Duk Su Koh

Relative abundance of subunit mRNAs determines gating and Ca2+ permeability of AMPA receptors in principal neurons and interneurons in rat CNS

Recording of glutamate-activated currents in membrane patches was combined with RT-PCR-mediated AMPA receptor (AMPAR) subunit mRNA analysis in single identified cells of rat brain slices. Analysis of AMPARs in principal neurons and interneurons of hippocampus and neocortex and in auditory relay neurons and Bergmann glial cells indicates that the GluR-B subunit in its flip version determines formation of receptors with relatively slow gating, whereas the GluR-D subunit promotes assembly of more rapidly gated receptors. The relation between Ca2+ permeability of AMPAR channels and the relative GluR-B mRNA abundance is consistent with the dominance of this subunit in determining the Ca2+ permeability of native receptors. The results suggest that differential expression of GluR-B and GluR-D subunit genes, as well as splicing and editing of their mRNAs, account for the differences in gating and Ca2+ permeability of native AMPAR channels.

Early-onset epilepsy and postnatal lethality associated with an editing-deficient GluR-B allele in mice.

The arginine residue at position 586 of the GluR-B subunit renders heteromeric α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-sensitive glutamate receptor channels impermeable to calcium. The codon for this arginine is introduced at the precursor messenger RNA (pre-mRNA) stage by site-selective adenosine editing of a glutamine codon. Heterozygous mice engineered by gene targeting to harbor an editing-incompetent GluR-B allele synthesized unedited GluR-B subunits and, in principal neurons and interneurons, expressed AMPA receptors with increased calcium permeability. These mice developed seizures and died by 3 weeks of age, showing that GluR-B pre-mRNA editing is essential for brain function.

Block of native Ca(2+)‐permeable AMPA receptors in rat brain by intracellular polyamines generates double rectification.

1. The influence of intracellular factors on current rectification of different subtypes of native alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleproprionate receptors (AMPARs) was studied in rat brain slices by combining fast application of glutamate with patch pipette perfusion. 2. The peak current‐voltage (I‐V) relation of the AMPARs expressed in Bergmann glial cells of cerebellum and dentate gyrus (DG) basket cells of hippocampus was weakly rectifying in outside‐out patches and nystatin‐perforated vesicles, but showed a doubly rectifying shape with a region of reduced slope between 0 and +40 mV in nucleated patches. The I‐V relation of AMPARs expressed in hippocampal CA3 pyramidal neurones was linear in all recording configurations. 3. Intracellular application of 25 microM spermine, a naturally occurring polyamine, blocked outward currents in outside‐out patches from Bergmann glial cells and DG basket cells in a voltage‐dependent manner, generating I‐V relations with a doubly rectifying shape which were similar to those recorded in nucleated patches. AMPARs in CA3 pyramidal cell patches were unaffected by 25 microM spermine. 4. The half‐maximal blocking concentration of spermine at +40 mV was 0.3 microM in Bergmann glial cell patches and 1.5 microM in DG basket cell patches, whereas it was much higher (>> 100 microM) for CA3 pyramidal cell patches. Spermidine also affected current rectification, but with lower affinity. The block of outward current by polyamines following voltage jumps developed within < 0.5 ms. 5. We conclude that current rectification, rather than being an intrinsic property of the Ca(2+)‐permeable AMPAR channel, is generated by polyamine block.

Ca(2+)-permeable AMPA and NMDA receptor channels in basket cells of rat hippocampal dentate gyrus

1. Glutamate receptor (GluR) channels were studied in basket cells in the dentate gyrus of rat hippocampal slices. Basket cells were identified by their location, dendritic morphology and high frequency of action potentials generated during sustained current injection. 2. Dual‐component currents were activated by fast application of glutamate to outside‐out membrane patches isolated from basket cell somata (10 microM glycine, no external Mg2+). The fast component was selectively blocked by 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX), the slow component by D‐2‐amino‐5‐phosphonopentanoic acid (D‐AP5). This suggests that the two components were mediated by alpha‐amino‐3‐ hydroxy‐5‐methyl‐4‐isoxazolepropionate receptor (AMPAR)/kainate receptor and N‐methyl‐D‐aspartate receptor (NMDAR) channels, respectively. The mean ratio of the peak current of the NMDAR component to that of the AMPAR/kainate receptor component was 0.22 (1 ms pulses of 10 mM glutamate). 3. The AMPAR/kainate receptor component, which was studied in isolation in the presence of D‐AP5, was identified as AMPAR mediated on the basis of the preferential activation by AMPA as compared with kainate, the weak desensitization of kainate‐activated currents, the cross‐desensitization between AMPA and kainate, and the reduction of desensitization by cyclothiazide. 4. Deactivation of basket cell AMPARs following 1 ms pulses of glutamate occurred with a time constant (tau) of 1.2 +/‐ 0.1 ms (mean +/‐ S.E.M.). During 100 ms glutamate pulses AMPARs desensitized with a tau of 3.7 +/‐ 0.2ms. 5. The peak current‐voltage (I‐V) relation of AMPAR‐mediated currents in Na(+)‐rich extracellular solution showed a reversal potential of ‐4.0 +/‐ 2.6 mV and was characterized by a a doubly rectifying shape. The conductance of single AMPAR channels was estimated as 22.6 +/‐ 1.6 pS using non‐stationary fluctuation analysis. AMPARs expressed in hippocampal basket cells were highly Ca2+ permeable (PCa/PK = 1.79). 6. NMDARs in hippocampal basket cells were studied in isolation in the presence of CNQX. Deactivation of NMDARs activated by glutamate pulses occurred bi‐exponentially with mean tau values of 266 +/‐ 23 ms (76%) and 2620 +/‐ 383 ms (24%). 7. The peak I‐V relation of the NMDAR‐mediated component in Na(+)‐rich extracellular solution showed a reversal potential of 1.5 +/‐ 0.6 mV and a region of negative slope at negative membrane potentials in the presence of external Mg2+, due to voltage‐dependent block by these ions. The conductance of single NMDAR channels in the main open state was 50.2 +/‐ 1.8 pS.(ABSTRACT TRUNCATED AT 400 WORDS)

Trypsin activates pancreatic duct epithelial cell ion channels through proteinase-activated receptor-2

Proteinase-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is cleaved by trypsin within the NH2-terminus, exposing a tethered ligand that binds and activates the receptor. We examined the secretory effects of trypsin, mediated through PAR-2, on well-differentiated nontransformed dog pancreatic duct epithelial cells (PDEC). Trypsin and activating peptide (AP or SLIGRL-NH2, corresponding to the PAR-2 tethered ligand) stimulated both an 125I- efflux inhibited by Ca2+-activated Cl- channel inhibitors and a 86Rb+ efflux inhibited by a Ca2+-activated K+ channel inhibitor. The reverse peptide (LRGILS-NH2) and inhibited trypsin were inactive. Thrombin had no effect, suggesting absence of PAR-1, PAR-3, or PAR-4. In Ussing chambers, trypsin and AP stimulated a short-circuit current from the basolateral, but not apical, surface of PDEC monolayers. In monolayers permeabilized basolaterally or apically with nystatin, AP activated apical Cl- and basolateral K+ conductances. PAR-2 agonists increased [Ca2+]i in PDEC, and the calcium chelator BAPTA inhibited the secretory effects of AP. PAR-2 expression on dog pancreatic ducts and PDEC was verified by immunofluorescence. Thus, trypsin interacts with basolateral PAR-2 to increase [Ca2+]i and activate ion channels in PDEC. In pancreatitis, when trypsinogen is prematurely activated, PAR-2-mediated ductal secretion may promote clearance of toxins and debris.

Stimulation of exocytosis without a calcium signal

More than 30 years ago, Douglas ( Douglas & Rubin, 1961 ; Douglas, 1968 ) proposed that intracellular Ca2+ controls stimulus‐secretion coupling in endocrine cells, and Katz & Miledi ( 1967 ; Katz, 1969 ) proposed that intracellular Ca2+ ions control the rapid release of neurotransmitters from synapses. These related hypotheses have been amply confirmed in subsequent years and for students of excitable cells, they dominate our teaching and research. Calcium controls regulated exocytosis. On the other hand, many studies of epithelial and blood cell biology emphasize Ca2+‐independent regulation of secretion of mucin, exocytotic delivery of transporters and degranulation. The evidence seems good. Are these contrasting conclusions somehow mistaken, or are the dominant factors controlling exocytosis actually different in different cell types? In this essay, we try to reconcile these ideas and consider classes of questions to ask and hypotheses to test in seeking a more integrated understanding of excitation‐secretion coupling. Our review is conceptual and narrowly selective of a few examples rather than referring to a broader range of useful studies in the extensive literature. The examples are taken from mammals and are documented principally by citing other reviews and two of our own studies. The evidence shows that protein phosphorylation by kinases potentiates Ca2+‐dependent exocytosis and often suffices to induce exocytosis by itself. Apparently, protein phosphorylation is the physiological trigger in a significant number of examples of regulated exocytosis. We conclude that although sharing many common properties, secretory processes in different cells are specialized and distinct from each other.

ATP-sensitive and Ca-activated K channels in vertebrate axons : novel links between metabolism and excitability

Two types of metabolically regulated K channels have been identified for the first time in enzymatically demyelinated fibres of amphibian sciatic nerve using the patch-clamp technique. A maxi K channel with a single-channel conductance of 132 pS (105 mM K on both sides of the membrane, 15 °C) is activated both by micromolar concentrations of internal Ca and by depolarization. A second type of K channel with a conductance of 44 pS is inhibited by intracellular adenosine 5′-triphosphate (ATP) with a half-maximal inhibitory concentration (IC50) of 35 μM. It is blocked by submicromolar concentrations of external glibenclamide. Both channels are sensitive to external tetraethylammonium chloride (IC50 = 0.2 mM for the maxi K channel and 4.2 mM for the ATP-sensitive channel). They may be part of a complex feedback system regulating axonal excitability under various metabolic conditions.

Na(+)-activated K+ channels localized in the nodal region of myelinated axons of Xenopus.

1. A potassium channel activated by internal Na+ ions (K+Na channel) was identified in peripheral myelinated axons of Xenopus laevis using the cell‐attached and excised configurations of the patch clamp technique. 2. The single‐channel conductance for the main open state was 88 pS with [K+]o = 105 mM and pS with [K+]o = 2.5 mM ([K+]i = 105 mM). The channel was selectively permeable to K+ over Na+ ions. A characteristic feature of the K+Na channel was the frequent occurrence of subconductance states. 3. The open probability of the channel was strongly dependent on the concentration of Na+ ions at the inner side of the membrane. The half‐maximal activating Na+ concentration and the Hill coefficient were 33 mM and 2.9, respectively. The open probability of the channel showed only weak potential dependence. 4. The K+Na channel was relatively insensitive to external tetraethylammonium (TEA+) in comparison with voltage‐dependent axonal K+ channels; the half‐maximal inhibitory concentration (IC50) was 21.3 mM (at ‐90 mV). In contrast, the channel was blocked by low concentrations of external Ba2+ and Cs+ ions, with IC50 values of 0.7 and 1.1 mM, respectively (at ‐90 mV). The block by Ba2+ and Cs+ was more pronounced at negative than at positive membrane potentials. 5. A comparison of the number of K+Na channels in nodal and paranodal patches from the same axon revealed that the channel density was about 10‐fold higher at the node of Ranvier than at the paranode. Moreover, a correlation between the number of K+Na channels and voltage‐dependent Na+ channels in the same patches was found, suggesting co‐localization of both channel types. 6. As weakly potential‐dependent (‘leakage’) channels, axonal K+Na channels may be involved in setting the resting potential of vertebrate axons. Simulations of Na+ ion diffusion suggest two possible mechanisms of activation of K+Na channels: the local increase of Na+ concentration in a cluster of Na+ channels during a single action potential or the accumulation in the intracellular axonal compartment during a train of action potentials.

A TEA-insensitive flickering potassium channel active around the resting potential in myelinated nerve

SummaryA novel potassium-selective channel which is active at membrane potentials between — 100 mV and +40 mV has been identified in peripheral myelinated axons of Xenopus laevis using the patch-clamp technique. At negative potentials with 105 mm-K on both sides of the membrane, the channel at 1 kHz resolution showed a series of brief openings and closings interrupted by longer closings, resulting in a flickery bursting activity. Measurements with resolution up to 10 kHz revealed a single-channel conductance of 49 pS with 105 mm-K and 17 pS with 2.5 mm-K on the outer side of the membrane. The channel was selective for K ions over Na ions (PNa/PK = 0.033). The probability of being within a burst in outside-out patches varied from patch to patch (>0.2, but often >0.9), and was independent of membrane potential. Open-time histograms were satisfactorily described with a single exponential (τo= 0.09 msec), closed times with the sum of three exponentials (τc= 0.13, 5.9, and 36.6 msec). Sensitivity to external tetraethylammonium was comparatively low (IC50 = 19.0 mm). External Cs ions reduced the apparent unitary conductance for inward currents at Em= −90 mV (IC50 = 1.1 mm). Ba and, more potently, Zn ions lowered not only the apparent singlechannel conductance but also open probability. The local anesthetic bupivacaine with high potency reduced probability of being within a burst (IC50 = 165 nm). The flickering K channel is clearly different from the other five types of K channels identified so far in the same preparation. We suggest that this channel may form the molecular basis of the resting potential in vertebrate myelinated axons.

Effect of the flavoid phloretin on Ca2+-activated K+ channels in myelinated nerve fibres of Xenopus laevis

Abstract The effects of the flavonoid phloretin on K+ channels in amphibian myelinated nerve were studied by patch clamping. The open probability of Ca2+-activated K+ channels was greatly increased by external phloretin (10–200 μM) due to a shift of the membrane potential for half-maximal activation, E50, of −63.9 mV (80 μM phloretin). Open times were prolonged and closed times shortened. Channel activation by phloretin developed slowly (τon = 33.4 s) and its washout was even slower (τoff,1 = 4.7 and τoff,2 = 183.2 s). In contrast, submillimolar phloretin blocked two delayed rectifier K+ channels (I and F) whereas the gating of the ATP-sensitive and the flickering K+ channel were unaffected. Phloretin may directly affect the voltage sensor of K+ channels.