Effects of K+ channel blockers on inwardly and outwardly rectifying whole-cell K+ currents in sheep parotid secretory cells

Single-channel patch-clamp techniques as well as standard and perforated-patch whole cell voltage-clamp techniques have been applied to the study of ionic channels in the corneal endothelium of several species. These studies have revealed two major K+ currents. One is due to an anion- and temperature-stimulated channel that is blocked by Cs+ but not by most other K+ channel blockers, and the other is similar to the family of A-currents found in excitable cells. The A-current is transient after a depolarizing voltage step and is blocked by both 4-aminopyridine and quinidine. These two currents are probably responsible for setting the -50 to -60 mV resting voltage reported for these cells. A Ca(2+)-activated ATP-inhibited nonselective cation channel and a tetrodotoxin-blocked Na+ channel are possible Na+ inflow pathways, but, given their gating properties, it is not certain that either channel works under physiological conditions. A large-conductance anion channel has also been identified by single-channel patch-clamp techniques. Single corneal endothelial cells have input resistances of 5-10 G omega and have steady-state K+ currents that are approximately 10 pA at the resting voltage. Pairs or monolayers of cells are electrically coupled and dye coupled through gap junctions.

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Acetylcholine potentiates acetylcholine-induced increases in K+ current in cat atrial myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1995.268.3.h1313 ◽

1995 ◽

Vol 268 (3) ◽

pp. H1313-H1321 ◽

Cited By ~ 24

Author(s):

Y. G. Wang ◽

S. L. Lipsius

Keyword(s):

Reversal Potential ◽

Membrane Conductance ◽

K Channel ◽

Channel Blockers ◽

Atrial Myocytes ◽

Initial Exposure ◽

Recovery Interval ◽

Cell Recording ◽

K Current ◽

K Currents

A nystatin-perforated patch whole cell recording method was used to study the effects of acetylcholine (ACh) on ACh-induced K+ currents in atrial myocytes isolated from cat hearts. The general protocol involved an initial 4-min exposure to ACh (ACh1), followed by a 4-min washout in ACh-free Tyrode solution and then a second 4-min ACh exposure (ACh2). Voltage ramps (40 mV/s) between -130 and +30 mV were used to assess changes in total membrane conductance. ACh2 (10 microM) induced an increase in K+ conductance that was significantly larger than that induced by ACh1 (10 microM) at voltages both negative and positive to the reversal potential. The potentiated current induced by ACh2 reversed at about -80 mV and inwardly rectified at voltages positive to the reversal potential. External Ba2+ (5 mM) or tetraethylammonium (10 mM) abolished all ACh2-induced increases in membrane conductance. The sensitivity to K+ channel blockers, reversal potential, and the rectifying properties indicate that the current potentiated by ACh2 is a K+ current. Atropine (1 microM) blocked all effects of ACh on K+ currents. Potentiation of K+ current by ACh2 required 1) ACh1 concentrations > or = 1 microM, 2) ACh1 duration > or = 2 min, and 3) recovery interval > or = 2 min. We conclude that an initial exposure to ACh potentiates subsequent ACh-induced increases in K+ current. ACh-induced potentiation depends on the concentration and duration of the initial ACh exposure and the recovery interval between consecutive ACh exposures.(ABSTRACT TRUNCATED AT 250 WORDS)

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The ACh-induced whole-cell currents in sheep parotid secretory cells. Do BK channels really carry the ACh-evoked whole-cell K+ current?

The Journal of Membrane Biology ◽

10.1007/bf00232801 ◽

1995 ◽

Vol 144 (2) ◽

Cited By ~ 7

Author(s):

T. Hayashi ◽

C. Hirono ◽

J.A. Young ◽

D.I. Cook

Keyword(s):

Bk Channels ◽

Secretory Cells ◽

Whole Cell ◽

K Current ◽

Sheep Parotid

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Calcium-mediated agonists activate an inwardly rectified K+ channel in colonic secretory cells

AJP Cell Physiology ◽

10.1152/ajpcell.1993.265.5.c1271 ◽

1993 ◽

Vol 265 (5) ◽

pp. C1271-C1280 ◽

Cited By ~ 49

Author(s):

D. C. Devor ◽

R. A. Frizzell

Keyword(s):

Single Channel ◽

Partial Substitution ◽

K Channel ◽

Secretory Cells ◽

Selectivity Ratio ◽

Channel Activity ◽

T84 Cells ◽

Outward Currents ◽

K Currents

Single-channel recording techniques were used to identify and characterize the K+ channel activated by Ca(2+)-mediated secretory agonists in T84 cells. Carbachol (CCh; 100 microM) and taurodeoxycholate (TDC; 0.75 mM) stimulated oscillatory outward K+ currents. With K gluconate in bath and pipette, cell-attached single-channel K+ currents stimulated by CCh and ionomycin (2 microM) were inwardly rectified and reversed at 0 mV. The single-channel chord conductance was 32 pS at -90 mV and 14 pS at +90 mV. Similar properties were observed in excised inside-out patches in symmetric K+, permitting further characterization of channel properties. Partial substitution of bath or pipette K+ with Na+ gave a K(+)-to-Na+ selectivity ratio of 5.5:1. Channel activity increased with increasing bath Ca2+ concentration in the physiological range of 50-800 nM. Maximal channel activity occurred at intracellular pH 7.2 and decreased at more acidic or alkaline pH values. Extracellular charybdotoxin (CTX; 50 nM) blocked inward but not outward currents. Extracellular tetraethylammonium (TEA; 10 mM) reduced single-channel amplitude at all voltages. No apparent block of the channel was observed with extracellular Ba2+ (1 mM), apamin (1 microM), 4-aminopyridine (4-AP; 4 mM), quinine (500 microM), or glyburide (10 microM). Cytosolic quinine and 4-AP blocked both inward and outward currents, whereas Ba2+ blocked only outward currents. Apamin, CTX, TEA, and glyburide did not affect channel activity. The agonist activation and pharmacological profile of this inwardly rectified K+ channel indicate that it is responsible for the increase in basolateral K+ conductance stimulated by Ca(2+)-mediated agonists in T84 cells.

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Cation channels in basolateral membranes of sheep parotid secretory cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1992.263.5.g786 ◽

1992 ◽

Vol 263 (5) ◽

pp. G786-G794 ◽

Cited By ~ 5

Author(s):

E. A. Wegman ◽

T. Ishikawa ◽

J. A. Young ◽

D. I. Cook

Keyword(s):

Membrane Potential ◽

Relative Permeability ◽

Basolateral Membrane ◽

Cation Channels ◽

K Channel ◽

Secretory Cells ◽

K Channels ◽

Basolateral Membranes ◽

Sheep Parotid ◽

Relative Conductance

We observed 240-pS K+ channels in 63% of cell-attached patches, and 30-pS K+ channels were observed in 95% of cell-attached patches. The 240-pS K+ channel had the relative permeability sequence of K+ (1) = Rb+ (1) > Cs+ (0.3) >> Na+ (0.03) and the relative conductance sequence of K+ (1) > Rb+ (0.22) > Cs+ (0.05) > Na+ (0). It was activated by intracellular free Ca2+ and by depolarization. It was blocked by 10 mmol/l tetraethylammonium (TEA) applied extracellularly. The 30-pS K+ channel had the relative permeability sequence of K+ (1) = Rb+ (1) > Cs+ (> Na+ (< 0.09) and the relative conductance sequence of K+ (1) > Rb+ (0.45) > Cs+ (0) = Na+ (0). Its activity was not sensitive to cytosolic free Ca2+ or membrane potential, and it was not blocked by 10 mmol/l TEA extracellularly. Acetylcholine (10 mumol/l) activated the 240-pS voltage-activated and Ca(2+)-activated K+ channels but did not activate the 30-pS K+ channels. We conclude that the 30-pS K+ channel probably determines the properties of the basolateral membrane in unstimulated sheep parotid secretory cells, whereas the 240-pS voltage-activated and Ca(2+)-activated K+ channel may be important during parasympathomimetic stimulation.

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A role for potassium currents in the generation of the swimming motor pattern of Xenopus embryos

Journal of Neurophysiology ◽

10.1152/jn.1994.72.1.337 ◽

1994 ◽

Vol 72 (1) ◽

pp. 337-348 ◽

Cited By ~ 11

Author(s):

M. J. Wall ◽

N. Dale

Keyword(s):

Nicotinic Acetylcholine Receptors ◽

Action Potentials ◽

Motor Pattern ◽

K Channel ◽

Cycle Period ◽

Channel Blockers ◽

Dependent Manner ◽

Spinal Neurons ◽

K Current ◽

K Currents

1. To assess the role that K+ currents play in the production of the swimming motor pattern in the Xenopus embryo, we have used low doses of the K+ channel blockers, 3,4-diaminopyridine (3,4-DAP; 25–100 microM) and tetraethylammonium (TEA; 500 microM), to reduce K+ currents and investigated the effects on motor output. 2. To confirm that 3,4 -DAP and TEA block K+ currents and characterize their actions, we made whole -cell voltage -clamp recordings from acutely isolated spinal neurons. Both 25–100 microM 3,4 -DAP and 100–500 microM TEA blocked the sustained K+ current in a dose -dependent manner. 3. Because TEA can block acetylcholine nicotinic receptors on autonomic ganglia, and nicotinic acetylcholine receptors have recently been shown to be present on Xenopus spinal neurons, we have tested both 3,4 -DAP and TEA for antagonist action against the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP). Although 500 microM TEA blocked the DMPP -induced depolarization, 25 microM 3,4-DAP did not. 4. In the intact embryo, application of 25–100 microM 3,4 -DAP or 500 microM TEA disrupted both the left and right alternation of ventral root discharge and the motor pattern recorded intracellularly from spinal neurons during swimming. Both blockers allowed the firing of an extra action potential at midcycle, which led to a number of different patterns. These patterns were categorized as follows: type A, cycles with midcycle action potentials; type B, the simultaneous firing of neurons on both sides of the cord; and type C, in which one side was active, whereas the other side was inhibited. In both 3,4-DAP and TEA these abnormalities tended to occur at the beginning of swimming episodes. Both blockers also caused a significant increase in the cycle period. Because both 3,4-DAP and TEA produced very similar affects to the motor pattern, we conclude that the perturbations are probably a result of reducing K+ current amplitude. 5. To investigate whether 3,4-DAP and TEA were producing disruptions in the motor pattern by increasing synaptic drive through the broadening of action potentials, we made measurements of spike width, tonic depolarization, and midcycle inhibitory postsynaptic potential (IPSP) amplitude during swimming. Both 3,4-DAP and TEA caused significant but modest spike broadening (20.8 and 29.8%, respectively); however, their effects on tonic depolarization were inconsistent although both blockers increased midcycle IPSP amplitude. 6. To test whether a reduction in K+ currents could plausibly produce the specific motor pattern perturbations that were seen, we have made computer simulations of simplified spinal networks.(ABSTRACT TRUNCATED AT 400 WORDS)

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Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling

AJP Cell Physiology ◽

10.1152/ajpcell.1992.262.2.c501 ◽

1992 ◽

Vol 262 (2) ◽

pp. C501-C509 ◽

Cited By ~ 38

Author(s):

R. E. Yantorno ◽

D. A. Carre ◽

M. Coca-Prados ◽

T. Krupin ◽

M. M. Civan

Keyword(s):

Regulatory Volume Decrease ◽

Membrane Conductance ◽

Cell Swelling ◽

K Channel ◽

Ciliary Epithelium ◽

Channel Blockers ◽

Channel Activity ◽

Patch Clamping ◽

Whole Cell ◽

Cl Channel

Anisosmotic cell swelling triggers a regulatory volume decrease (RVD) in cell lines derived from human nonpigmented ciliary epithelium. Measurements of cell volume have indicated that the RVD reflects activation of K+ and/or Cl- channels. We have begun to characterize the putative channels by whole cell patch clamping. The results obtained by altering the external K+ and Cl- concentrations and by adding 20-50 microM quinidine or 1 mM Ba2+ indicate that K+ conductances contribute substantially and Cl- conductances contribute very little to the total membrane conductance (GT) under baseline isotonic conditions. Reducing the external osmolality by 20-50% reversibly and reproducibly increased GT by an order of magnitude. Data obtained from ion substitutions and the channel blockers quinidine and 5-nitro-2-(3-phenylpropylamino)-benzoate indicate that most of the hypotonicity-induced conductance reflects stationary Cl(-)-channel activity. The contribution of new K(+)-channel activity was small at intracellular free Ca2+ concentrations of 10 or 200 nM. We conclude that the RVD triggered by bath hypotonicity primarily reflects increased Cl(-)-channel activity.

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Transient K current in the somatic membrane of cultured central neurons of embryonic rat

Journal of Neurophysiology ◽

10.1152/jn.1992.68.5.1708 ◽

1992 ◽

Vol 68 (5) ◽

pp. 1708-1719 ◽

Cited By ~ 6

Author(s):

M. A. Rizzo ◽

W. Nonner

Keyword(s):

Spinal Cord ◽

Steady State ◽

K Channel ◽

Kinetic Properties ◽

Spinal Cord Neurons ◽

Whole Cell ◽

Component Size ◽

Steady State Inactivation ◽

K Current ◽

K Currents

1. Somatic K currents of cultured hippocampal, striatal, and spinal cord neurons of embryonic rat were recorded under voltage clamp in membrane spheres ("blebs") excised by means of a tight-seal pipette. 2. The somatic K current in blebs was subject to rapid and near complete inactivation during 300-ms depolarizations, whereas whole-cell K currents included a substantial maintained component. Size and kinetic properties of bleb and whole-cell currents were stable throughout the recording period. 3. The steady-state inactivation of somatic A current was steeply voltage dependent and complete near voltage levels that activated current, whereas peak conductances did not saturate during depolarizations up to +90 mV. Activation started with a delay. Half-times of activation decreased with depolarization, but half-times of inactivation varied little with depolarization. Recovery from inactivation followed a sigmoidal time course with half-times of approximately 50 ms. 4. Half-times of activation and inactivation varied over more than an order of magnitude between individual neurons. Midpoint potentials of inactivation and peak conductance varied over approximately 40 mV. The parameter ranges of hippocampal, striatal, and spinal cord neurons overlapped. 5. Individual soma membranes revealed signs of K channel heterogeneity in their 4-aminopyridine block, current fluctuations, and current kinetics. On the other hand, currents elicited after conditioning pulses that established varied degrees of steady-state inactivation or of recovery from full inactivation had superimposable time courses. 6. The described characteristics of the somatic A channels are compared with those reported for the RCK4, Raw3, and mShal products expressed in Xenopus oocytes. Whereas the ranges of voltage dependencies and of most kinetic characteristics are compatible among native and cloned channels, these three cloned channels recover much more slowly from inactivation. In addition, inactivation in native channels, unlike that in RCK4 and Raw3 channels, was stable after excision in a subcellular fragment.

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