The catalytic subunit of cyclic AMP-dependent protein kinase directly inhibits sodium channel activities in guinea-pig ventricular myocytes

We investigated the effects of the purified catalytic subunit (C subunit) of the cAMP-dependent protein kinase (A-kinase) on the cardiac Na⁺ channel currents. Single Na⁺ channel currents in guinea-pig ventricular myocytes were recorded using the patch clamp technique of the inside-out configuration. Application of C subunit decreased the peak average current and slowed the current decay, effects which were caused by decrease in the open probability of Na⁺ channels and increase in the first latency, whereas the unitary current amplitude and mean open times were not affected. We conclude that the cardiac Na⁺ channel is directly modulated by phosphorylation process through A-kinase.     

Responses to moving slits by single units in cat striate cortex

Summary A quantitative study has been made of the responses to moving slit stimuli by single units in the cat striate cortex whose receptive fields lay within 5° of the visual axis. Special attention was given to finding the optimal stimulus parameters including slit width, length, orientation and speed. The analysis was largely based on averaged response vs. time histograms. Using the classification of simple and complex responses types, the units were further subdivided on the basis of the number of modes in the response and on the presence or absence of directional selectivity. Simple unimodal units with directional selectivity (SUDS) had the most specific stimulus requirements and nearly always had zero background activity. Complex units usually had a high level of background activity. SUDS units also showed a preference for horizontally- and vertically ****-orientated stimuli. Whenever the response survived reversal of contrast the directional selectivity remained independent of the change. Optimal stimulus speeds varied widely from unit to unit with a mean at 4°/sec: simple bimodal units and complex units tended to have higher optimal stimulus speeds and responded over a wider range of speeds than did simple unimodal units. While SUDS units with very small receptive fields tended to prefer slowly moving stimuli, in general there was no correlation between receptive field size and optimal stimulus speed.Selby Fellow of the Australian Academy of Sciences.     

Aromatic residues affecting permeation and gating in dSlo BK channels

 Structural determinants of permeation in large unit conductance calcium-activated potassium channels (BK channels) were investigated. Y293 and F294 in the P-region of dSlo were substituted by tryptophans. Compared to wild-type channels, Y293W channels displayed reduced inward unitary currents while F294W channels exhibited normal inward current amplitudes but flickery kinetics. Both mutations produced changes in current/voltage relations under bi-ionic conditions. Sensitivity to block by external tetraethylammonium (TEA) was affected in both channels, and the voltage dependence of TEA block was increased in F294W channels. Both mutations also affected gating by shifting the half-maximal activation voltage of macroscopic conductance/voltage relations to more positive potentials, and eliminating a slow component of deactivation. The double mutant did not produce ionic currents. These data are consistent with a model in which Y293 contributes to a potassium-binding site close to the outer mouth of the dSlo pore, while F294 contributes to an energy barrier near this site. Received: 16 September 1997 / Received after revision: 20 November 1997 / Accepted: 21 November 1997     

Effects of ATP antagonists on purinoceptor-operated inward currents in rat phaeochromocytoma cells

The effects of suramin, reactive blue 2 (RB2) and d-tubocurarine (d-TC) were investigated electrophysiologically to elucidate the mechanisms underlying their antagonism of P₂ purinoceptor-mediated responses. All three compounds inhibited an adenosine triphosphate (ATP)-activated inward current in rat phaeochromocytoma PC12 cells in a concentration-dependent manner. The order of potency was RB2 > suramin > d-TC. The inhibition induced by suramin or RB2 was reversible, whereas that induced by d-TC was not reversed after a 5-min rinse. The inactivation of the ATP-activated current was accelerated by d-TC but not by suramin or RB2. RB2 administered simultaneously with ATP exerted much weaker inhibition compared to that induced by prior administration, suggesting that RB2 is a slowly acting antagonist. This was not observed for suramin or d-TC. Suramin and RB2 caused a parallel shift in the concentration/response curve for the ATP-activated current. With d-TC the maximal response of ATP was decreased but the concentration producing half-maximal response was unchanged. The voltage dependency of the ATP-activated current showed less inward rectification in the presence of d-TC. Suramin or RB2 did not affect the voltage dependency. These results suggest that suramin and RB2 reversibly block binding of ATP to receptors, whereas d-TC blocks ion permeability through the ATP-activated channel.     

Chemosensory Cell-Derived Acetylcholine Drives Tracheal Mucociliary Clearance in Response to Virulence-Associated Formyl Peptides

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Sodium nitroprusside and cGMP decrease Ca2+ channel availability in basilar artery smooth muscle cells

 We studied the effect of the nitric oxide (NO) donor, sodium nitroprusside (SNP), on the macroscopic and single-channel currents due to the 22-pS Ca²⁺ channel in smooth muscle cells from guinea pig basilar artery. In nystatin-perforated whole-cell recordings, 50 nM SNP decreased the macroscopic current to 63±12% of control values, without changing the voltage dependence of the current. In cell-attached patches with BAY-K8644 in the pipette, SNP caused a comparable decrease in single-channel availability (n ·P _o) that was dose dependent over the range of 10 nM to 10 μM SNP. SNP had no effect on single-channel properties, including slope conductance, voltage dependence of activation, the number of open states, the time constants of the open states, and the proportion of time spent in each open state. The effect of SNP (50 nM) on single Ca²⁺ channel openings was reproduced by 8-Br-cGMP (100 μM), which also reduced channel availability without altering channel properties. The protein kinase inhibitor H-8 (1.5 μM), which exhibits relative specificity for cGMP-dependent protein kinase, completely inhibited the decrease in single-channel availability expected with SNP. The dose-dependent decrease in Ca²⁺ channel availability caused by SNP was not altered by prior application of 8-Br-cAMP or forskolin, both of which cause an increase in Ca²⁺ channel availability in these cells. Our findings suggest that NO decreases openings of Ca²⁺ channels in basilar artery smooth muscle cells without altering channel properties, and that it does so by a mechanism likely to involve cGMP-dependent protein kinase. Received: 2 July 1996 / Received after revision: 30 September 1996 / Accepted: 2 October 1996     

Effects of cGMP-dependent phosphorylation on rat and human connexin43 gap junction channels

The effects of 8-bromoguanosine 3:5-cyclic monophosphate (8Br-cGMP), a membrane-permeant activator of protein kinase G (PKG), were studied on rat and human connexin43 (Cx43), the most abundant gap junction protein in mammalian heart, which were exogenously expressed in SKHep1 cells. Under dual whole-cell voltage-clamp conditions, 8Br-cGMP decreased gap junctional conductance (g_j) in rat Cx43-transfected cells by 24.0±3.7% (mean±SEM, n=5), whereas g_j was not affected in human Cx43-transfected cells by the same treatment. The relaxation of g_j in response to steps in transjunctional voltage observed in rat Cx43 transfectants was best fitted with three exponentials. Time constants and amplitudes of the decay phases changed in the presence of 8Br-cGMP. Single rat and human Cx43 gap junction channels were resolved in the presence of halothane. Under control conditions, three single-channel conductance states (_j) of about 20, 40–45 and 70 pS were detected, the events of the intermediate size being most frequently observed. In the presence of 8Br-cGMP, the _j distribution shifted to the lower size in rat Cx43 but not in human Cx43 transfectants. Immunoblot analyses of Cx43 in subconfluent cultures of rat Cx43 or human Cx43 transfectants showed that 8Br-cGMP did not induce changes in the electrophoretic mobility of Cx43 in either species. However, the basal incorporation of [³²P] into rat Cx43 was significantly altered by 8Br-cGMP, whereas this incorporation of [³²P] into human Cx43 was not affected. We conclude that 8Br-cGMP modulates phosphorylation of rat Cx43 in SKHep1 cells, but not of human Cx43. This cGMP-dependent phosphorylation of rat Cx43 is associated with a decreased g_j, which results from both an increase in the relative frequency of the lowest conductance state and a change in the kinetics of these channels.     

Non-cholinergic effects of acetylcholinesterase in the substantia nigra: a possible role for an ATP-sensitive potassium channel

Summary Within the substantia nigra acetylcholinesterase has non-cholinergic actions that can be demonstrated at both behavioural and cellular levels: the aim of this study was, thus, to explore, in the in vitro guinea pig substantia nigra, the ionic mechanisms which mediate these non-classical phenomena. Acetylcholinesterase had a reversible hyperpolarizing action, via an opening of potassium channels, on a selective population of nigral neurons. These neurons could be identified by an ability to generate bursts of action potentials and by a sensitivity to either amphetamine or to a reduction of glucose in the perfusing medium. The acetylcholinesterase-induced hyperpolarization could not be attributed to a contaminant in the exogenous solution, since a highly purified preparation was even more potent. Furthermore, enzymatic action of any kind could be eliminated as boiled acetylcholinesterase was equally efficacious. The effect of acetylcholinesterase was not subject to tachyphylaxis and was resistant to blockade of potassium channels with tetraethylammonium: since both these phenomena are features of the D₂ autoreceptor for dopamine within the substantia nigra, it seems unlikely that acetylcholinesterase is operating on the same target as dendritically released local dopamine. On the other hand, the actions of acetylcholinesterase were enhanced by low glucose and blocked by the sulfonylurea, tolbutamide. These results strongly suggest that acetylcholinesterase can exert a nonenzymatic action and that this action, in the substantia nigra, is mediated by an ATP-sensitive potassium channel.     

HLA-DR residues accessible under the peptide-binding groove contribute to polymorphic antibody epitopes

Many residues involved in polymorphic antibody-binding epitopes on class II molecules are located on the -helix of DRβ chains. Although they have received less attention, residues in the peptide-binding groove and second domain of the DRβ chain may also be critical for polymorphic anti-DR antibody epitopes. In this study, we used transfectants expressing site-directed mutations at positions in the HLA-DR β₁ and β₂ domains and flow cytometry to define the epitopes of several polymorphic anti-DR antibodies. Both DR(β 1^*0403) residues 14 and 25 were shown to be involved in the epitopes of mAbs DA6. 164, HU-20, Q5/6, and 50D6, and DR(β 1^*0701) residue 14 was shown to be critical for the epitopes of two DR7-specific mAbs, SFR16-DR7M and TAL 13. 1. Unlike most other residues shown to be important in antibody-binding epitopes, residue 14 is located in the floor of the peptide-binding groove and residue 25 is in an outer loop, each with their side chains pointing down, such that antibodies may directly contact these residues from below the binding groove. Two residues in the β₂ domain, β180 and β181, were also shown to be involved in the epitopes of three polymorphic anti-DR mAbs, NFLD.D1, NFLD.M1, and LY9. Although these two residues are close to the transmembrane domain in the linear sequence, their solvent accessibility in the DR1 structures is quite impressive. Our data provide new evidence that residues accessible under the peptidebinding groove contribute to polymorphic antibody-binding epitopes.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.