Transient Ca2+-channel current characterized by a low-threshold voltage in zona glomerulosa cells of rat adrenal cortex

Voltage-gated Ca²⁺-current was identified in single isolated cells of the zona glomerulosa of adrenal cortex and its properties were studied by the tight-seal whole cell recording technique. The Ca²⁺-channel current was dissected from the net current by dialyzing the cells with CsCl. The identified Ca²⁺-current was found to be activated by a relatively small depolarization only when the cells were held at a large negative holding potential, but it was inactivated within 10–30 ms. The time course of activation and inactivation was voltage-dependent and became faster when the amplitude of depolarization was increased. The transmembrane potential of the glomerulosa cells was highly sensitive to [K⁺]_e, the slope of the potential change per tenfold change in [K⁺]_e being 48 mV. An increase in [K⁺]_e from 4.7 to 10 mM induce a membrane depolarization by 15 mV, which was sufficient to cause the membrane to reach the threshold potential (–60 mV) for activation of the Ca²⁺-current at physiological concentration of [Ca²⁺]_e (2.5 mM –CaCl₂). The observed properties of the Ca²⁺-current and K⁺-dependence of the membrane potential may give reasonable explanation for the mechanism of Ca²⁺-uptake and consequent aldosterone secretion induced by a small increase in [K⁺]_e, which is known to be one of the major stimulations for aldosterone secretion.     

Voltage-gated potassium conductances in Gymnotus electrocytes(AB)

Electrocytes are muscle-derived cells that generate the electric organ discharge (EOD) in most gymnotiform fish. We used an in vitro preparation to determine if the complex EOD of Gymnotus carapo was related to the membrane properties of electrocytes. We discovered that in addition to the three Na⁺-mediated conductances described in a recent paper [Sierra F, Comas V, Buño W, Macadar O (2005) Sodium-dependent plateau potentials in electrocytes of the electric fish Gymnotus carapo. J Comp Physiol A 191:1–11] there were four K⁺-dependent conductances. Membrane depolarization activated a delayed rectifier (I_K) and an A-type (I_A) current. I_A displayed fast voltage-dependent activation-inactivation kinetics, was blocked by 4-aminopyridine (1 mM) and played a major role in action potential (AP) repolarization. Its voltage dependence and kinetics shape the brief AP that typifies Gymnotus electrocytes. The I_K activated by depolarization contributed less to AP repolarization. Membrane hyperpolarization uncovered two inward rectifiers (IR1 and IR2) with voltage dependence and kinetics that correspond to the complex “hyperpolarizing responses” (HRs) described under current-clamp. IR1 shows “instantaneous” activation, is blocked by Ba²⁺ and Cs⁺ and displays a voltage and time dependent inactivation that matches the hyperpolarizing phase of the HR. The activation of IR2 is slower and at more negative potentials than IR1 and is resistant to Ba²⁺ and Cs⁺. This current fits the depolarizing phase of the HR.     

Properties of voltage-gated currents of microglia developed using macrophage colony-stimulating factor

Microglia were isolated from a murine neonatal brain cell culture in which their development had been stimulated by supplementation with the macrophage/microglial growth factor macrophage colony-stimulating factor (M-CSF). Using the whole-cell configuration of the patch-clamp technique, voltage-gated membrane currents were recorded from these microglial cells. Hyperpolarization induced inward rectifying K⁺ currents, as described for microglia from untreated cultures. These currents activated negative to the K⁺ equilibrium potential and, with a strong hyperpolarization, displayed time-dependent inactivation. The inactivation was abolished when extracellular NaCl was replaced by N-methyl-d-glucamine (NMG), thereby indicating a partial block of this K⁺ conductance by Na⁺. Inward rectifying currents were also blocked by extracellularly applied Cs⁺ or Ba²⁺. They were slightly diminished following treatment with extracellular tetraethylammonium chloride (TEA) but were not affected by 4-aminopyridine (4-AP). Upon long lasting depolarizing voltage pulses to potentials positive to 0 mV, the cells exhibited a slowly activating H⁺ current which could be reduced by application of inorganic polyvalent cations (Ba²⁺, Cd²⁺, Co²⁺, La³⁺, Ni²⁺, Zn²⁺) as well as by 4-AP or TEA. Based on their kinetics and pharmacological characteristics, both currents detected on M-CSF-grown microglia are suggested to correspond to the inward rectifier and the H⁺ current of macrophages.     

Current profiles of astrocytes from the corpus callosum of newborn and 28-day-old rats

In astrocytes, ion currents are predominantly carried by K⁺ ions, and their potassium channel expression changes during development. Here, we studied ion current generated by voltage-ramp protocols in cultured astrocytes from the corpus callosum of newborn (P0) and 28-day-old (P28) rats. Inward currents measured at −140 mV and chord conductances measured from −140 to −75 mV, were smaller in P0-astrocytes than in P28-astrocytes, and in P28-astrocytes were affected by 100 μM Ba²⁺, indicating the presence of an inward rectifier K⁺ (Kir) current. On the other hand, P0-astrocytes showed higher outward current measured at 80 mV and a higher chord conductance, between 0 and 80 mV, than P28-astrocytes. The outward current was more potently reduced by 2 mM Ba²⁺ in P0-astrocytes than in P28-astrocytes, and slightly reduced at both ages using low concentrations of Ba²⁺. Moreover, outward current was partially blocked by iberiotoxin in P0-astrocytes, indicating the presence of big-conductance Ca²⁺-activated K⁺ (BK) channels. In addition, 4-aminopyridine inhibited the outward current in P0- and P28-astrocytes. In summary, P0-astrocytes exhibited the BK current, a major density of delayed rectifier K⁺ (K_DR) current, and a low density of the Kir current, whereas P28-astrocytes presented a major density of Kir current, a low density of the K_DR current, and the absence of BK current. These results could contribute to a better understanding of the role of K⁺ currents in the corpus callosum.     

K+ channel antisense oligodeoxynucleotides inhibit cytokine-induced expansion of human hemopoietic progenitors

Primitive human hemopoietic progenitor cells identified by surface membrane markers CD33^–CD34⁺ are capable of expansion into lineage-restricted precursors following in vitro stimulation by hemopoietic regulators such as stem cell factor (SCF) and interleukin-3 (IL-3). In search of ionic currents involved in cytokine-induced progenitor cell growth and differentiation, human umbilical cord blood CD33^–CD34⁺ cells were subjected to perforated patch-clamp recordings following overnight incubation with SCF and/or IL-3. An inward rectifying potassium channel (K_ir) was found in 33% of control unstimulated cells, in 34% of cells incubated with IL-3, in 31 % of cells incubated with SCF and in 75% of cells incubated with IL-3 plus SCE K_ir activity increased with elevation of extracellular potassium and was blocked by extracellular Cs⁺ or Ba²⁺. Antisense oligodeoxynucleotides directed against K_ir blocked both mRNA and functional expression of K_ir channels. K_ir antisense also inhibited the in vitro expansion of cytokine-stimulated CD33^–CD34⁺ cells into erythroid (BFU-E) and myeloid (GM-CFU) progenitors in 7-day suspension cultures. Extracellular Cs⁺ or Ba²⁺ induced a similar degree of inhibition (40–60%) of progenitor cell generation. These findings strongly suggest an essential role for K_ir in the process of cytokine-induced primitive progenitor cell growth and differentiation.     

Impact of Amyloid-β Peptide (1–42) on Voltage-Gated Ion Currents in Molluscan Neurons

Different types of voltage-gated ion currents were recorded in isolated neurons of snail Helix pomatia using the two-microelectrode voltage-clamp technique. Application of amyloid-β peptide (1–42, 1–10 μM) in the bathing solution did not change delayed rectifier K⁺-current and leakage current, but enhanced inactivation of Ca²⁺-current and blocked Ca²⁺-dependent К⁺-current.     

Barium block of the muscarinic potassium current in guinea-pig atrial cells

Block of the muscarinic K⁺ current (i _K,ACh) by Ba²⁺ has been studied in guinea-pig atrial cells using the whole-cell patch-clamp technique. The dose-response curve for the block of i _K,ACh can be fitted assuming that a muscarinic K⁺ channel is blocked when a single Ba²⁺ ion binds to it (apparent dissociation constant, K _d=125 M at 0 mV). Block was voltage and time dependent. The voltage dependence can be explained by Ba²⁺ binding to a site within the pore of the channel, 36% across the width of the membrane electric field (from the outside). Raising the bathing K⁺ concentration reduced Ba²⁺ block of i _K,ACh which suggests that Ba²⁺ and K⁺ compete for a common binding site. When Ba²⁺ was added during an exposure to ACh (muscarinic K⁺ channel open), block of i _K,ACh developed rapidly, but when Ba²⁺ was added prior to an exposure to ACh (muscarinic K⁺ channel closed), little block of i _K,ACh was evident when ACh was first applied. This suggests that when the muscarinic K⁺ channel is closed in the absence of ACh, Ba²⁺ does not have access to the binding site within the pore of the channel. In conclusion, Ba²⁺ block of i _K,ACh is concentration, voltage, time, K⁺ and state dependent.     

Whole-cell currents in olfactory receptor cells of Xenopus laevis

Summary Olfactory mucosae of Xenopus laevis were dissociated without enzymatic treatment and the isolated olfactory neurones were studied with the tight-seal whole-cell recording configuration of the patch clamp technique. In the voltage clamp, five current components could be distinguished: a fast, TTX-sensitive Na⁺-current, a small and slow inward current carried by Ca²⁺ ions, a Ca²⁺ dependent K⁺-current, a K⁺-current which activates rapidly at voltages more positive than-20 mV and quickly inactivates, and a slowly activating and very slowly inactivating K⁺-current. Some of the characteristics of the whole-cell currents herein reported contradict previous findings while others verify them, thereby allowing a tentative interpretation of their physiological role in the transduction process.     

Voltage-activated ion channels and Ca2+-induced Ca2+ release shape Ca2+ signaling in Merkel cells

Ca²⁺ signaling and neurotransmission modulate touch-evoked responses in Merkel cell–neurite complexes. To identify mechanisms governing these processes, we analyzed voltage-activated ion channels and Ca²⁺ signaling in purified Merkel cells. Merkel cells in the intact skin were specifically labeled by antibodies against voltage-activated Ca²⁺ channels (Ca_V2.1) and voltage- and Ca²⁺-activated K⁺ (BK_Ca) channels. Voltage-clamp recordings revealed small Ca²⁺ currents, which produced Ca²⁺ transients that were amplified sevenfold by Ca²⁺-induced Ca²⁺ release. Merkel cells’ voltage-activated K⁺ currents were carried predominantly by BK_Ca channels with inactivating and non-inactivating components. Thus, Merkel cells, like hair cells, have functionally diverse BK_Ca channels. Finally, blocking K⁺ channels increased response magnitude and dramatically shortened Ca²⁺ transients evoked by mechanical stimulation. Together, these results demonstrate that Ca²⁺ signaling in Merkel cells is governed by the interplay of plasma membrane Ca²⁺ channels, store release and K⁺ channels, and they identify specific signaling mechanisms that may control touch sensitivity.     

Analogies and differences between ω-conotoxins MVIIC and MVIID: binding sites and functions in bovine chromaffin cells

 The characteristics of the binding sites for the Conus magus toxins ω-conotoxin MVIIC and ω-conotoxin MVIID, as well as their effects on K⁺-evoked ⁴⁵Ca²⁺ entry and whole-cell Ba²⁺ currents (I _Ba), and K⁺-evoked catecholamine secretion have been studied in bovine adrenal chromaffin cells. Binding of [¹²⁵I] ω-conotoxin GVIA to bovine adrenal medullary membranes was displaced by ω-conotoxins GVIA, MVIIC and MVIID with IC₅₀ values of around 0.1, 4 and 100 nM, respectively. The reverse was true for the binding of [¹²⁵I] ω-conotoxin MVIIC, which was displaced by ω-conotoxins MVIIC, MVIID and GVIA with IC₅₀ values of around 30, 80 and 1.200 nM, respectively. The sites recognized by ω-conotoxins MVIIC and MVIID in bovine brain exhibited higher affinities (IC₅₀ values of around 1 nM). Both ω-conotoxin MVIIC and MVIID blocked I _Ba by 70–80%; the higher the [Ba²⁺]_o of the extracellular solution the lower the blockade induced by ω-conotoxin MVIIC. This was not the case for ω-conotoxin MVIID; high Ba²⁺ (10 mM) slowed down the development of blockade but the maximum blockade achieved was similar to that obtained in 2 mM Ba²⁺. A further difference between the two toxins concerns their reversibility; washout of ω-conotoxin MVIIC did not reverse the blockade of I _Ba while in the case of ω-conotoxin MVIID a partial, quick recovery of current was produced. This component was irreversibly blocked by ω-conotoxin GVIA, suggesting that it is associated with N-type Ca²⁺ channels. Blockade of K⁺-evoked ⁴⁵Ca²⁺ entry produced results which paralleled those obtained by measuring I _Ba. Thus, 1 μM of each of ω-conotoxin GVIA and MVIIA inhibited Ca²⁺ uptake by 25%, while 1 μM of each of ω-conotoxin MVIIC and MVIID caused a 70% blockade. K⁺-evoked catecholamine secretory responses were not reduced by ω-conotoxin GVIA (1 μM). In contrast, at 1 μM both ω-conotoxin MVIIC and MVIID reduced the exocytotic response by 70%. These data strengthen the previously established conclusion that Q-type Ca²⁺ channels that contribute to the regulation of secretion and are sensitive to ω-conotoxins MVIIC and MVIID are present in bovine chromaffin cells. These channels, however, seem to possess binding sites for ω-conotoxins MVIIC and MVIID whose characteristics differ considerably from those described to occur in the brain; they might represent a subset of Q-type Ca²⁺ channels or an entirely new subtype of voltage-dependent high-threshold Ca²⁺ channel. Received: 16 April 1997 / Received after revision: 10 July 1997 / Accepted: 23 July 1997     

Patch-clamp studies of K+ and Cl− channel currents in canine pancreatic acinar cells

K⁺ and Cl^– channel currents in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp single-channel and whole-cell current recording techniques. In excised inside-out patches, we found a Ca⁺-activated (control range 0.01–0.4 M) and voltage-activated K²⁺-selective channel with a unit conductance of approximately 40 pS in symmetrical K⁺-rich solutions. In intact cells, addition of acetylcholine (1 M) or bombesin tetradecapeptide (0.1 nM) to the bath evoked an increase in frequency of K⁺ channel opening. In whole-cell recordings on cells dialyzed with K⁺-rich and Ca²⁺-free solution containing 0.5 mM EGTA, the resting potential was about –40mV. Depolarizing voltage pulses activated outward K⁺ currents, which were blocked by 10 mM tetraethylammonium, whereas hyperpolarizing pulses evoked smaller inward currents. Acetylcholine or bombesin activated the K⁺ current and enhanced the inward current, which was reduced by a low Cl^– (10 mM) intracellular solution at –90 mV holding potential. These results suggest that both Ca²⁺- and voltage-activated K⁺ channels and Ca²⁺-activated Cl^– channels exist in the plasma membrane of canine pancreatic acinar cells.     

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca²⁺-activated K⁺ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na⁺, 5.4 mM K⁺) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca²⁺ concentration (10^–7 to 10^–6 M). No voltage dependence of the channel activity was observed. Internal Na⁺ blocked the outward K⁺ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba²⁺ (0.1 mM) and tetracthylammonium (TEA⁺, 20–50 mM). Ba²⁺ reduced the open probability but not the single channel conductance, whereas TEA⁺ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K⁺ solution (150 mM K⁺) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca²⁺-activated K⁺ channels observed in many other cells.     

Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH

With the single-channel patch-clamp technique we have identified Ca²⁺-sensitive, high-conductance (maxi) K⁺ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K⁺ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K⁺. In excised inside-out patches and with symmetrical 140 mmol/l K⁺, single-channel conductance was 200 pS and the channel exhibited a high selectivity for K⁺ over Na⁺. Depolarization of the BLM increased maxi K⁺ channel activity. Increasing cytosolic free Ca²⁺ (by addition of 100 nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca²⁺ directly activated channel activity in a voltage-dependent manner. Maxi K⁺ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K⁺ channel outward current was reversibly blocked by micromolar concentrations of Ba²⁺ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches.     

The Ba2+ block of the ATP-sensitive K+ current of mouse pancreaticβ-cells

We studied the block of whole-cell ATP-sensitive K⁺ (K_ATP) currents in mouse pancreatic-cells produced by external Ba²⁺. Ba²⁺ produced a time- and voltage-dependent block of K_ATP currents, both the rate and extent of the block increasing with hyperpolarization. With 5.6 mM [K⁺]_o, the relationship between the steady-state KATP current and [Ba²⁺]_o, was fit by the Hill equation with aK _d of 12.5 ± 2.8 M at –123 mV and of 0.18 ± 0.02 mM at –62 mV The Hill coefficient (n) was close to 1 at all potentials indicating that binding of a single Ba²⁺ ion is sufficient to block the channel. When [K⁺]_o was raised to 28 mM the K_d was little changed (12.4 ± 4.1 gM at –123 mV 0.27 ± 0.05 mM at –62 mV) and n was unaffected, suggesting that K⁺ does not interact with the Ba²⁺ binding site. The kinetics of Ba²⁺ block were slow, 10 M Ba²⁺ blocking the K_ATP current with a time constant of 20 ms at –123 mV in 28 mM [K⁺]_o. The blocking rate constant was calculated as 1.7 mM^–1 ms^–1 and the unblocking rate as 0.02 ms^–1, at –123 mV The data are discussed in terms of a model in which Ba²⁺ binds to a site at the external mouth of the channel to inhibit the K_ATP channel.     

Characteristics and modulation by thyrotropin-releasing hormone of an inwardly rectifying K+ current in patch-perforated GH3 anterior pituitary cells

Hyperpolarization of patch-perforated GH₃ rat anterior pituitary cells in high-K⁺ Ca²⁺-free medium reveals an inwardly rectifying K⁺ current. This current showed potential-dependent activation and inactivation kinetics, complete inactivation during strong hyperpolarization and rectification at depolarized potentials. The current was blocked by millimolar concentrations of external Cs⁺, Ba²⁺, Cd²⁺ and Co²⁺, but it was almost insensitive to tetraethylammonium, 4-aminopyridine and two dihydropyridines, nisoldipine and nitrendipine. Verapamil and methoxyverapamil produced a strong and reversible inhibition of the current. In the presence of 100 nM thyrotropin-releasing hormone (TRH), the current was reduced. This reduction was increased by holding the cell at more negative potentials and was accompanied by a shift in steady-state voltage dependence of inactivation towards more positive voltages. Furthermore, the current slowly returned to the initial levels upon washout. Treatment of the cell with the protein phosphatase inhibitor okadaic acid increased the magnitude of the inhibition caused by TRH. Moreover, the current did not return towards the control level during a 30-min washout period. It is concluded that protein phosphatases participate in modulation of the GH₃ cell inwardly rectifying K⁺ channels by TRH. Furthermore, these data indicate that either a protein phosphatase or a factor necessary for its activation is lost under whole-cell mode, which could account for the permanent reduction of the current in response to TRH.     

Differential modulation of pharmacologically distinct components of Ca2+ currents by protein kinase C activators and phosphatase inhibitors in nerve-growth-factor-differentiated rat pheochromocytoma (PC12) cells

We have studied the effects of protein kinase C (PKC) activators 4-phorbol 12-myristate 13-acetate (4-PMA) and 1-oleoyl-2-acetylglycerol (OAG) and of phosphatase inhibitors (okadaic acid and calyculin A) on voltage-activated Ca²⁺ and K⁺ channels in nerve growth factor-(NGF)-differentiated pheochromocytoma (PC12) cells. Whole-cell Ba²⁺ and K⁺ currents were recorded at room temperature with the patch-clamp technique. By using -conotoxin (CgTX) and isradipine, two specific Ca²⁺ channel blockers, we found three types of Ba²⁺ currents (I _Ba): (1) a -CgTX-sensitive I _Ba; (2) an isradipine-sensitive I _Ba; and (3) a -CgTX plus isradipine-resistant I _Ba. The external application of 4 -PMA or OAG down-modulated the isradipine-sensitive I _Ba whereas the two other I _Ba were not affected. 4-PMA-induced inhibition of I _Ba was prevented by staurosporine (a protein kinase inhibitor) and PKC (19–31) (a specific PKC inhibitor). The delayed rectifier K⁺ current (I _K) was unaffected by PKC activators. Both okadaic acid and calyculin A affected the components of the I _Ba in different manners. The presence of okadaic acid decreased the isradipine-sensitive I _Ba more than the -CgTX-sensitive I _Ba. The -CgTX plus isradipine-resistant I _Ba was not affected. Calyculin A down-modulated all three components of I _Ba to a similar degree. Our results suggest a differential modulation of voltage-activated Ca²⁺ and K⁺ channels by the PKC signalling pathway in NGF-differentiated PC12 cells.     

High-conductance K+ channel in apical membranes of principal cells cultured from rabbit renal cortical collecting duct anlagen

Using the patch clamp technique, one type of K⁺ channel was identified in the apical cell membrane of cultured principal cells of rabbit renal collecting ducts in the cell-attached or excised-patch configuration. The channel was highly selective for K⁺ over Na⁺ (typically 30-70-fold) and had a conductance of 180, SD±39 pS (n=6), referred to a situation of 140 mmolar K⁺-Ringer solution present on either surface of the patch membrane. Channel activity was completely blocked by Ba²⁺ (5 mmol/l) and partially inhibited by Na⁺. The latter was evidenced by a deviation from Goldman rectification at high cytoplasm-positive membrane potentials, which was observed when Na⁺ competed with K⁺ for channel entrace from the cytoplasmic surface. Channel open probability depended strongly on membrane voltage and cytoplasmic Ca²⁺ concentration. Open-close kinetics exhibited double exponential behaviour, with a strong voltage dependence of the slow open time constant. Infrequently also a substate conductance level was identified. The voltage and calcium dependence suggest that the channel plays a role in adjusting K⁺ secretion to Na⁺ absorption in the fine regulation of cation excretion in renal collecting ducts.     

Blockade of the delayed rectifier K+ currents, I Kr, in rabbit sinoatrial node cells by external divalent cations

We investigated the actions of various divalent cations on the delayed rectifier K⁺ currents (I _Kr) in rabbit sinoatrial node cells using the whole-cell voltage-clamp technique in isotonic K⁺ solutions. External divalent cations decreased the amplitude of currents, accelerated the time course of deactivation and shifted the activation to positive potentials in a dose-dependent manner. The concentrations for half-maximum inhibition of the steady-state currents (K _M) obtained at 0 mV were 0.63, 1.36, 1.65 and 2.16 mM for Ni²⁺, Co²⁺, Mn²⁺ and Ba²⁺, respectively. The effect was voltage dependent (K _M decreased e-fold for 12.2–16.8 mV hyperpolarization), but the dependence did not vary significantly among different cations. Acceleration of the time course of current deactivation by the increase of divalent cation concentration was well fitted by the voltage-dependent block model, and the binding rate constant (k ₁) was obtained. The binding rates for the ions took the following order: Ni²⁺ >Co²⁺ >Mn²⁺ >Ba²⁺. The degree of the shift of activation occurred in the same order: Ni²⁺ >Co²⁺ >Mn²⁺ >Ba²⁺. From these results, we conclude that I _Kr channels are non-selectively blocked by most divalent cations from the external side and that the binding site is located deep inside the channel, resulting in a steep voltage dependence of the blockade. Received: 26 January 1999 / Received after revision: 16 March 1999 / Accepted: 18 March 1999     

Mechanisms of K(+) induced renal vasodilation in normo- and hypertensive rats in vivo

Aim: We investigated the mechanisms behind K⁺‐induced renal vasodilation in vivo in normotensive Sprague–Dawley (SD) rats and spontaneously hypertensive rats (SHR). Methods: Renal blood flow (RBF) was measured utilizing an ultrasonic Doppler flow probe. Renal vascular resistance (RVR) was calculated as the ratio of mean arterial pressure (MAP) and RBF (RVR = MAP/RBF). Test drugs were introduced directly into the renal artery. Inward rectifier K⁺ (K_ir) channels and Na⁺,K⁺‐ATPase were blocked by Ba²⁺ and ouabain (estimated plasma concentrations ～20 and ～7 μm ) respectively. Results: Confocal immunofluorescence microscopy demonstrated K_ir 2.1 channels in pre‐glomerular vessels of SD and SHR. Ba²⁺ caused a transient (6–13%) increase in baseline RVR in both SD and SHR. Ouabain had a similar effect. Elevated renal plasma [K⁺] (～12 mm ) caused a small and sustained decrease (5–13%) in RVR in both strains. This decrease was significantly larger in SHR than in SD. The K⁺‐induced vasodilation was attenuated by Ba²⁺ in control SD and SHR and by ouabain in SD. Nitric oxide (NO) blockade using l ‐NAME treatment increased MAP and decreased RBF in both rat strains, but did not affect the K⁺‐induced renal vasodilation. Conclusion: K⁺‐induced renal vasodilation is larger in SHR, mediated by K_ir channels in SD and SHR, and in addition, by Na⁺,K⁺‐ATPase in SD. In addition, NO is not essential for K⁺‐induced renal vasodilation.     

Small and maxi K+ channels in the basolateral membrane of isolated crypts from rat distal colon: single-channel and slow whole-cell recordings

The patch-clamp technique was used to characterize K⁺ channel activity in the basolateral membrane of isolated crypts from rat distal colon. In cell-attached patches with KCl in the pipette, channels with conductances ranging from 6 pS to 80 pS appeared. With NaCl in the pipette and KCl in the bath in excised inside-out membrane patches a small-conductance channel with a mean conductance of 12±6 pS (n=18) was observed. The channel has been identified as K⁺ channel by its selectivity for K⁺ over Na⁺ and by its sensitivity to conventional K⁺ channel blockers, Ba²⁺ and tetraethylammonium (TEA⁺). Changes of cytosolic pH did not attenuate channel activity. Activity of the 12-pS channel was increased by membrane depolarization and elevated cytosolic Ca²⁺ concentration. In addition, a maxi K⁺ channel with a mean conductance of 187±15 pS (n=4) in symmetrical KCl solutions was only occasionally recorded. The maxi K⁺ channel could be blocked by Ba²⁺ (5 mmol/l) on the cytosolic side. Using the slow-whole cell recording technique under control conditions, a cell membrane potential of –70±10mV (n=18) was measured. By application of various K⁺ channel blockers such as glibenclamide, charybdotoxin, apamin, risotilide, Ba²⁺ and TEA⁺ in the bath, only Ba²⁺ and TEA⁺ depolarized the cell membrane. The present data suggest that the small K⁺ channel (12 pS) is involved in the maintenance of the cell membrane resting potential.