, potentially, in vivo.

Differences in the actions of some blockers of the calcium‐activated potassium permeability in mammalian red cells

The actions of some inhibitors of the Ca2+‐activated K+ permeability in mammalian red cells have been compared. Block of the permeability was assessed from the reduction in the net loss of K+ that followed the application of the Ca2+ ionophore A23187 (2 μM) to rabbit red cells suspended at a haematocrit of 1% in a low potassium solution ([K]0 0.12–0.17 mM) at 37°C. Net movement of K+ was measured using a K+‐sensitive electrode placed in the suspension. The concentrations (μM±s.d.) of the compounds tested causing 50% inhibition of K+ loss were: quinine, 37±3; cetiedil, 26±1; the cetiedil congeners UCL 1269, UCL 1274 and UCL 1495, ∼150, 8.2±0.1, 0.92±0.03 respectively; clotrimazole, 1.2±0.1; nitrendipine, 3.6±0.5 and charybdotoxin, 0.015±0.002. The characteristics of the block suggested that compounds could be placed in two groups. For one set (quinine, cetiedil, and the UCL congeners), the concentration‐inhibition curves were steeper (Hill coefficient, nH, 2.7) than for the other (clotrimazole, nitrendipine, charybdotoxin) for which nH∼amp;1. Compounds in the first set alone became less active on raising the concentration of K+ in the external solution to 5.4 mM. The rate of K+ loss induced by A23187 slowed in the presence of high concentrations of cetiedil and its analogues, suggesting a use‐dependent component to the inhibitory action. This was not seen with clotrimazole. The blocking action of the cetiedil analogue UCL 1274 could not be overcome by an increase in external Ca2+ and its potency was unaltered when K+ loss was induced by the application of Pb2+ (10 μM) rather than by A23187. These results, taken with the findings of others, suggest that agents that block the red cell Ca2+‐activated K+ permeability can be placed in two groups with different mechanisms of action. The differences can be explained by supposing that clotrimazole and charybdotoxin act at the outer face of the channel whereas cetiedil and its congeners may block within it, either at or near the K+ binding site that determines the flow of K+.

The Synthesis and Some Pharmacological Actions of the Enantiomers of the K+‐Channel Blocker Cetiedil

Cetiedil ((±)‐2−cyclohexyl‐2‐(3−thienyl)ethanoic acid 2‐(hexahydro‐1H‐azepin‐1−yl) ethyl ester) possesses anti‐sickling and analgesic, antispasmodic, local anaesthetic and vasodilator activities. A total synthesis and circular dichroism spectra of the enantiomers of cetiedil is described, together with a comparison of their effectiveness as blockers of the Ca2+‐activated K+ permeability of rabbit erythrocytes; the contractile response of intestinal smooth muscle to acetylcholine; the Ca2+‐dependent contraction of depolarized intestinal muscle; and the cell volume‐sensitive K+ permeability (Kvol) of liver cells.

The Relationship between Functional Inhibition and Binding for KCa2 Channel Blockers

Small conductance calcium-activated potassium channels (KCa2.1,2.2,2.3) are blocked with high affinity by both peptide toxins (e.g. apamin) and small molecule blockers (e.g. UCL 1848). In electrophysiological experiments, apamin shows subtype selectivity with IC50s of ∼100 pM and ∼1 nM for block KCa2.2 and KCa2.3 respectively. In binding studies, however, apamin appears not to discriminate between KCa2.2 and 2.3 and is reported to have a significantly higher (∼20–200-fold) affinity (∼5 pM). This discrepancy between binding and block has been suggested to reflect an unusual mode of action of apamin. However, these binding and electrophysiological block experiments have not been conducted in the same ionic conditions, so it is also possible that the discrepancy arises simply because of differences in experimental conditions. We have now examined this latter possibility. Thus, we measured 125I-apamin binding to intact HEK 293 cells expressing KCa2 channels under the same ionic conditions (i.e. normal physiological conditions) that we also used for current block measurements. We find that binding and block experiments agree well if the same ionic conditions are used. Further, the binding of apamin and other blockers showed subtype selectivity when measured in normal physiological solutions (e.g.125I-apamin bound to KCa2.2 with K L 91±40 pM and to KCa2.3 with K L 711±126 pM, while inhibiting KCa2.2 current at IC50 103±2 pM). We also examined KCa2 channel block in Ca2+ and Mg2+ free solutions that mimic conditions reported in the literature for binding experiments. Under these (non-physiological) conditions the IC50 for apamin block of KCa2.2 was reduced to 20±3 pM. Our results therefore suggest that the apparent discrepancy between blocking and binding reported in the literature can be largely accounted for by the use of non-physiological ionic conditions in binding experiments.

The effects of cetiedil and its congeners on levcromakalim-stimulated 86Rb efflux from smooth and skeletal muscle

The effects of cetiedil on levcromakalim-stimulated 86Rb efflux from rat aorta, rat anococcygeus and frog sartorius muscle have been investigated. In experiments on rat aorta, cetiedil inhibited the tracer efflux stimulated by 10 microM levcromakalim with an IC50 of 1.3 +/- 0.4 microM. In the rat anococcygeus and frog sartorius 10 microM cetiedil caused 67 +/- 11% and 84 +/- 4% inhibition of the responses to 10 microM and 50 microM levcromakalim respectively. The effect of two analogues of cetiedil, UCL 1285 (cetiedil methiodide) and UCL 1495 (triphenyl acetic acid-2-N[5-ethyl-2-methylpiperidinoethyl] ester) were also tested on rat aorta. UCL 1285 caused inhibition of the response to 10 microM levcromakalim with an IC50 of approximately 6 microM. In contrast, UCL 1495 (10 microM) had no significant effect. It is concluded that cetiedil is an effective blocker of the action of levcromakalim in smooth and skeletal muscle but does not distinguish between tissues. The relative activities of cetiedil, UCL 1285 and UCL 1495 are discussed in relation to their activity at other cetiedil-sensitive K+ channels.