Regulation of ion channels in rat hepatocytes

 Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K⁺ channel with a mean inward conductance of 55 ± 6.5 pS (n = 20) and a mean outward conductance of 25 ± 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl^– channel with a mean outward conductance of 30 ± 4.5 pS (n = 8) and a mean inward conductance of 10 ± 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca²⁺ activity on the intracellular side. Accordingly, the Ca²⁺ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K⁺ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K⁺ channel is inhibited by progesterone, while the outwardly rectifying Cl^– channel is insensitive to progesterone. Received: 21 May 1997 / Received after revision: 7 August 1997 / Accepted: 19 August 1997     

Tetraethylammonium block of Slowpoke calcium-activated potassium channels expressed in Xenopus oocytes: Evidence for tetrameric channel formation

Unitary currents were recorded from insideout membrane patches pulled from Xenopus oocytes that had been injected with RNA transcribed from a cDNA encoding the Drosophila maxi-K channel (Slowpoke). Site-directed mutagenesis was used to make cDNAs encoding channel subunits with single amino acid substitutions (Y308V and C309P). The extracellular side of the patch was exposed to tetraethylammonium (TEA) in the pipette solution; unitary currents in the presence of TEA were compared with currents in the absence of TEA to compute the inhibition. Amplitude distributions were fit by functions to estimate the blocking and unblocking rate constants. For wild-type channels, TEA blocked with an apparent K _d of 80 M at 0 mV and sensed 0.18 of the membrane electric field; the voltage dependence lay entirely in the blocking rate constant. TEA blocked currents through C309P channels with a similar affinity to wild-type at 0 mV, but this was not voltage-dependent. Currents through Y308V channels were very insensitive to any block by TEA; the apparent K _d at 0 mV was 26 mM and the blockade sensed 0.18 of the electric field. Oocytes injected with a mixture of RNAs encoding wild-type and Y308V channels showed unitary currents of four discrete amplitudes in the presence of 3 mM TEA; at 40 mV these corresponded to inhibitions of approximately 80%, 55%, 25% and 10%. These values agreed well with those expected for inhibition by TEA of currents through channels containing 3, 2, 1 and 0 tyrosine residues at the channel mouth, assuming that a tyrosine residue from each of four subunits contributes equally to the binding of the TEA ion. This indicates that Slowpoke channels form as tetramers.     

Enhancement of Ca2+-dependent outward current in sheep bladder myocytes by Evans blue dye

 Whole-cell and inside-out patch-clamp techniques were used to assess the action of a well-known dye, Evans blue, on membrane currents in bladder isolated smooth muscle cells from sheep. In whole cells Evans blue dose-dependently increased the outward current by up to fivefold. In contrast, Evans blue had no effect on inward Ca²⁺ current. The effect on outward current was abolished or reduced if the cells were bathed in Ca²⁺-free solution, iberiotoxin (5 × 10^–8 M), or charybdotoxin (5 × 10^–8 M), but was unaffected by externally applied caffeine (5 mM) or in cells exposed to heparin (1 mg/ml) via the patch pipette. In inside-out patches bathed in a Ca²⁺ concentration of 5 × 10^–7 M, Evans blue (10^–4 M) increased the open probability of large-conductance (298-pS) Ca²⁺-dependent K⁺ channels (BK channels), shifting the half maximal-activation voltage by –70 mV. We conclude that Evans blue dye acts as an opener of BK channels. Received: 1 September 1997 / Received after revision: 18 November 1997 / Accepted: 20 November 1997     

Mg2+ block and inward rectification of mechanosensitive channels in Xenopus oocytes

 The effects of Mg²⁺ on single mechanosensitive (MS) channel currents recorded from Xenopus oocytes were studied using cell-attached and inside-out patch configurations. Mg²⁺ both permeates and blocks MS channels. Under symmetrical ionic conditions, the blocking effects of Mg²⁺ can be described by a Hill coefficient of 0.9 at ±100 mV and IC₅₀s of 0.12 mM (–100 mV) and 0.60 mM at (+100 mV). Although block by intracellular Mg²⁺ may contribute to inward MS channel rectification, significant current rectification is retained even under symmetrical KCl concentrations and in the complete absence of Mg²⁺. The observed voltage dependencies of the IC₅₀ for Mg²⁺ block and the K_m for K⁺ current saturation indicate asymmetries in the MS channel pore. In addition, the absence of K⁺ self block and anomalous mole fraction effects with K⁺/Tl⁺ mixtures indicate a single site pore model. Received: 11 September 1997 / Received after revision: 21 November 1997 / Accepted: 24 November 1997     

Single-channel analysis of fast transient outward K+ currents in frog skeletal muscle

 The patch-clamp method was employed to examine the voltage-dependent gating mechanism of A-type K⁺ channels, which generate the transient outward K⁺ currents described previously in a study of vesicles derived from the sarcolemma of frog skeletal muscle. Channels were activated by depolarizing pulses. There is evidence for non-random grouping of records with channel openings and blank records when depolarizations were repeated at brief intervals, suggesting a slow process similar to slow inactivation. Binomial analysis was consistent with independent behaviour of the channels. Ensemble average currents obtained from multichannel patches had kinetics similar to those of the macroscopic A-type K⁺ current, I _A. The rate of activation, fitted to n ⁴ kinetics, was fast and voltage dependent. The rate of inactivation had an exponential time course with a voltage-independent time constant. The mean open time and the probability of a channel being open increased with depolarization. The histograms of latency to first opening revealed the presence of more than two voltage-dependent closed states. Channel openings occurred in bursts and the closed-time histograms could be fitted by the sum of two or three exponentials. These results suggest a gating scheme with at least three closed states, probably two open states, and two inactivated states. Received: 4 November 1997 / Received after revision: 9 January 1998 / Accepted: 12 January 1998     

Ionic currents and current-clamp depolarisations of type I and type II hair cells from the developing rat utricle

 Ionic currents and the voltage response to injected currents were studied in an acutely dissected preparation of the rat utricle between birth and postnatal day 12 (PN12). Based upon morphological criteria, the sensory cells examined were divided into two classes, ”type I” and ”type 2 category,” the latter of which may include some immature type I cells. The former group comprises a clearly defined electrophysiological population, with one large outwardly rectifying potassium conductance that is sensitive to 4-aminopyridine (4-AP), insensitive to tetraethylammonium (TEA) and displays voltage-dependent activation kinetics. In the absence of enzymatic dissociation procedures, and with the epithelium left largely intact, the mean half activation of this conductance was –30.3 mV at PN3, and –37.5 mV at PN12. At both stages it was almost entirely turned off at –74 mV. Omission of ATP from the intracellular solution appeared to prevent rundown of this conductance. Type II category hair cells formed a more heterogeneous population, exhibiting a distinct TEA-sensitive delayed rectifier potassium conductance; the rapidly activating and inactivating I _A; an inward rectifier; and inward sodium currents at around PN3. Both cell types depolarised strongly in response to injected currents, with time courses reflecting the activation kinetics of their major outward conductances. Received: 11 July 1998 / Received after revision: 19 January 1999 / Accepted: 16 February 1999     

Characterization with barium of potassium currents in turtle retinal Müller cells

Müller cells are highly permeable to potassium ions and play a crucial role in maintaining potassium homeostasis in the vertebrate retina. The potassium current found in turtle Müller cells consists of two components: an inwardly rectifying component and a linear, passive component. These currents are insensitive to broadband potassium channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP) and well blocked by barium. Differential block by the polyamine spermine suggests that these currents flow through different channels. In this study, we used barium ions as a probe to investigate the properties of these currents by whole cell, voltage-clamp recordings from isolated cells. Current-voltage (I-V) relationships generated from current responses to short (35 ms) and long (3.5 s) voltage pulses were fit with the Hill equation. With extracellular barium, the time course of block and unblock was voltage and concentration dependent and could be fit with single exponential functions and time constants larger than 100 ms. Blocking effects by extracellular barium on the two types of currents were indistinguishable. The decrease of the outward current originates in part due to charge effects. We also found that intracellular barium was an effective blocker of the potassium currents. The relative block of the inward rectifier by intracellular barium suggests the existence of two "apparent" binding sites available for barium within the channel. Under depolarizing conditions favoring the block by internal polyamines, the Hill coefficient for barium binding was 1, indicating a single apparent binding site for barium within the pore of the passive linear conductance. The difference in the steepness of the blocking functions suggests that the potassium currents flow through two different types of channels, an inward rectifier and a linear passive conductance. Last, we consider the use of barium as an intracellular K(+) channel blocker for voltage-clamp experiments.     

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells

We have shown previously that secretagogues acting via the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl⁻ conductance, a K⁺ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K⁺ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 μmol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K⁺ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency ( f _c) of 308 ± 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K⁺ selective. 293B reduced the power density of the noise (S _o) to 46 ± 8.7% of its control value and shifted f _c from 291 ± 26 to 468 ± 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5′-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba²⁺ and was completely absent when K⁺ in the bath was replaced by Na⁺. These data suggest that forskolin activates a K⁺ channel of very small conductance which can be inhibited directly and reversibly by 293B. Received: 1 October 1995/Received after revision: 28 December 1995/Accepted: 28 December 1995     

pH regulation of voltage-dependent K+ channels in canine pulmonary arterial smooth muscle cells

 Voltage-dependent K⁺ currents (K_v) may play a role in hypoxic pulmonary vaso constriction. The effects of changes in extracellular pH (pH_o) and intracellular pH (pH_i) on K_v currents in smooth muscle cells isolated from canine pulmonary artery were studied using the amphotericin B perforated-patch technique for whole-cell recording. Under these conditions, cellular mechanisms for pH_i regulation remain intact, and the effects of pH_o were examined by directly changing the pH of external solutions and changes in pH_i were produced by external application of weak extracellular acids and bases and the cation/H⁺ ionophore, nigericin. Ca²⁺-free external solutions were used to isolate whole-cell K_v currents from contaminating Ca²⁺-activated K⁺ currents. Extracellular acidification (pH_o = 6.4–7.0) reduced K_v currents, produced a positive voltage shift in steady-state activation and reduced maximum K_v conductance (-g _K). Extracellular alkalinization (pH_o = 8.0–8.4) increased K_v currents, produced a small negative voltage shift in steady-state activation, and increased -g _K. Intracellular acidification produced by exposure of cells to external sodium butyrate (20 mM) or nigericin (5 μg/ml) increased K_v currents, produced a negative voltage shift in steady-state activation, and increased -g _K. Intracellular alkalinization produced by exposure of cells to external trimethylamine (20 mM) reduced K_v currents, produced a small positive voltage shift in steady-state activation and reduced -g _K. These results suggest that the effects of pH_o and pH_i on K_v currents are distinctly different, but are consistent with reported effects of pH_o and pH_i on hypoxic pulmonary vasoconstriction, suggesting that such modulation may be mediated in part by pH-induced alterations in K_v channel activity. Received: 1 November 1996 / Received after revision: 19 December 1996 / Accepted: 3 January 1997     

Potassium channels of adult locust (Schistocerca gregaria) muscle

Two types of K⁺ channels have been identified in patches of plasma membrane of metathoracic extensor tibiae muscle fibres of adult locust, Schistocerca gregaria. One channel had a maximum conductance of 170 pS, fast open-closed kinetics, and a linear current/ voltage relationship. In inside-out patches it was activated by ‘‘internally applied’’ Ca²⁺, but at unexpectedly low levels (between 10⁻¹⁰ and 10⁻⁹M). The other channel had a maximum conductance of 35 pS, slower open-closed kinetics, and was not activated by Ca²⁺. In cell-attached patches, its channel conductance measured in symmetrical salines was about three times greater for hyperpolarisations than for depolarisations. This inward rectification was proved to be due to block by intracellular Mg²⁺. For both channels, open probability (P _o) and mean open time increased during depolarisations and decreased during hyperpolarisations, resulting in outward rectifications in terms of net current (I _n , product of the single-channel current and P _o). For both channels, the K⁺ conductance was 10 times greater than that for Na⁺. Internally applied tetraethylammonium or tetramethylammonium ions blocked both channels. Received: 12 June 1995/Received after revision and accepted: 30 January 1996     

Membrane currents in immature oocytes of the Rana perezi frog

 Immature oocytes of the Rana perezi frog were studied electrophysiologically to see if some of the unusual ionic channels found in Xenopus oocytes were also expressed in these cells. Growing oocytes showed a fairly linear current/voltage relationship (from –200 to +60 mV), whereas fully grown cells had several voltage-dependent conductances. Depolarizing pulses elicited a potassium current blocked by tetraethylammonium (TEA) and two kinetically different Ca²⁺-dependent Cl^–currents (I _Cl(Ca)), both sensitive to niflumic acid. I _Cl(Ca), which have not been previously observed in Rana immature oocytes, were also found in response to acetylcholine or rabbit serum superfusion or intracellular injection of Ca²⁺. In addition, three different Cl^–currents were activated in these cells by hyperpolarization: (1) a transient inward current dependent on a critical intracellular Ca²⁺ concentration; (2) an inward rectifier Cl^–current, which was Ca²⁺ independent; and (3) a high threshold (over –140 mV), slow Cl^–current, blocked by several divalent cations, 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanatostilbene-2,2′-disulphonic acid (SITS). The presence of most of these infrequent currents in immature oocytes of several frogs and toads suggests that they are not merely the result of random genomic expression but a programmed decision, probably related to a definite functional role. Received: 27 November 1996 / Received after revision and accepted: 9 April 1997     

cAMP Stimulation of CFTR-expressing Xenopus oocytes activates a chromanol-inhibitable K+ conductance

 Cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl^–channel in a large variety of cells expressing this protein. Recently evidence has accumulated that it also regulates other ion channels. A coordinated increase in Cl^–and K⁺ conductances is necessary in many Cl^–-secreting epithelia. This has, for example, recently been demonstrated for the colonic crypt, for which a new type of K⁺ channel and a specific inhibitor of this channel, the chromanol 293B, have been described. In the present study we have examined whether the cAMP-evoked activation of CFTR, overexpressed in Xenopus oocytes, in addition to its known activation of a Cl^–conductance, also upregulates endogenous K⁺ channels. It is shown that CFTR-cRNA-injected but not water-injected oocytes possess a cAMP-activated Cl^–conductance. Of the cAMP-induced whole-cell current increase, 15–25% was due to a 293B-, Ba²⁺and TEA⁺-inhibitable K⁺ conductance. The cRNA of the mutated CFTR (ΔF508 CFTR) had no such effect. We conclude that cAMP activated CFTR and an endogenous IsK-type and 293B-sensitive K⁺ conductance. Similar events, occurring, for example, in the colonic crypt possessing CFTR and 293B-sensitive K⁺ channels, might explain the coordinated cAMP-mediated increase in Cl^–and K⁺ conductances. Received: 12 March 1996 / Accepted: 10 April 1996     

Antisense oligodeoxynucleotides of sulfonylurea receptors inhibit ATP-sensitive K+ channels in cultured neonatal rat ventricular cells

 To identify the functional sulfonylurea receptor (SUR), a subunit of the adenosine 5′-triphosphate (ATP)-sensitive K⁺ (K_ATP) channels, in neonatal rat ventricular cells, such cells in primary culture were treated for 6 days with antisense (AS) oligodeoxynucleotides (ODNs) complementary to the mRNA for SURs. For quantification, single-channel (inside-out patches) and whole-cell currents were measured using the patch-clamp technique. The maximal K_ATP currents (at 0 mV) induced by metabolic inhibition were 48.9±2.8 pA/pF in control (n=48), 34.3±3.5 pA/pF in AS-SUR1 (n=21, P<0.05 vs control), and 23.5±3.4 pA/pF in AS-SUR2 (n=17, P<0.01 vs control). As a control, scramble oligonucleotides had no effect. The fast Na⁺ current and inward-rectifying K⁺ current were not affected by AS-SURs. Treatment with both AS-SUR1 and AS-SUR2 had no additive effects on inhibition of K_ATP currents compared with AS-SUR2 alone. The single-channel conductance, open probability, and kinetics (in ATP-free solution) were not significantly different between control, AS-SUR1, and AS-SUR2. These results suggest that treatment with AS-ODN for SUR1 or SUR2 reduced the number of functional K_ATP channels. Furthermore, in four out of seven control cells tested, outward K⁺ currents were stimulated by diazoxide, which is a potent K⁺ channel-opening drug for the constructed SUR1/Kir6.2 and SUR2B/Kir6.2 channels, but not for the SUR2A/Kir6.2 channel. Therefore, in neonatal rat ventricular cells, both SUR2 and SUR1 subtypes could be integral components of the functional K_ATP channels. The larger population of K_ATP channels may be constructed with SUR2, whereas a smaller population may be constructed with a combination of SUR1 and SUR2. Received: 29 May 1998 / Received after revision: 8 September 1998 / Accepted: 13 October 1998     

The basolateral Ca2+-dependent K+ channel in rat colonic crypt cells

 Previous studies have indicated that a 16-pS K⁺ channel (KC_ca) in the basolateral membrane is responsible for the acetylcholine-induced whole-cell K⁺ conductance in these cells. In the present study we have examined this channel in excised inside-out patches of the basolateral membrane. Over a wide voltage range this channel showed inward rectification. The Ca²⁺ sensitivity was very marked, with a Hill coefficient of three and with half-maximal activation at 330 nmol/l. After several minutes most channels showed a slow run-down. Channel activity could be refreshed by addition of ATP (1 mmol/l) to the bath solution. The non-metabolizable derivative 5’-adenylylimidodiphosphate (AMP-PNP) had no such effect. In contrast, it inhibited channel activity by some 50%. ATP and its derivatives had no effect on the Ca²⁺ sensitivity. Channels activated by ATP were subsequently studied in the presence of alkaline (10 kU/l) or acidic (1 kU/l) phosphatase. Both phosphatases reduced channel activity significantly. These data suggest that the 16-pS K⁺ channel is directly controlled by cytosolic Ca²⁺. This regulatory step is probably distal to an activation produced by protein-kinase-C-dependent phosphorylation. As is the case for several other K⁺ channels, high concentrations of non-metabolizable ATP analogues inhibit this channel. Received: 23 July 1997 / Accepted: 17 September 1997     

Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts

 It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca²⁺ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K⁺ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P _o, N=number, P _o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K⁺ channel is controlled by cytosolic Ca²⁺ ([Ca²⁺]_i). Therefore we measured [Ca²⁺]_i by the fura-2 method and found that hypertonic solution reduced [Ca²⁺]_i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca²⁺]_i falls in hypertonic solution leading to a reduction in the value of K⁺ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell. Received: 21 July 1997 / Received after revision: 24 November 1997 / Accepted: 15 December 1997     

Functional expression of a large-conductance Ca-activated K channel in mouse substantia nigra pars compacta dopaminergic neurons

The existence of large-conductance Ca²⁺-activated K⁺ (BK) channels in substantia nigra pars compacta (SNc) has been a matter of debate. Using the patch-clamp technique in the inside-out configuration, we have recorded BK channel currents in SNc dopaminergic neurons. The channel has a conductance of 301 pS with a slight inward rectification and is both voltage- and calcium-dependent. Paxilline, a specific BK channel blocker, can completely block the channel, while tetraethylammonium (TEA), a nonspecific blocker of voltage-gated potassium channels, reduces its conductance and a high concentration of TEA (30 mM) inhibits its activity. ATP and GTP reduce the channel activity, while ADP is less potent, and AMP has no effect. The channel is also sensitive to changes in intracellular pH. Our results indicate that functional BK channels are expressed in SNc and suggest the possibility that the BK channel may be involved in the response of SNc dopaminergic neurons to metabolic stress.     

Amiloride-inhibitable Na+ conductance in rat proximal tubule

 Previous single-channel recordings from the luminal membrane of the rabbit proximal tubule have revealed amiloride-inhibitable Na⁺ channels of a characteristic conductance range. The present study aimed to pursue this issue in rat proximal tubule. Control rats were compared to those put on a low-Na⁺ diet or pretreated by triamcinolone injections (s.c.). Stimulation of Na⁺ absorption by glucocorticoids was verified by examining the transepithelial voltage in Ussing chamber studies of the distal colon. The membrane voltage (V _m) of isolated, in-vitro-perfused proximal tubule segments was measured in patch-clamp and impalement studies. It was found that amiloride hyperpolarized V _m significantly by 2.1 ± 0.9 mV (n = 26) in tubules of control rats, by 3.9 ± 0.7 mV (n = 12) in rats put on a low-Na⁺ diet and by 3.7 ± 1.0 mV (n = 17) in rats treated with glucocorticoids. The effect of amiloride was concentration dependent with a half-maximal effect at < 1 μmol/l. RT-PCR techniques were used to search for the presence of the α-, β- and γ-subunits of the epithelial Na⁺ channel in isolated oximal tubule segments. The presence of the respective mRNAs was verified. These data indicate that: (1) amiloride-inhibitable Na⁺ channels are present in rat proximal tubules; (2) the Na⁺ conductance may be upregulated by Na⁺ deprivation but is still very limited when compared to total cell conductance; (3) therefore, the contribution of Na⁺-channel-mediated absorption to total proximal Na⁺ absorption is probably small. Received: 5 August 1996 / Received after revision: 22 January 1997 / Accepted: 28 January 1997     

Modulation of a steady-state Ca2+-activated, K+ current in tail sensory neurons of Aplysia: role of serotonin and cAMP

1. The modulation of membrane currents by serotonin (5-HT) was studied in isolated clusters of tail sensory neurons. Serotonin was applied by micropressure ejection onto the somata of sensory neurons voltage-clamped at fixed holding potentials. The range of holding potentials tested in this study was selected to produce a steady-state Ca2+-activated K+ current (IK,Ca). Serotonin induced an inward shift in the holding current associated with a decrease in slope conductance. 2. Intracellular injection of adenosine 3',5'-cyclic monophosphate (cAMP) mimicked the response to 5-HT and induced an inward current associated with a decrease in slope conductance. The responses to 5-HT and cAMP had similar voltage dependencies and both responses were due to an apparent decrease in K+ current. Responses to cAMP were markedly reduced when generated at the peak of a response to 5-HT. The nonsummation of the maximal current responses indicated that 5-HT and cAMP utilize a common, saturable mechanism. 3. In contrast to the consistent decrease in steady-state K+ conductance elicited by cAMP, injection of guanosine 3',5'-cyclic monophosphate (cGMP) produced variable responses. In most cells, cGMP induced outward shifts in holding current that were associated with an increase in slope conductance. 4. Several lines of evidence indicated that IK,Ca contributed to the holding current at the level of membrane potentials that were examined. Inward shifts in holding current associated with a decrease in slope conductance were produced in the presence of agents that block Ca2+ channels, such as Co2+, Cd2+ or Ni2+ and by replacement of extracellular Ca2+ with Ba2+. Reducing the concentration of cytoplasmic Ca2+ through intracellular injection of EGTA had similar effects. Furthermore, inward shifts in holding current were produced by 5 mM tetraethylammonium chloride (TEA), which is known to block IK,Ca in neurons of Aplysia. This concentration of TEA also attenuated the outward current produced in response to direct intracellular injection of Ca2+. 5. Serotonin appears to modulate the IK,Ca that contributes to the steady-state holding current. The same manipulations that block the steady-state IK,Ca (see above) also attenuated the response to 5-HT. Furthermore, K+ currents activated by intracellular injection of Ca2+ were attenuated by 5-HT. 6. These results indicate that the changes in holding current produced by 5-HT are mediated, at least in part, by cAMP. In addition, it appears that 5-HT modulates a steady-state calcium-activated K+ current in addition to the previously described S-current (40, 58) and delayed K+ current (8, 9).(ABSTRACT TRUNCATED AT 250 WORDS)     

Non-typical K+-current in cesium-loaded guinea pig type I vestibular hair cell

Isolated guinea pig type I vestibular hair cells were voltage clamped at HP-110 mV in whole cell clamp configuration and depolarized up to +20 mV. Increasing depolarizations elicited large outward currents. These currents were replaced, in cesium-loaded cells, by inward/outward currents that reversed at membrane potentials between –55 and –30 mV. The reversal potential varied from cell to cell, and appeared to depend on the intracellular potassium cesium ratio. The current remaining in the presence of intracellular cesium was essentially due to a non-typical potassium conductance, which decreased in the presence of 4-AP and was blocked by 4-AP plus TEA. This current appeared as soon as the membrane was depolarized, showing the high potassium permeability of type I vestibular hair cells. A small part of this current was a strictly calcium inward current, sensitive to flunarizine, with a leakage component in the hyperpolarized state and a voltage component when the cell was depolarized.     

Increase of the single-channel conductance of KvLQT1 potassium channels induced by the association with minK

 Gating of the delayed rectifier K⁺ channel KvLQT1 is drastically slowed by the association with the small membrane protein minK and it is thought that the KvLQT1/minK complex underlies the slow delayed rectifier K⁺ current of cardiac cells. There is controversy about the effects of the association between KvLQT1 and minK on the single-channel conductance. Here, nonstationary fluctuation analysis was applied to inward K⁺ tail currents recorded with a high-time resolution (5 kHz bandwidth) from macropatches of homomeric KvLQT1 and heteromeric KvLQT1/minK channels expressed in Xenopus oocytes to estimate their single-channel conductance. It was found that heteromers have a threefold larger conductance (5.8 pS) compared to homomeric channels (1.8 pS) in symmetrical high-K⁺ solutions. The larger conductance of heteromers explains in part their larger macroscopic conductance in heterologous expression systems. The molecular mechanism underlying the conductance increase remains to be identified. Received: 1 September 1998 / Received after revision 9 October 1998 / Accepted: 12 October 1998