cAMP Increases Apical IsK Channel Current and K+ Secretion in Vestibular Dark Cells

Adenosine 3′,5′-cyclic monophosphate (cAMP) is known to stimulate exogenous I_sK channel current in the Xenopus oocyte expression system. The present study was performed to determine whether elevation of cytosolic cAMP in a native mammalian epithelium known to secrete K⁺ through endogenously expressed I_sK channels would stimulate K⁺ secretion through these channels. The equivalent short circuit current (I _sc ) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current (I _IsK ) and conductance (g _IsK ) of I_sK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has previously been shown that I _sc can be accounted for by transepithelial K⁺ secretion and that the apical I_sK channels constitute a significant pathway for K⁺ secretion. The identification of the voltage-dependent whole-cell currents in vestibular dark cells was strengthened by the finding that a potent blocker of I_sK channels, chromanol 293B, strongly reduced I _IsK from 646 ± 200 to 154 ± 22 pA (71%) and g _IsK from 7.5 ± 2.6 to 2.8 ± 0.4 nS (53%). Cytoplasmic cAMP was elevated by applying dibutyryl cyclic AMP (dbcAMP), or the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-20-1724. dbcAMP (1 mm) increased I _sc and I _IsK from 410 ± 38 to 534 ± 40 μA/cm² and from 4.3 ± 0.8 to 11.4 ± 2.2 pA, respectively. IBMX (1 mm) caused transient increases of I _sc from 415 ± 30 to 469 ± 38 μA/cm² and Ro-20-1724 (0.1 mm) from 565 ± 43 to 773 ± 58 μA/cm². IBMX increased I _IsK from 5.5 ± 1.5 to 16.9 ± 5.8 pA in on-cell experiments and from 191 ± 31 to 426 ± 53 pA in whole-cell experiments. The leak conductance due to all non-I_sK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced I _IsK by 84% and g _IsK by 62%, similar to unstimulated conditions. These results demonstrate that the cAMP pathway is constitutively active in vestibular dark cells and that the cAMP pathway stimulates transepithelial K⁺ secretion by increasing I_sK channel current rather than by altering another transport pathway. Received: 9 June 1995/Revised: 17 October 1996     

II. Voltage-activated Proton Currents in Human THP-1 Monocytes

Depolarization-activated H⁺-selective currents were studied using whole-cell and excised-patch voltage clamp methods in human monocytic leukemia THP-1 cells, before and after being induced by phorbol ester to differentiate into macrophage-like cells. The H⁺ conductance, g _H, activated slowly during depolarizing pulses, with a sigmoidal time course. Fitted by a single exponential following a delay, the activation time constant, τ_act was roughly 10 sec at threshold potentials, decreasing at more positive potentials. Tail currents upon repolarization decayed mono-exponentially at all potentials. The tail current time constant, τ_tail, was voltage dependent, decreasing with hyperpolarization from 2–3 sec at 0 mV to ∼200 msec at −100 mV. Surprisingly, although τ_act depended strongly on pH _o , τ_tail was completely independent of pH _o . H⁺ currents were inhibited by Zn²⁺. Increasing pH _o or decreasing pH _i shifted the voltage-activation relationship to more negative potentials, tending to activate the g _H at any given voltage. Studied in excised, inside-out membrane patches, H⁺ currents were larger and activated much more rapidly at lower bath pH (i.e., pH _i ). In THP-1 cells differentiated into macrophages, the H⁺ current density was reduced by one-half, and τ_act was slower by about twofold. The properties of H⁺ channels in THP-1 cells and in other macrophage-related cells are compared. Received: 19 September 1995/Revised: 14 March 1996     

A Membrane-delimited Effect of Internal pH on the K+ Outward Rectifier of Vicia Faba Guard Cells

ABA stimulation of outward K⁺ current (I _K,out) in Vicia faba guard cells has been correlated with a rise in cytosolic pH (pH _i ). However, the underlying mechanism by which I _K,out is affected by pH _i has remained unknown. Here, we demonstrate that pH _i regulates outward K⁺ current in isolated membrane patches from Vicia faba guard cells. The stimulatory effect of alkalinizing pH _i was voltage insensitive and independent of the two free calcium levels tested, 50 nm and 1 μm. The single-channel conductance was only slightly affected by pH _i . Based on single-channel measurements, the kinetics of time-activated whole-cell current, and the analysis of current noise in whole-cell recordings, we conclude that alkaline pH _i enhances the magnitude of I _K,out by increasing the number of channels available for activation. The fact that the pH _i effect is seen in excised patches indicates that signal transduction pathways involved in the regulation of I _K,out by pH _i , and by implication, components of hormonal signal transduction pathways that are downstream of pH _i , are membrane-delimited. Received: 5 June 1996/Revised: 1 August 1996     

Comparison of Amphibian and Human ClC-5: Similarity of Functional Properties and Inhibition by External pH

Loss of function mutations of the renal chloride channel, ClC-5, have been implicated in Dent's disease, a genetic disorder characterized by low weight proteinuria, hypercalciuria, nephrolithasis and, in some cases, eventual renal failure. Recently, our laboratory used an RT-PCR/RACE cloning strategy to isolate an amphibian cDNA from the renal epithelial cell line A6 that had high homology to human ClC-5. We now report a full-length native ClC-5 clone (xClC-5, containing 5′ and 3′ untranslated regions) isolated by screening a cDNA library from A6 cells that was successfully expressed in Xenopus oocytes. In addition, we compared the properties of xClC-5 and hClC-5 using isogenic constructs of xClC-5 and hClC-5 consisting of the open reading frame subcloned into an optimized Xenopus expression vector. Expression of the full-length ``native' xClC-5 clone resulted in large, strongly rectifying, outward currents that were not significantly affected by the chloride channel blockers DIDS, DPC, and 9AC. The anion conductivity sequence was NO⁻ ₃ > Cl⁻= I⁻ > HCO⁻ ₃ >> glutamate for xClC-5 and NO⁻ ₃ > Cl⁻ > HCO⁻ ₃ > I⁻ >> glutamate for hClC-5. Reduction of the extracellular pH (pH _o ) from 7.5 to 5.7 inhibited outward ClC-5 currents by 27 ± 9% for xClC-5 and 39 ± 7% for hClC-5. The results indicate that amphibian and mammalian ClC-5 have highly similar functional properties. Unlike hClC-5 and most other ClC channels, expression of xClC-5 in oocytes does not require the removal of its untranslated 5′ and 3′ regions. Acidic solutions inhibited both amphibian and human ClC-5 currents, opposite to the stimulatory effects of low external pH on other ClC channels, suggesting a possibly distinct regulatory mechanism for ClC-5 channels. Received: 28 August 1998/Revised: 13 January 1999     

Single-Channel Characteristics of Wild-Type IKs Channels and Channels formed with Two MinK Mutants that Cause Long QT Syndrome

I_Ks channels are voltage dependent and K⁺ selective. They influence cardiac action potential duration through their contribution to myocyte repolarization. Assembled from minK and KvLQT1 subunits, I_Ks channels are notable for a heteromeric ion conduction pathway in which both subunit types contribute to pore formation. This study was undertaken to assess the effects of minK on pore function. We first characterized the properties of wild-type human I_Ks channels and channels formed only of KvLQT1 subunits. Channels were expressed in Xenopus laevis oocytes or Chinese hamster ovary cells and currents recorded in excised membrane patches or whole-cell mode. Unitary conductance estimates were dependent on bandwidth due to rapid channel “flicker.” At 25 kHz in symmetrical 100-mM KCl, the single-channel conductance of I_Ks channels was ∼16 pS (corresponding to ∼0.8 pA at 50 mV) as judged by noise-variance analysis; this was fourfold greater than the estimated conductance of homomeric KvLQT1 channels. Mutant I_Ks channels formed with D76N and S74L minK subunits are associated with long QT syndrome. When compared with wild type, mutant channels showed lower unitary currents and diminished open probabilities with only minor changes in ion permeabilities. Apparently, the mutations altered single-channel currents at a site in the pore distinct from the ion selectivity apparatus. Patients carrying these mutant minK genes are expected to manifest decreased K⁺ flux through I_Ks channels due to lowered single-channel conductance and altered gating.     

III. Ion Channel Expression in PMA-differentiated Human THP-1 Macrophages

Ion channel expression was studied in THP-1 human monocytic leukemia cells induced to differentiate into macrophage-like cells by exposure to the phorbol ester, phorbol 12-myristate 13-acetate (PMA). Inactivating delayed rectifier K⁺ currents, I _DR, present in almost all undifferentiated THP-1 monocytes, were absent from PMA-differentiated macrophages. Two K⁺ channels were observed in THP-1 cells only after differentiation into macrophages, an inwardly rectifying K⁺ channel (I _IR) and a Ca²⁺-activated maxi-K channel (I _BK). I _IR was a classical inward rectifier, conducting large inward currents negative to E _K and very small outward currents. I _IR was blocked in a voltage-dependent manner by Cs⁺, Na⁺, and Ba²⁺, block increasing with hyperpolarization. Block by Na⁺ and Ba²⁺ was time-dependent, whereas Cs⁺ block was too fast to resolve. Rb⁺ was sparingly permeant. In cell-attached patches with high [K⁺] in the pipette, the single I _IR channel conductance was ∼30 pS and no outward current could be detected. I _BK channels were observed in cell-attached or inside-out patches and in whole-cell configuration. In cell-attached patches the conductance was ∼200–250 pS and at potentials positive to ∼100 mV a negative slope conductance of the unitary current was observed, suggesting block by intracellular Na⁺. I _BK was activated at large positive potentials in cell-attached patches; in inside-out patches the voltage-activation relationship was shifted to more negative potentials by increased [Ca²⁺]. Macroscopic I _BK was blocked by external TEA⁺ with half block at 0.35 mm. THP-1 cells were found to contain mRNA for Kv1.3 and IRK1. Levels of mRNA coding for these K⁺ channels were studied by competitive PCR (polymerase chain reaction), and were found to change upon differentiation in the same direction as did channel expression: IRK1 mRNA increased at least 5-fold, and Kv1.3 mRNA decreased on average 7-fold. Possible functional correlates of the changes in ion channel expression during differentiation of THP-1 cells are discussed. Received: 19 September 1995/Revised: 14 March 1996     

Characterization of Volume-activated Chloride Currents in Endothelial Cells from Bovine Pulmonary Artery

We have measured the kinetic and pharmacological properties of volume-activated Cl⁻ currents (I _Cl,vol) in endothelial cells, and tried to correlate them with those of the already described volume-activated current I _Cln. Both conductances show a similar permeability sequence for monovalent anions, and they are blocked by extracellular ATP. In the present report, we demonstrate by Western blot and RT-PCR that cultured endothelial cells from bovine pulmonary artery (CPAE) contain pI _Cln. The expression of this protein has been shown to be closely associated with the I _Cln current. I _Cl,vol showed however, in contrast with I _Cln, no striking inactivation at positive potentials. This property is also at variance with that of the volume-activated current related to MDR-1. Activation of I _Cl,vol at potentials more negative than −80 mV was not time dependent, which excludes a major contribution of a ClC-2 related current. The antiviral nucleoside analogue AZT (3′-azido-3′-deoxythymidine) inhibited I _Cl,vol by 21 ± 2.7% (n = 10), at a concentration of 100 μm. Another antiviral drug, acyclovir (ACV, 9-[(2-hydroxyethoxy)methyl]guanine) blocked I _Cl,vol by 27 ± 6.2% at 100 μm (n = 11). Both blocking effects are much smaller than those reported for I _Cln. The phenol derivative gossypol, which blocks I _Cln-related currents, efficiently inhibited I _Cl,vol in CPAE cells (67 ± 2.1% at 1 μm, n = 7, K _I = 0.4 μm). The presence of pI _Cln in CPAE cells and the similar qualitative pharmacological profile of I _Cl,vol and I _Cln support the hypothesis that pI _Cln is a good molecular candidate for I _Cl,vol in endothelial cells. The discrepant kinetic properties may indicate that these time-dependent currents at high positive or negative potentials are not intrinsic properties of the channels, but are caused by time-dependent depletion/accumulation phenomena due to the large amplitudes of these currents. Received: 8 May 1995/Revised: 12 October 1995     

A Calcium Homeostasis Mechanism Induced by Heterologous Expression of Total RNA from Chicory Leaves in Xenopus Oocytes

Xenopus oocytes were injected with total RNA from chicory leaf tissues and then examined by the voltage-clamp technique. A double-step voltage protocol was used, with an initial hyperpolarization step from the holding potential of −35 to −140 mV followed by a second depolarization step to +60 mV. Two different outward currents were observed, one noninactivating (I _ni ), and one inactivating (I _i ). Only the noninactivating outward current (I _ni ) could be induced by depolarization from −35 to +60 mV. The mean amplitude of I _ni was 2915 ± 848 nA (n= 11). This current, carried by chloride ions, declined nearly to the baseline in 153 ± 64 sec (n= 13), and was highly dependent on intracellular calcium. After the rundown of I _ni , the same oocyte was depolarized from −140 to +60 mV. This protocol induced an inactivating outward current (I _i ) with a mean amplitude of 4461 ± 1605 nA (n= 13). I _i was also carried by chloride ions and dependent on extracellular calcium. I _i was strongly inhibited by 100 μm extracellular La³⁺. These two types of chloride currents were also observed after IP₃ injection in control oocytes. I _ni and I _i were not observed in noninjected oocytes or water-injected oocytes. We suggest that the expression of total chicory leaf tissue RNA in Xenopus oocytes reveals a calcium homeostasis mechanism responsible for calcium mobilization from internal stores and subsequent calcium entry. Received: 22 May 1998/Revised: 2 October 1998     

Effects of ATP-sensitive Potassium Channel Regulators on Chloride Channels in the Sarcoplasmic Reticulum Vesicles from Rabbit Skeletal Muscle

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K⁺ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl⁻ channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] _cis or [glibenclamide] _cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] _cis and [glibenclamide] _cis highlights the therapeutic potential of these drugs in regulating the Ca²⁺-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998     

Separate Swelling- and Ca2+-activated Anion Currents in Ehrlich Ascites Tumor Cells

A Ca²⁺-activated (I _Cl,Ca) and a swelling-activated anion current (I _Cl,vol) were investigated in Ehrlich ascites tumor cells using the whole cell patch clamp technique. Large, outwardly rectifying currents were activated by an increase in the free intracellular calcium concentration ([Ca²⁺] _i ), or by hypotonic exposure of the cells, respectively. The reversal potential of both currents was dependent on the extracellular Cl⁻ concentration. I _Cl,Ca current density increased with increasing [Ca²⁺] _i , and this current was abolished by lowering [Ca²⁺] _i to <1 nm using 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid (BAPTA). In contrast, activation of I _Cl,vol did not require an increase in [Ca²⁺] _i . The kinetics of I _Cl,Ca and I _Cl,vol were different: at depolarized potentials, I _Cl,Ca as activated in a [Ca²⁺] _i - and voltage-dependent manner, while at hyperpolarized potentials, the current was deactivated. In contrast, I _Cl,vol exhibited time- and voltage-dependent deactivation at depolarized potentials and reactivation at hyperpolarized potentials. The deactivation of I _Cl,vol was dependent on the extracellular Mg²⁺ concentration. The anion permeability sequence for both currents was I ⁻ > Cl⁻ > gluconate. I _Cl,Ca was inhibited by niflumic acid (100 μm), 5-Nitro-2-(3-phenylpropylamino)benzoic acid (NPPB, 100 μm) and 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS, 100 μm), niflumic acid being the most potent inhibitor. In contrast, I _Cl,vol was unaffected by niflumic acid (100 μm), but abolished by tamoxifen (10 μm). Thus, in Ehrlich cells, separate chloride currents, I _Cl,Ca and I _Cl,vol, are activated by an increase in [Ca²⁺] _i and by cell swelling, respectively. Received: 12 November 1997/Revised: 5 February 1998     

Extracellular GTP Causes Membrane-Potential Oscillations through the Parallel Activation of Mg2+ and Na+ Currents in Paramecium tetraurelia

Paramecium tetraurelia responds to extracellular GTP (≥ 10 nm) with repeated episodes of prolonged backward swimming. These backward swimming events cause repulsion from the stimulus and are the behavioral consequence of an oscillating membrane depolarization. Ion substitution experiments showed that either Mg²⁺ or Na⁺ could support these responses in wild-type cells, with increasing concentrations of either cation increasing the extent of backward swimming. Applying GTP to cells under voltage clamp elicited oscillating inward currents with a periodicity similar to that of the membrane-potential and behavioral responses. These currents were also Mg²⁺- and Na⁺-dependent, suggesting that GTP acts through Mg²⁺-specific (I _Mg) and Na⁺-specific (I _Na) conductances that have been described previously in Paramecium. This suggestion is strengthened by the finding that Mg²⁺ failed to support normal behavioral or electrophysiological responses to GTP in a mutant that specifically lacks I _Mg (``eccentric'), while Na<sup>+</sup> failed to support GTP responses in ``fast-2,' a mutant that specifically lacks I _Na. Both mutants responded normally to GTP if the alternative cation was provided. As I _Mg and I _Na are both Ca²⁺-dependent currents, the characteristic GTP behavior could result from oscillations in intracellular Ca²⁺ concentration. Indeed, applying GTP to cells in the absence of either Mg²⁺ or Na⁺ revealed a minor inward current with a periodicity similar to that of the depolarizations. This current persisted when known voltage-dependent Ca²⁺ currents were blocked pharmacologically or genetically, which implies that it may represent the activation of a novel purinergic-receptor–coupled Ca²⁺ conductance. Received: 28 October 1996/Revised: 24 December 1996     

Ni2+ Slows the Activation Kinetics of High-Voltage-Activated Ca2+ Currents in Cortical Neurons: Evidence for a Mechanism of Action Independent of Channel-Pore Block

The effects of Ni²⁺ were evaluated on slowly-decaying, high-voltage-activated (HVA) Ca²⁺ currents expressed by pyramidal neurons acutely dissociated from guinea-pig piriform cortex. Whole-cell, patch-clamp recordings were performed with Ba²⁺ as the charge carrier. Ni²⁺ blocked HVA Ba²⁺ currents (I _Bas) with an EC₅₀ of approximately 60 μm. Additionally, after application of nonsaturating Ni²⁺ concentrations, residual currents activated with substantially slower kinetics than both total and Ni²⁺-sensitive I _Bas. None of the pharmacological components of slowly decaying, HVA currents activated with kinetics significantly different from that of total currents, indicating that the effect of Ni²⁺ on I _Bas kinetics cannot be attributed to the preferential inhibition of a fast-activating component. The effect of Ni²⁺ on I _Ba amplitude was voltage-independent over the potential range normally explored in our experiments (−60 to +20 mV), hence the Ni²⁺-dependent decrease of I _Ba activation rate is not due to a voltage- and time-dependent relief from block. Moreover, Ni²⁺ significantly reduced I _Ba deactivation speed upon repolarization, which also is not compatible with a depolarization-dependent unblocking mechanism. The dependence on Ni²⁺ concentration of the I _Ba activation-rate reduction was remarkably different from that found for I _Ba block, with an EC₅₀ of ∼20 μm and a Hill coefficient of ∼1.73 vs.∼1.10. These results demonstrate that Ni²⁺, besides inhibiting the I _Bas under study probably by exerting a blocking action on the pore of the underlying Ca²⁺ channels, also interferes with Ca²⁺-channel gating kinetics, and strongly suggest that the two effects depend on Ni²⁺ occupancy of binding sites at least partly distinct. Received: 13 July 2000/Revised: 9 November 2000     

Two Kinds of Transient Outward Currents, I A and I Adepol , in F76 and D1 Soma Membranes of the Subesophageal Ganglia of Helix aspersa

Transient outward currents were characterized with twin electrode voltage clamp techniques in isolated F76 and D1 neuronal membranes (soma only) of Helix aspersa subesophageal ganglia. In this study, in addition to the transient outward current (A-current, I _A ) described by Connor and Stevens (1971b), another fast outward current, referred to as I _Adepol here, is described for the first time. This is similar to the current component characterized in Aplysia (Furukawa, Kandel & Pfaffinger, 1992). The separation of these two current components was based on activation and steady-state inactivation curves, holding potentials and sensitivity to 4-aminopyridine (4-AP). In contrast to I _A , I _Adepol did not require hyperpolarizing conditioning pulses to remove inactivation; it was evoked from a holding potential of −40 mV, at which I _A is completely inactivated. I _Adepol shows noticeable activation at around −5 mV, whereas I _A activates at around −50 mV. The time courses of I _Adepol activation and inactivation were similar but slower than I _A . It was found that I _Adepol was more sensitive than I _A to 4-AP. 4-AP at a concentration of 1 mm blocked I _Adepol completely, whereas 5–6 mm 4-AP was needed to block I _A completely. This current is potentially very important because it may, like other A currents, regulate firing frequency but notably, it does not require a period of hyperpolarization to be active. Received: 12 May 2000/Revised: 12 October 2000     

A Long-Term Blockade of L-Type Calcium Currents Upregulates the Number of Ca2+ Channels in Skeletal Muscle

The effects of a long-term blockade of L-type Ca²⁺ channels on membrane currents and on the number of dihydropyridine binding sites were investigated in skeletal muscle fibers. Ca²⁺ currents (I _Ca) and intramembrane charge movement were monitored using a voltage-clamp technique. The peak amplitude of I _Ca increased by more than 40% in fibers that were previously incubated for 24 hr in solutions containing the organic Ca²⁺ channel blocker nifedipine or in Ca²⁺-free conditions. A similar incubation period with Cd²⁺, an inorganic blocker, produced a moderate increase of 20% in peak I _Ca. The maximum mobilized charge (Q _max) increased by 50% in fibers preincubated in Ca²⁺-free solutions or in the presence of Cd²⁺. Microsomal preparations from frog skeletal muscle were isolated by differential centrifugation. Preincubation with Cd²⁺ prior to the isolation of the microsomal fraction doubled the number of ³H-PN200-110 binding sites and produced a similar increase in the values of the dissociation constant. The increase in the number of binding sites is consistent with the increase in the peak amplitude of I _Ca as well as with the increase in Q _max. Received: 31 August 1998/Revised: 7 December 1998     

Temperature-Dependent Expression of a Squid Kv1 Channel in Sf9 Cells and Functional Comparison with the Native Delayed Rectifier

SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) α-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (I _K) if cultured at the standard postinfection temperature of 27°C. At 18°C, less SqKv1A protein is produced than at 27°C, but cells with I _K currents are much more numerous and can survive for at least 20 days postinfection (vs. ∼5 days at 27°C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (∼3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of I _K inactivation in 20 mm external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL I _K is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.     

Cl− Currents Activated by Extracellular Nucleotides in Human Bronchial Cells

The perforated-patch technique was used to study the response of human bronchial cells to extracellular nucleotides. ATP or UTP (100 μm) elicited a complex response consisting of a large transient membrane current increase followed by a relatively small sustained level. These two phases were characterized by different current kinetics. Throughout the transient phase (2–3 min) the membrane current (I _p ) displayed slow activation and deactivation kinetics at depolarizing and hyperpolarizing potentials respectively. At steady-state (I _s ) the relaxation at hyperpolarizing potential disappeared whereas at positive membrane potentials the current became slightly deactivating. The I _s amplitude was dependent on the extracellular Ca²⁺ concentration, being completely inhibited in Ca²⁺-free medium. Cell pre-incubation with the membrane-permeable chelating agent BAPTA/AM prevented completely the response to nucleotides, thus suggesting that both I _p and I _s were dependent on intracellular Ca²⁺. The presence of a hypertonic medium during nucleotide stimulation abolished I _s leaving I _p unchanged. On the contrary, niflumic acid, a blocker of Ca²⁺-activated Cl⁻ channels, prevented completely I _p without reducing significantly I _s . 1,9-dideoxyforskolin fully inhibited I _s but also reduced I _p . Replacement of extracellular Cl⁻ with aspartate demonstrated that the currents activated by nucleotides were Cl⁻ selective. I _p resulted five times more Cl⁻ selective than I _s with respect to aspartate. Taken together, our results indicate that ATP and UTP activate two types of Cl⁻ currents through a Ca²⁺-dependent mechanism. Received: 15 August 1996/Revised: 6 December 1996     

Effect of Trace Levels of Nigericin on Intracellular pH and Acid-Base Transport in Rat Renal Mesangial Cells

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pH _i ) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pH _i in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH _i decrease elicited by removing extracellular Na⁺, (ii) an increase in acid extrusion during the pH _i increase caused by elevating extracellular [K⁺], and (iii) an acid shift in the pH _i dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pH _i dependence of Na-H exchange, computed by adding the pH _i dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment. Received: 14 October 1998/Revised: 2 March 1999     

Calcium Channel Current in Rat Dental Pulp Cells

Voltage-gated Ca²⁺ currents in early-passage rat dental pulp cells were studied using whole-cell patch-clamp techniques. With Ba²⁺ as the charge carrier, two prominent inwardly-directed currents, I _f and I _s , were identified in these cells that could be distinguished on the basis of both kinetics and pharmacology. I _f was activated by membrane depolarizations more positive than −30 mV, and displayed fast inactivation kinetics, while I _s was activated by steeper depolarizations and inactivated more slowly. At peak current, time constants of inactivation for I _f and I _s were ∼17 vs.∼631 msec. Both I _f and I _s could be blocked by lanthanum. By contrast, only I _s was sensitive to either Bay-K or nifedipine, a specific agonist and antagonist, respectively, of L-type Ca²⁺ channels. I _s was also blocked by the peptide omega-Conotoxin GVIA. Taken together, results suggested that I _f was mediated by divalent cation flow through voltage-gated T-type Ca²⁺ channels, whereas I _s was mediated by L- and N-type Ca²⁺ channels in the pulp cell membrane. The expression of these prominent, voltage-gated Ca²⁺ channels in a presumptive mineral-inductive phenotype suggests a functional significance vis a vis differentiation of dental pulp cells for the expression and secretion of matrix proteins, and/or formation of reparative dentin itself. Received: 29 November 1999/Revised: 24 April 2000     

pH-Modulation of Chloride Channels from the Sarcoplasmic Reticulum of Skeletal Muscle

The understanding of the role of cytoplasmic pH in modulating sarcoplasmic reticulum (SR) ion channels involved in Ca²⁺ regulation is important for the understanding of the function of normal and adversely affected muscles. The dependency of the SR small chloride (SCl) channel from rabbit skeletal muscle on cytoplasmic pH (pH _cis ) and luminal pH (pH _trans ) was investigated using the lipid bilayer-vesicle fusion technique. Low pH _cis 6.75–4.28 modifies the operational mode of this multiconductance channel (conductance levels between 5 and 75 pS). At pH _cis 7.26–7.37 the channel mode is dominated by the conductance and kinetics of the main conductance state (65–75 pS) whereas at low pH _cis 6.75–4.28 the channel mode is dominated by the conductance and kinetics of subconductance states (5–40 pS). Similarly, low pH _trans 4.07, but not pH _trans 6.28, modified the activity of SCl channels. The effects of low pH _cis are pronounced at 10⁻³ and 10⁻⁴ m [Ca²⁺] _cis but are not apparent at 10⁻⁵ m [Ca²⁺] _cis , where the subconductances of the channel are already prominent. Low pH _cis -induced mode shift in the SCl channel activity is due to modification of the channel proteins that cause the uncoupling of the subconductance states. The results in this study suggest that low pH _cis can modify the functional properties of the skeletal SR ion channels and hence contribute, at least partly, to the malfunction in the contraction-relaxation mechanism in skeletal muscle under low cytoplasmic pH levels. Received: 20 May 1998/Revised: 24 September 1998