Ammonium transport in medullary thick ascending limb of rabbit kidney: Involvement of the Na+, K+, Cl−-cotransporter

Summary In order to investigate the question whether ammonium reabsorption in the thick ascending limb of Henle's loop (TALH) proceeds via the Na⁺,K⁺,Cl^–-cotransporter, plasma membrane vesicles were prepared from TALH cells isolated from rabbit kidney outer medulla and the effect of NH ₄ ⁺ on their transport properties was investigated. It was found that, in the presence of a 78-mmol/liter NaCl gradient, 5 mmol/liter NH ₄ ⁺ inhibited bumetanide-sensitive rubidium flux by 86%; a similar decrease was observed for 5 mmol/liter, K⁺. Inhibition of bumetanide-sensitive rubidium uptake by NH ₄ ⁺ was competitive and an apparentK _i of 1.9 mmol/liter was found Bumetanide-sensitive sodium uptake measured in the presence of a 83 mmol/liter KCl gradient was not inhibited by 5 mmol/liter NH ₄ ⁺ . A 100-mmol/liter NH₄Cl gradient was, however, capable of stimulating bumetanide-sensitive sodium uptake to the same extent as a KCl gradient. These data suggest that NH ₄ ⁺ is accepted by the K⁺ site of the Na⁺,K⁺,Cl-cotransport system and that the transporter can function in a Na⁺, NH ₄ ⁺ ,2Cl mode. Since the affinity of the transporter for NH ₄ ⁺ lies in the concentration range found in the TALH lumen in vivo, it is concluded that Na⁺, NH ₄ ⁺ 2Cl-cotransport can contribute to the NH ₄ ⁺ reabsorption in this tubular segment.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: II. Determinants of the ADH-mediated increases in transepithelial voltage and in net Cl− absorption

Summary Cellular impalements were used in combination with standard transepithelial electrical measurements to evaluate some of the determinants of the spontaneous lumen-positive voltage,V_e, which attends net Cl^– absorption,J_Cl^net, and to assess how ADH might augment bothJ_Cl^met andV_e in the mouse medullary thick ascending limb of Henle microperfusedin vitro. Substituting luminal 5mm Ba⁺⁺ for 5mm K⁺ resulted in a tenfold increase in the apical-to-basal membrane resistance ratio,R_c/R_bl, and increasing luminal K⁺ from 5 to 50mm in the presence of luminal 10^–4m furosemide resulted in a 53-mV depolarization of apical membrane voltage,V_a. Thus K⁺ accounted for at least 85% of apical membrane conductance. Either with or without ADH. 10^–4m luminal furosemide reducedV_e andJ_Cl^net to near zero values and hyperpolarized bothV_a andV_bl, the voltage across basolateral membranes; however, the depolarization ofV_bl was greater in the presence than in the absence of hormone while the hormone had no significant effect on the depolarization ofV_a, Thus ADH-dependent increases inV_b were referable to greater depolarizations ofV_bl in the presence of ADH than in the absence of ADH 68% of the furosemide-induced hyperpolarization ofV_a was referable to a decrease in the K⁺ current across apical membranes, but, at a minimum, only 19% of the hyperpolarization ofV_bl could be accounted for by a furosemide-induced reduction in basolateral membrane Cl^– current. Thus an increase in intracellular Cl^– activity may have contributed to the depolarization ofV_bl during net Cl^– absorption, and the intracellular Cl^– activity was likely greater with ADH than without hormone. Since ADH increases apical K⁺ conductance and since the chemical driving force for electroneutral Na⁺,K⁺,2Cl^– cotransport from lumen to cell may have been less in the presence of ADH than in the absence of hormone, the cardinal effects of ADH may have been to increase the functional number of both Ba⁺⁺-sensitive conductance K⁺ channels and electroneutral Na⁺,K⁺,2Cl^– cotransport units in apical plasma membranes.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Evidence for a K+ channel in bovine chromaffin granule membranes: single-channel properties and possible bioenergetic significance

A K⁺ channel was incorporated into voltage-clamped planar lipid bilayers from bovine chromaffin granules and resealed granule membranes (ghosts). It was not incorporated from plasma membrane-rich fractions from the adrenal medulla. The channel had a conductance of 400 pS in symmetric 450 mM KCI, with the permeability sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺, and was insensitive to both Ca²⁺ and charybdotoxin. It exhibited complex gating kinetics, consistent with the presence of multiple open and closed states, and its gating was voltage-dependent. The channels appeared to incorporate into bilayers with the same orientation, and were blocked from one side (the side of vesicle addition) by 0.2-1 mM TEA'. The block was slightly voltage-dependent. Acidification of resealed granule membranes in response to external ATP (which activated the vacuolartype ATPase) was significantly reduced in the presence of 1 mM intralumenal TEACI (with 9 mM KCl), and parallel measurements with the potential-sensitive dye Oxonol V showed that such vesicles tended to develop higher internal-positive membrane potentials than control vesicles containing only 10 mM KCI. 1 mM TEA⁺ had no effect on proton-pumping activity when applied externally, and did not directly affect either the proton-pumping or ATP hydrolytic activity of the partially-purified ATPase. These results suggest that chromaffin granule membranes contain a TEA⁺-sensitive K⁺ channel which may have a role in regulating the vesicle membrane potential. Correspondence to: R. H. Ashley     

Identification and reconstitution of a Na/K/Cl cotransporter and K channel from luminal membranes of renal red outer medulla

Electrophysiological studies on renal thick ascending limb segments indicate the involvement of a luminal Na⁺/K⁺/Cl⁻ cotransport system and a K⁺ channel in transepithelial salt transport. Sodium reabsorption across this segment is blocked by the diuretics furosemide and bumetanide. The object of our study has been to identify in intact membranes and reconstitute into phospholipid vesicles the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel, as an essential first step towards purification of the proteins involved and characterization of their roles in the regulation of transepithelial salt transport. Measurements of ⁸⁶Rb⁺ uptake into membrane vesicles against large opposing KCl gradients greatly magnify the ratio of specific compared to non-specific isotope flux pathways. Using this sensitive procedure, it has proved possible to demonstrate in crude microsomal vesicle preparations from rabbit renal outer medulla two ⁸⁶Rb⁺ fluxes. (A) A furosemide-inhibited ⁸⁶Rb⁺ flux in the absence of Na⁺ (K⁺-K⁺ exchange). This flux is stimulated by an inward Na⁺ gradient (Na⁺/K⁺ cotransport) and is inhibited also by bumetanide. (B) A Ba²⁺-inhibited ⁸⁶Rb⁺ flux, through the K⁺ channel. Luminal membranes containing the Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channels, and basolateral membranes containing the Na⁺/K⁺ pumps were separated from the bulk of contaminant protein by metrizamide density gradient centrifugation. The Na⁺/K⁺/Cl⁻ cotransporter and K⁺ channel were reconstituted in a functional state by solubilizing both luminal membranes and soybean phospholipid with octyl glucoside, and then removing detergent on a Sephadex column.     

Mechanisms underlying modulation of neuronal KCNQ2/KCNQ3 potassium channels by extracellular protons

Changes in extracellular pH occur during both physiological neuronal activity and pathological conditions such as epilepsy and stroke. Such pH changes are known to exert profound effects on neuronal activity and survival. Heteromeric KCNQ2/3 potassium channels constitute a potential target for modulation by H+ ions as they are expressed widely within the CNS and have been proposed to underlie the M-current, an important determinant of excitability in neuronal cells. Whole-cell and single-channel recordings demonstrated a modulation of heterologously expressed KCNQ2/3 channels by extracellular H+ ions. KCNQ2/3 current was inhibited by H+ ions with an IC50 of 52 nM (pH 7.3) at -60 mV, rising to 2 microM (pH 5.7) at -10 mV. Neuronal M-current exhibited a similar sensitivity. Extracellular H+ ions affected two distinct properties of KCNQ2/3 current: the maximum current attainable upon depolarization (Imax) and the voltage dependence of steady-state activation. Reduction of Imax was antagonized by extracellular K+ ions and affected by mutations within the outer-pore turret, indicating an outer-pore based process. This reduction of Imax was shown to be due primarily to a decrease in the maximum open-probability of single KCNQ2/3 channels. Single-channel open times were shortened by acidosis (pH 5.9), while closed times were increased. Acidosis also recruited a longer-lasting closed state, and caused a switch of single-channel activity from the full-conductance state ( approximately 8 pS) to a subconductance state ( approximately 5 pS). A depolarizing shift in the activation curve of macroscopic KCNQ2/3 currents and single KCNQ2/3 channels was caused by acidosis, while alkalosis caused a hyperpolarizing shift. Activation and deactivation kinetics were slowed by acidosis, indicating specific effects of H+ ions on elements involved in gating. Contrasting modulation of homomeric KCNQ2 and KCNQ3 currents revealed that high sensitivity to H+ ions was conferred by the KCNQ3 subunit.     

Diclofenac-induced peripheral antinociception is associated with ATP-sensitive K+ channels activation

In order to investigate to the contribution of K+ channels on the peripheral antinociception induced by diclofenac, we evaluated the effect of several K+ channel blockers, using the rat paw pressure test, in which sensitivity is increased by intraplantar injection (2 microg) of prostaglandin E2. Diclofenac administered locally into the right hindpaw (25, 50, 100 and 200 microg) elicited a dose-dependent antinociceptive effect which was demonstrated to be local, since only higher doses produced an effect when injected in the contralateral paw. This blockade of PGE2 mechanical hyperalgesia induced by diclofenac (100 microg/paw) was antagonized in a dose-dependent manner by intraplantar administration of the sulphonylureas glibenclamide (40, 80 and 160 microg) and tolbutamide (80, 160 and 320 microg), specific blockers of ATP-sensitive K+ channels, and it was observed even when the hyperalgesic agent used was carrageenin, while the antinociceptive action of indomethacin (200 microg/paw), a typical cyclo-oxygenase inhibitor, over carrageenin-induced hyperalgesia was not affected by this treatment. Charybdotoxin (2 microg/paw), a blocker of large conductance Ca2+-activated K+ channels and dequalinium (50 microg/paw), a selective blocker of small conductance Ca2+-activated K+ channels, did not modify the effect of diclofenac. This effect was also unaffected by intraplantar administration of non-specific voltage-dependent K+ channel blockers tetraethylammonium (1700 microg) and 4-aminopyridine (100 microg) or cesium (500 microg), a non-specific K+ channel blocker. The peripheral antinociceptive effect induced by diclofenac was antagonized by NG-Nitro L-arginine (NOarg, 50 microg/paw), a NO synthase inhibitor and methylene blue (MB, 500 microg/paw), a guanylate cyclase inhibitor, and this antagonism was reversed by diazoxide (300 microg/paw), an ATP-sensitive K+ channel opener. We also suggest that an endogenous opioid system may not be involved since naloxone (50 microg/paw) did not affect diclofenac-induced antinociception in the PGE2-induced hyperalgesia model. This study provides evidence that the peripheral antinociceptive effect of diclofenac may result from activation of ATP-sensitive K+ channels, possible involving stimulation of L-arginine/NO/cGMP pathway, while Ca2+-activated K+ channels, voltage-dependent K+ channels as well as endogenous opioids appear not to be involved in the process.     

Whole-cell K+ current activation in response to voltages and carbachol in gastric parietal cells isolated from guinea pig

Summary Patch-clamp studies of whole-cell ionic currents were carried out in parietal cells obtained by collagenase digestion of the gastric fundus of the guinea pig stomach. Applications of positive command pulses induced outward currents. The conductance became progressively augmented with increasing command voltages, exhibiting an outwardly rectifying current-voltage relation. The current displayed a slow time course for activation. In contrast, inward currents were activated upon hyperpolarizing voltage applications at more negative potentials than the equilibrium potential to K⁺ (E _K). The inward currents showed time-dependent inactivation and an inwardly rectifying current-voltage relation. Tail currents elicited by voltage steps which had activated either outward or inward currents reversed at nearE _K, indicating that both time-dependent and voltagegated currents were due to K⁺ conductances. Both outward and inward K⁺ currents were suppressed by extracellular application of Ba²⁺, but little affected by quinine. Tetraethylammonium inhibited the outward current without impairing the inward current, whereas Cs⁺ blocked the inward current but not the outward current. The conductance of inward K⁺ currents, but not outward K⁺ currents, became larger with increasing extracellular K⁺ concentration. A Ca²⁺-mobilizing acid secretagogue, carbachol, and a Ca²⁺ ionophore, ionomycin, brought about activation of another type of outward K⁺ currents and voltage-independent cation currents. Both currents were abolished by cytosolic Ca²⁺ chelation. Quinine preferentially inhibited this K⁺ current. It is concluded that resting parietal cells of the guinea pig have two distinct types of voltage-dependent K⁺ channels, inward rectifier and outward rectifier, and that the cells have Ca²⁺-activated K⁺ channels which might be involved in acid secretion under stimulation by Ca²⁺-mobilizing secretagogues.     

Effects of changes in extracellular pH and potassium concentration on Kv1.3 inactivation

The Kv1.3 channel inactivates via the P/C-type mechanism, which is influenced by a histidine residue in the pore region (H399, equivalent of Shaker 449). Previously we showed that the electric field of the protonated histidines at low extracellular pH (pHe) creates a potential barrier for K+ ions just outside the pore that hinders their exit from the binding site controlling inactivation (control site) thereby slowing inactivation kinetics. Here we examined the effects of extracellular potassium [K+]e and pHe on the rate of inactivation of Kv1.3 using whole-cell patch-clamp. We found that in 150 mM [K+]e inactivation was accelerated upon switching to pHe 5.5 as opposed to the slowing at 5 mM [K+]e. The transition from slowing to acceleration occurred at 40 mM [K+]e, whereas this "turning point" was at 20 mM [K+]e for inward currents. The rate of entry of Ba(2+) ions from the extracellular space to the control site was significantly slowed by low pHe in wild-type hKv1.3, but it was insensitive to pH(e) in H399K and H399L mutants. Based on these observations we expanded our model and propose that the potential barrier created by the protonated histidines impedes the passage of K+ ions between the extracellular medium and the control site in both directions and the effect on inactivation rate (acceleration or slowing) depends on the relative contribution of filling from the extracellular and intracellular sides.     

Functional Consequences of Various Leucine Mutations in the M3/M4 Loop of the Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase α-Subunit

Leucines were mutated within the sequence L³¹¹ILGYTWLE³¹⁹ of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na⁺,K⁺-ATPase α-subunit. Replacement of Leu³¹¹ with Glu resulted in a considerable loss of Na⁺,K⁺-ATPase activity. Replacement of Leu³¹³ with Glu shifted the equilibrium of E₁P and E₂P toward E₁P and reduced the rate of the E₁P to E₂P transition. The reduction of the transition rate and stronger inhibition of Na⁺,K⁺-ATPase activity by Na⁺ at higher concentrations together suggest that there is interference of Na⁺ release on the extracellular side in the Leu³¹³ mutant. Thus, Leu³¹³ could be in the pathway of Na⁺ exit. Replacement of Leu³¹⁸ with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K⁺, with an equilibrium shifted toward E₂P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K⁺ ₂]E₂ caused by inhibition of dephosphorylation through reduction of the K⁺ affinity of E₂P. Thus, Leu³¹⁸ may be a critical position in guiding external K⁺ to its binding site.     

Modulation of gating of a metabolically regulated,ATP-dependent K+ channel by intracellular pH in B cells of the pancreatic islet

Summary Membrane-permeant weak acids and bases, when applied to the bath, modulate the resting membrane potential and the glucose-induced electrical activity of pancreatic B cells, as well as their insulin secretion. These substances alter the activity of a metabolite-regulated. ATP-sensitive K⁺ channel which underlies the B-cell resting potential. We now present several lines of evidence indicating that the channel may be directly gated by pH _i . (1) The time course of K⁺(ATP) channel activity during exposure to and washout of NH₄Cl under a variety of experimental conditions, including alteration of the electrochemical gradient for NH₄Cl entry and inhibition of the Na _o ⁺ H _i ⁺ exchanger, resembles the time course of pH _i measured in other cell types that have been similarly treated. (2) Increasing pH _o over the range 6.25–7.9 increases K⁺(ATP) channel activity in cell-attached patches where the cell surface exposed to the bath has been permeabilized to H⁺ by the application of the K⁺/H⁺ exchanger nigericin. (3) Increasing pH _i over a similar range produces similar effects on K⁺(ATP) channels in inside-out excised patches exposed to small concentrations of ATP _i . The physiological role of pH _i in the metabolic gating of this channel remains to be explored.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Interactions of K+ ATP channel blockers with Na+/K+-ATPase

Two K⁺ _ATP channel blockers, 5-hydroxydecanoate (5-HD) and glyburide, are often used to study cross-talk between Na⁺/K⁺-ATPase and these channels. The aim of this work was to characterize the effects of these blockers on purified Na⁺/K⁺-ATPase as an aid to appropriate use of these drugs in studies on this cross-talk. In contrast to known dual effects (activating and inhibitory) of other fatty acids on Na⁺/K⁺-ATPase, 5-HD only inhibited the enzyme at concentrations exceeding those that block mitochondrial K⁺ _ATP channels. 5-HD did not affect the ouabain sensitivity of Na⁺/K⁺-ATPase. Glyburide had both activating and inhibitory effects on Na⁺/K⁺-ATPase at concentrations used to block plasma membrane K⁺ _ATP channels. The findings justify the use of 5-HD as specific mitochondrial channel blocker in studies on the relation of this channel to Na⁺/K⁺-ATPase, but question the use of glyburide as a specific blocker of plasma membrane K⁺ _ATP channels, when the relation of this channel to Na⁺/K⁺-ATPase is being studied.     

Mechanism of aldosterone-induced increase of K+ conductance in early distal renal tubule cells of the frog

Summary Isolated early distal tubule cells (EDC) of frog kidney were incubated for 20–28 hr in the presence of aldosterone and then whole-cell K⁺ currents were measured at constant intracellular pH by the whole-cell voltage-clamp technique. Aldosterone increased barium-inhibitable whole-cell K⁺ conductance (gK⁺) threefold. This effect was reduced by amiloride and totally abolished by ouabain. However, aldosterone could still raisegK⁺ in ouabain-treated cells in the presence of furosemide.We tested whether changes in intracellular pH (pH_i) could be a signal for cells to regulategK⁺. After removal of aldosterone, the increase ingK⁺ was preserved by subsequent incubation for 8 hr at pH 7.6 but abolished at pH 6.6. In the complete absence of aldosterone, incubation of cells at pH 8.0 for 20–28 hr raised pH_i and doubledgK⁺.Using the patch-clamp technique, three types of K⁺-selective channels were identified, which had conductances of 24, 45 and 59 pS.Aldosterone had no effect on the conductance or open probability (P_o) of any of the three types of channels. However, the incidence of observing type II channels was increased from 4 to 22%. Type II channels were also found to be pH sensitive,P_o was increased by raising pH.These results indicate that prolonged aldosterone treatment raises pH_i and increasesgK⁺ by promoting insertion of K⁺ channels into the cell membrane. Channel insertion is itself triggered by raising both pH_i and increasing the activity of the Na⁺/K⁺ pump in early distal cells of frog kidney.Present address: Department of Physiology, The University of Leeds, Leeds, LS2 9NQ, England     

Vernakalant activates human cardiac K2P17.1 background K channels

Atrial fibrillation (AF) contributes significantly to cardiovascular morbidity and mortality. The growing epidemic is associated with cardiac repolarization abnormalities and requires the development of more effective antiarrhythmic strategies. Two-pore-domain K⁺ channels stabilize the resting membrane potential and repolarize action potentials. Recently discovered K_2P17.1 channels are expressed in human atrium and represent potential targets for AF therapy. However, cardiac electropharmacology of K_2P17.1 channels remains to be investigated. This study was designed to elucidate human K_2P17.1 regulation by antiarrhythmic drugs.     

Patch clamp analysis of chemically activated and modulated ionic channels in isolated mammalian cardiomyocytes

Techniques routinely utilized in this laboratory for recording currents through single ionic channels of isolated atrial and ventricular rat cardiomyocytes are described. Emphasis is placed in two main areas: first, on methods for obtaining a sufficient yield of Ca⁺⁺-tolerant myocytes suitable for patch clamp experiments, and secondly, on methods for analyzing the temporal characteristics of patched ionic channels. These methods were used on acetylcholine activated K⁺ channels in isolated atrial myocytes and on an inwardly-rectifying K⁺ channel in ventricular myocytes. The latter is an example of a hormonally modulated K⁺ channel, since its activity could be substantially increased by norepinephrine. Analysis of the closed and open time distributions suggested that one of the closed states of this channel is markedly abbreviated by norepinephrine, whereas the open state is nearly unaffected. Norepinephrine was effective when channel activity was recorded from on-cell patches and the hormone was added to the solution bathing the cell membrane outside of the patched area. This indicates that a second messenger substance is probably mediating the action of norepinephrine.