Voltage-independent Currents Research Articles

Abstract 922: Cardiac-specific Deletion of Orai3 Channel Causes Dilated Cardiomyopathy

Background: Calcium (Ca2+) signalling is a vital regulator of cardiac myocyte function. Defective cellular calcium handling is widely recognized as a significant patho-physiological event in the contractile dysfunction of the failing heart. Orai3 is a newly discovered calcium channels that participate in the voltage-independent calcium currents in the mycardium. However, its role in the heart remain uncharacterized. Methods and Results: Here, we show that disruption of Orai3 function in murine cardiomyocytes leads to decompensated dilated cardiomyopathy. Echocardiographic analysis supported by histological examinations revealed that 4-month-old Orai3 knockout (KO) mice have signs of dilated cardiomyopathy denoted by a loss of EF and a thinner left ventricular posterior wall and septum, with significant fibrosis, whereas in the younger animals only a thinner septum could be observed. In both genders, most mice died within six months after birth. Myocardial samples that underwent electron microscopy and immunohistochemical analysis showed myofilament changes in Orai3 KO mice with misexpression of cardiac contractile proteins and profound sarcomere disarray. Transverse aortic constriction (TAC) was used as a chronic stressor on the younger mice to determine whether the ability to compensate against a pathologic insult is compromised in the Orai3 KO heart. Orai3 KO mice presented with significantly reduced systolic function and ventricular dilation that deteriorated into congestive HF within 4 weeks post-surgery, while constricted WT hearts remained well-adapted throughout. Conclusions: Thus, our results identify Orai3 critical role in adult cardiac function and demonstrate a novel cardioprotective role of Orai3 against cardiac pressure overload-induced heart failure.

Abstract 198: Stim/orai Channel Complex In Normal And Hypertrophied Adult Cardiac Myocytes

Cardiac myocytes use Ca2+ not only in excitation-contraction coupling but also as a signaling molecule for cardiac growth. It is however unclear how Ca2+ triggers signaling in cardiac myocytes in the presence of the rapid and large Ca2+ fluctuations that occur during excitation-contraction coupling. We have recently reported that stromal interaction molecule 1 (STIM1), a calcium sensor in the sarcoplasmic reticulum, is a critical element in promoting cardiomyocyte growth through the control of a previously unrecognized sarcolemmal voltage-independent current. Our goal was to characterize the plasma membrane partners that contribute to STIM1-dependent currents in normal and hypertrophied adult cardiac myocytes. We firstly characterized the expression profile of TRPCs and ORAIs proteins in both normal and hypertrophied ventricular myocytes from abdominal aortic banded rats. The expression of TRPC1, 3, 4, 5 and 6 and of ORAI1 - 3 was identified with an up-regulation of TRPC1 in hypertrophied cells. We then used a non-viral method to deliver cy3-tagged siRNAs to ventricular myocytes to knock-down the candidate channels. The cardiac myocytes were isolated and whole cell patch-clamp technique as well as Fura-2 imaging were performed to measure the currents in cy3-labelled and in control cardiac myocytes. Our data demonstrated that the drug-inducible STIM1-dependent store-operated calcium entry was marginal in normal myocytes but was enhanced in hypertrophied myocytes. In addition, a basal inwardly rectifying current was observed in hypertrophied but not in control myocytes. This spontaneous current was recorded in the absence of calcium store depletion, required STIM1 expression and meets the characteristics of Arachidonate-Regulated Calcium selective (ARC) channels. ORAI silencing completely inhibited both store-dependent and store-independent currents suggesting the critical implication of ORAIs whereas TRPCs were only marginally involved. In conclusion, ORAI channels are involved in STIM1-dependent currents observed in hypertrophied adult cardiac myocytes. In addition to store-operated calcium entry, the STIM1/ORAI complex provides an alternative, store-independent pathway for agonist-activated Ca2+ entry.

Expression of olfactory‐type cyclic nucleotide‐gated channels in rat cortical astrocytes

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cyclic AMP (cAMP) or cyclic GMP (cGMP). They were originally identified in retinal and olfactory receptors, but evidence has also emerged for their expression in several mammalian brain areas. Because cGMP and cAMP control important aspects of glial cell physiology, we wondered whether CNG channels are expressed in astrocytes, the most functionally relevant glial cells in the CNS. Immunoblot and immunofluorescence experiments demonstrated expression of the CNG channel olfactory-type A subunit, CNGA2, in cultured rat cortical astrocytes. In patch-clamp experiments, currents elicited in these cells by voltage ramps from -100 to +100 mV in the presence of the cGMP analogue, dB-cGMP, were significantly reduced by the CNG channel blockers, L-cis-diltiazem (LCD) and Cd(2+) . The reversal potentials of the LCD- and Cd(2+) -sensitive currents were more positive than that of K(+) , as expected for a mixed cation current. Noninactivating, voltage-independent currents were also elicited by extracellular application of the membrane permeant cGMP analogue, 8-Br-cGMP. These effects were blocked by LCD and were mimicked by natriuretic peptide receptor activation and inhibition of phosphodiesterase activity. Voltage-independent, LCD-sensitive currents were also elicited by 8-Br-cGMP in astrocytes of hippocampal and neocortical brain slices. Immunohistochemistry confirmed a broad distribution of CNG channels in astrocytes of the rat forebrain, midbrain, and hindbrain. These findings suggest that CNG channels are downstream targets of cyclic nucleotides in astrocytes, and they may be involved in the glial-mediated regulation of CNS functions under physiological and pathological conditions.

Differential Effects of ICA-27243 on Cloned KV7 Channels

Background/Aims: The neuronal K_V7 family members (K_V7.2–5) are important regulators of neuronal excitability. K_V7 channel openers are therefore attractive drug candidates for the treatment of several hyperexcitability disorders. While most described K_V7 channel openers discriminate poorly between K_V7.2–5, Icagen’s N-(6-chloropyridin- 3-yl)-3,4-difluorobenzamide (ICA-27243) is more potent at K_V7.2/3 than at K_V7.4 and K_V7.3/5 and offers some progress towards subtype selectivity. We have investigated its mode of action on K_V7.2 and K_V7.4, compared its effect to that of retigabine and studied the combinatorial effect of retigabine and ICA-27243, as these two compounds recognize different binding sites in the channels. Methods: The effects of ICA-27243 and retigabine were studied using voltage-clamp electrophysiology in Xenopus laevis oocytes and rubidium flux in Chinese hamster ovary cells. Results: We found that in contrast to retigabine’s voltage-dependent action on K_V7.2, ICA-27243 induced a voltage-independent current on this subtype, which was not observed on K_V7.4. Additionally, the combined treatment of K_V7.2 and K_V7.4 with retigabine and ICA-27243 revealed that the effect of ICA-27243 on K_V7.2 dominates that of retigabine, while the compounds act additively and synergistically on K_V7.4. Conclusions: These results offer further detailed insight into pharmacological activation of K_V7 channels and offer evidence of differential functional and subtype-specific effects by activation of different binding sites in the K_V7 channels.

Analysis of Astroglial K+ Channel Expression in the Developing Hippocampus Reveals a Predominant Role of the Kir4.1 Subunit

Astrocytes in different brain regions display variable functional properties. In the hippocampus, astrocytes predominantly express time- and voltage-independent currents, but the underlying ion channels are not well defined. This ignorance is partly attributable to abundant intercellular coupling of these cells through gap junctions, impeding quantitative analyses of intrinsic membrane properties. Moreover, distinct types of cells with astroglial properties coexist in a given brain area, a finding that confused previous analyses. In the present study, we investigated expression of inwardly rectifying (Kir) and two-pore-domain (K2P) K+ channels in astrocytes, which are thought to be instrumental in the regulation of K+ homeostasis. Freshly isolated astrocytes were used to improve space-clamp conditions and allow for quantitative assessment of functional parameters. Patch-clamp recordings were combined with immunocytochemistry, Western blot analysis, and semiquantitative transcript analysis. Comparative measurements were performed in different CA1 subregions of astrocyte-targeted transgenic mice. While confirming weak Ba2+ sensitivity in situ, our data demonstrate that in freshly isolated astrocytes, the main proportion of membrane currents is sensitive to micromolar Ba2+ concentrations. Upregulation of Kir4.1 transcripts and protein during the first 10 postnatal days was accompanied by a fourfold increase in astrocyte inward current density. Hippocampal astrocytes from Kir4.1-/- mice lacked Ba2+-sensitive currents. In addition, we report functional expression of K2P channels of the TREK subfamily (TREK1, TREK2), which mediate astroglial outward currents. Together, our findings demonstrate that Kir4.1 constitutes the pivotal K+ channel subunit and that superposition of currents through Kir4.1 and TREK channels underlies the "passive" current pattern of hippocampal astrocytes.

PH‐sensitive K+ current in glial cells of the retrotrapezoid nucleus (RTN)

The RTN contains pH‐sensitive neurons that sense changes in arterial pH to dive breathing (i.e., chemoreceptor). RTN glial cells are also thought to contribute to chemoreception by an unknown mechanism. We hypothesize that select glial cells in the RTN are intrinsically pH sensitive. To test this we made whole cell voltage clamp recordings from glial cells in transverse brain slices (300 ìm) from rats (P7‐12). Glial cells were defined based on a membrane potential <‐75 mV and an inability to generate action potentials. We found a subset of RTN glial cells feature a pH‐sensitive current; alkalization increased outward current whereas acidification decreased outward current. The I‐V relationship of the pH‐sensitive current reveals a voltage‐independent current which reverses near EK (‐90 mV, N=14). Duel current clamp recordings from pH‐sensitive glial and RTN chemoreceptors identify an interaction between glia cells and neurons (cross correlation coefficient, 0.8); glial cell depolarization initiates neuronal depolarization with a delay of ~50 ms. These results indicate that i) RTN glial cells can respond to physiologically relevant changes in pH; ii) a voltage‐independent K+ channel likely confers pH‐sensitivity to RTN glia; iii) pH‐sensitive glial cells can interact with RTN chemoreceptors. These results identify a potential mechanism by which RTN glial cells can contribute to central chemoreception.

Electrophysiological characterization of neural stem/progenitor cells during in vitro differentiation: Study with an immortalized neuroectodermal cell line

Despite the accumulating data on the molecular and cell biological characteristics of neural stem/progenitor cells, their electrophysiological properties are not well understood. In the present work, changes in the membrane properties and current profiles were investigated in the course of in vitro-induced neuron formation in NE-4C cells. Induction by retinoic acid resulted in neuronal differentiation of about 50% of cells. Voltage-dependent Na+ currents appeared early in neuronal commitment, often preceding any morphological changes. A-type K+ currents were detected only at the stage of network formation by neuronal processes. Flat, epithelial- like, nestin-expressing progenitors persisted beside differentiated neurons and astrocytes. Stem/progenitor cells were gap junction coupled and displayed large, symmetrical, voltage-independent currents. By the blocking of gap junction communication, voltage-independent conductance was significantly reduced, and delayed-rectifying K+ currents became detectable. Our data indicate that voltage-independent symmetrical currents and gap junction coupling are characteristic physiological features of neural stem and progenitor cells regardless of the developmental state of their cellular environment.

Novel 384-Well Population Patch Clamp Electrophysiology Assays for Ca2+-Activated K+ Channels

Planar array electrophysiology techniques were applied to assays for modulators of recombinant hIK and hSK3 Ca2+-activated K+ channels. In CHO-hIK-expressing cells, under asymmetric K+ gradients, small-molecule channel activators evoked time- and voltage-independent currents characteristic of those previously described by classical patch clamp electrophysiology methods. In single-hole (cell) experiments, the large cell-to-cell heterogeneity in channel expression rendered it difficult to generate activator concentration-response curves. However, in population patch clamp mode, in which signals are averaged from up to 64 cells, well-to-well variation was substantially reduced such that concentration-response curves could be easily constructed. The absolute EC50 values and rank order of potency for a range of activators, including 1-EBIO and DC-EBIO, corresponded well with conventional patch clamp data. Activator responses of hIK and hSK3 channels could be fully and specifically blocked by the selective inhibitors TRAM-34 and apamin, with IC50 values of 0.31 microM and 3 nM, respectively. To demonstrate assay precision and robustness, a test set of 704 compounds was screened in a 384-well format of the hIK assay. All plates had Z' values greater than 0.6, and the statistical cutoff for activity was 8%. Eleven hits (1.6%) were identified from this set, in addition to the randomly spiked wells with known activators. Overall, our findings demonstrate that population patch clamp is a powerful and enabling method for screening Ca2+-activated K+ channels and provides significant advantages over single-cell electrophysiology (IonWorks(HT)) and other previously published approaches. Moreover, this work demonstrates for the 1st time the utility of population patch clamp for ion channel activator assays and for non-voltage-gated ion channels.

Molecular Identification and Physiological Roles of Parotid Acinar Cell Maxi-K Channels

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca(2+)-activated Cl(-) and K(+) channels. Parotid acinar cells express two types of Ca(2+)-activated K(+) channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the K(Ca)3.1 gene, and the K(Ca)1.1 gene is a likely candidate for the maxi-K channel. To confirm the genetic identity of the maxi-K channel and to probe its specific roles, we studied parotid glands in mice with the K(Ca)1.1 gene ablated. Parotid acinar cells from these animals lacked maxi-K channels, confirming their genetic identity. The stimulated parotid gland fluid secretion rate was normal, but the sodium and potassium content of the secreted fluid was altered. In addition, we found that the regulatory volume decrease in acinar cells was substantially impaired in K(Ca)1.1-null animals. We examined fluid secretion from animals with both K(+) channel genes deleted. The secretion rate was severely reduced, and the ion content of the secreted fluid was significantly changed. We measured the membrane potentials of acinar cells from wild-type mice and from animals with either or both K(+) channel genes ablated. They revealed that the observed functional effects on fluid secretion reflected alterations in cell membrane voltage. Our findings show that the maxi-K channels are critical for the regulatory volume decrease in these cells and that they play an important role in the sodium uptake and potassium secretion process in the ducts of these fluid-secreting salivary glands.

Distinct Populations of HCN Pacemaker Channels Produce Voltage-dependent and Voltage-independent Currents

Hyperpolarization-activated HCN pacemaker channels are critical for the generation of spontaneous activity and the regulation of excitability in the heart and in many types of neurons. These channels produce both a voltage-dependent current (Ih) and a voltage-independent current (Iinst or VIC). In this study, we explored the molecular basis of the voltage-independent current. We found that for the spHCN isoform, VIC averaged ∼4% of the maximum HCN conductance that could be activated by hyperpolarization. Cyclic AMP increased the voltage-independent current in spHCN to ∼8% of maximum. In HCN2, VIC was ∼2% of the maximal current, and was little affected by cAMP. VIC in both spHCN and HCN2 was blocked rapidly both by ZD7288 (an HCN channel blocker that is thought to bind in the conduction pore) and by application of Cd2+ to channels containing an introduced cysteine in the pore (spHCN-464C or HCN2-436C). These results suggest that VIC flows through the main conduction pathway, down the central axis of the protein. We suspected that VIC simply represented a nonzero limiting open probability for HCN channels at positive voltages. Surprisingly, we found instead that the spHCN channels carrying VIC were not in rapid equilibrium with the channels carrying the voltage-dependent current, because they could be blocked independently; a single application of blocker at a depolarized potential essentially eliminated VIC with little change in Ih. Thus, VIC appears to be produced by a distinct population of HCN channels. This voltage-independent current could contribute significantly to the role of HCN channels in neurons and myocytes; VIC flowing through the channels at physiological potentials would tend to promote excitability by accelerating both depolarization and repolarization.

In vitro molecular interactions and distribution of KCNE family with KCNQ1 in the human heart

The voltage-gated K+ channel KCNQ1 associates with the small KCNE1 beta subunit to underlie the IKs repolarizing current in the heart. Based on sequence homology, the KCNE family is recognized to comprise five members. Controversial data have indicated their participation in several K+ channel protein complexes, including KCNQ1. The expression level and the putative functions of the different KCNE subunits in the human heart still require further investigation. We have carried out a comparative study of all KCNE subunits with KCNQ1 using the patch-clamp technique in mammalian cells. Real-time RT-PCR absolute quantification was performed on human atrial and ventricular tissue. While KCNQ1/KCNE1 heteromultimer reached high current density with slow gating kinetics and pronounced voltage dependence, KCNQ1/KCNE2 and KCNQ1/KCNE3 complexes produced instantaneous voltage-independent currents with low and high current density, respectively. Co-expression of KCNE4 or KCNE5 with KCNQ1 induced small currents in the physiological range of voltages, with kinetics similar to those of the KCNQ1/KCNE1 complex. However, co-expression of these inhibitory subunits with a disease-associated mutation (S140G-KCNQ1) led to currents that were almost undistinguishable from the KCNQ1/KCNE1 canonical complex. Absolute cDNA quantification revealed a relatively homogeneous distribution of each transcript, except for KCNE4, inside left atria and endo- and epicardia of left ventricular wall with the following abundance: KCNQ1 >> KCNE4 > or = KCNE1 > KCNE3 > KCNE2 > KCNE5. KCNE4 expression was twice as high in atrium compared to ventricle. Our data show that KCNQ1 forms a channel complex with 5 KCNE subunits in a specific manner but only interactions with KCNE1, KCNE2, and KCNE3 may have physiological relevance in the human heart.

Voltage-independent KCNQ4 currents induced by (+/-)BMS-204352.

The compound BMS-204352 has been targeted for use against acute ischemic stroke, due to its activation of the large-conductance Ca2+-activated K-channel (BK). We have previously described that the racemate (+/-)BMS-204352 reversibly modulates KCNQ4 voltage dependency. Here we show that (+/-)BMS-204352 also induces a voltage-independent KCNQ4 current. The channels were stably expressed in human embryonic kidney cells (HEK293), and investigated by use of the whole-cell mode of the patch-clamp technique. (+/-)BMS-204352 was applied extracellularly (10 microM) in order to precipitate the robust appearance of the voltage-independent current. The voltage-independent KCNQ4 currents were recorded as instantaneous increases in currents upon hyperpolarizing or depolarizing voltage steps elicited from holding potentials of -90 or -110 mV. The voltage-independent current reversed at the equilibrium potential for potassium ( E(K)), hence was carried by a K+ conductance, and was blocked by the selective KCNQ channel blockers XE991 and linopirdine. Similar results were obtained with KCNQ4 channels transiently transfected into Chinese hamster ovary cells (CHO). When (+/-)BMS-204352 was applied to stably expressed BK channels, only the voltage dependency was modulated. Retigabine, the classic activator of KCNQ channels, did not induce voltage-independent currents. Our data indicate that KCNQ4 channels may conduct voltage-dependent and voltage-independent currents in the presence of (+/-)BMS-204352.

Analysis of the K+ current profile of mature rat oligodendrocytes in situ.

Previous studies have reported that mature oligodendrocytes (OLGs) in vitro display various voltage-dependent K+ currents while in situ OLGs show only voltage-independent K+ currents. Given this discrepancy and the lack of information on myelinating OLG ion channel expression in situ, we characterized mature OLG currents in myelinating corpus callosum slices from 17 to 36-day old rats. OLGs were recorded in cell-attached and whole-cell patch-clamp configurations, displayed morphology typical of mature OLGs, and stained positive for myelin basic protein. OLGs displayed large voltage-independent currents that decayed during the voltage pulse and small voltage-activated outward currents. The latter were blocked by TEA, activated between -40 and -50 mV, and decayed slowly. The former were composed of large voltage-independent, time-dependent Ba2+ (1 mM)-sensitive currents, and voltage-dependent Cs+ (5 mM) and Ba2+ (100 mM)-sensitive currents that reversed near the K+ equilibrium potential and inactivated at hyperpolarized potentials, identifying them as inwardly rectifying K+ currents. Inwardly rectifying single-channel K+currents could be recorded in the cell-attached configuration. The estimated single-channel slope conductance was 30 pS. The steady-state open probability was voltage-dependent and declined from 0.9 to 0.5 between -80 and -150 mV. Overall, mature OLGs in situ possess time- and also voltage-dependent K+ currents, which may facilitate clearance of K+ released during axonal firing.

Differential transduction mechanisms underlying NaCl- and KCl-induced responses in mouse taste cells.

The transduction mechanism of salt-induced responses of mouse taste cells was investigated using the patch clamp and the local stimulation techniques under quasi-natural conditions. Apically applied NaCl induced a voltage-independent current, which was partially suppressed by amiloride and Cd2+. In contrast, apically applied 0.5 M KCl induced an inwardly rectifying current (KCl-induced Iir). The KCl-induced Iir was unaffected by amiloride. The Iir was suppressed not only by external Ba2+ and Cs+, but also by a Cl- channel blocker, niflumic acid. The Er of the KCl-induced response was independent of the apical ionic concentration, but rather was close to the equilibrium potential of Cl- (E(Cl)) at the basolateral membrane. The KCl-induced Iir displayed a fast run-down under the conditions of the conventional whole cell clamp method, but not under the perforated patch conditions. Immunohistochemical localization of an inwardly rectifying Cl- channel protein, ClC-2, was observed in taste bud cells of the fungiform papillae. It is concluded that the transduction mechanism of NaCl-induced responses is completely different from that of KCl-induced responses in mouse taste cells.

A Novel Pacemaker Mechanism Drives Gastrointestinal Rhythmicity.

Electric pacemaker activity drives peristaltic and segmental contractions in the gastrointestinal tract. Interstitial cells of Cajal (ICC) are responsible for spontaneous pacemaker activity. ICC remain rhythmic in culture and generate voltage-independent inward currents via a nonselective cation conductance. Ca(2+) release from endoplasmic reticulum and uptake by mitochondria initiates pacemaker currents. This novel mechanism provides the basis for electric rhythmicity in gastrointestinal muscles.

Functional and Structural Analysis of ClC-K Chloride Channels Involved in Renal Disease

ClC-K channels belong to the CLC family of chloride channels and are predominantly expressed in the kidney. Genetic evidence suggests their involvement in transepithelial transport of chloride in distal nephron segments; ClC-K1 gene deletion leads to nephrogenic diabetes insipidus in mice, and mutations of the hClC-Kb gene cause Bartter's syndrome type III in humans. Expression of rClC-K1 in Xenopus oocytes yielded voltage-independent currents that were pH-sensitive, had a Br(-) > NO(3)(-) = Cl(-) > I(-) conductance sequence, and were activated by extracellular calcium. A glutamate for valine exchange at amino acid position 166 induced strong voltage dependence and altered the conductance sequence of ClC-K1. This demonstrates that rClC-K1 indeed functions as an anion channel. By contrast, we did not detect currents upon hClC-Kb expression in Xenopus oocytes. Using a chimeric approach, we defined a protein domain that, when replaced by that of rClC-K1, allowed the functional expression of a chimera consisting predominantly of hClC-Kb. Its currents were linear and were inhibited by extracellular acidification. Contrasting with rClC-K1, they displayed a Cl(-) > Br(-)> I(-) > NO(3)(-) conductance sequence and were not augmented by extracellular calcium. Insertion of point mutations associated with Bartter's syndrome type III destroyed channel activity. We conclude that ClC-K proteins form constitutively open chloride channels with distinct physiological characteristics.

Lysophosphatidylcholine triggers intracellular calcium release and activation of non-selective cation channels in renal arterial smooth muscle cells.

The effects of a lipid component of oxidized low-density lipoproteins (ox-LDL), L-alpha-palmitoyl-lysophosphatidylcholine (LPC), on membrane currents of isolated canine renal artery smooth muscle cells (RASMC) were examined using the whole-cell configuration of the patch-clamp technique. In RASMC exposed to nominally Ca2+-free solutions and dialyzed with 0.1 mM EGTA and 140 mM K+, superfusion with LPC (10 microM) elicited spontaneous transient outward currents (STOCs) and/or spontaneous transient inward currents (STICs), followed by the activation of a large voltage-independent current with a reversal potential (Er) close to 0 mV. Buffering intracellular Ca2+ with 10 mM BAPTA prevented the appearance of STOCs and STICs, but not the activation of the voltage-independent current. Er of the LPC-induced voltage-independent current exhibited sensitivity to changes in [K+]o and [Na+]o in a manner consistent with a non-selective cation current (I(NSC)) and was blocked by gadolinium (Gd3+; 10 microM). Shifts in Er of the LPC-induced I(NSC) in response to changes in [Ca2+]o were used to estimate a relative Ca2+ to Na+ permeability ratio (P(Ca)/P(Na)) of 1.67. These results suggest that LPC causes abnormal sarcoplasmic reticulum Ca2+ regulation, leading to the appearance of STOCs and STICs, and the activation of I(NSC) in vascular smooth muscle cells. These effects may explain the ability of ox-LDLs to elevate [Ca2+]i in vascular smooth muscle and inhibit endothelium-dependent relaxation.

Ion Currents in Smooth Muscle Cells from Human Small Bronchioles: Presence of an Inward Rectifier K+ Current and Three Types of Large Conductance K+ Channel

Bronchoconstriction of small bronchioles plays a major role in the increase in airway resistance following agonist challenge. There is evidence that the airway smooth muscle (ASM) of small bronchioles differs functionally from that in larger airways. Little is known however about the electrophysiology of small bronchioles. Ion currents were therefore studied in airway smooth muscle cells freshly dissociated from human intralobular bronchioles, with a diameter between 0.3 and 1.0 mm. As previously reported for human large airways, the major outward current in these cells was due to activity of large conductance K+ (BK) channels, with a relatively minor component due to a voltage-gated delayed rectifier current (IDR), which was only observed in 30 % of cells. Three distinct types of iberiotoxin- and TEA-sensitive large conductance K+ channel contributed to large conductance K+ current (IBK). These included a highly voltage- and Ca2+-sensitive 200 pS channel previously reported in human large airways, and two smaller channels of 150 and 100 pS previously seen only in human fetal or cultured ASM. In contrast to large airways, ASM cells from bronchioles also demonstrated a voltage-gated inward rectifier current (IIR). IIR was activated by hyperpolarisation below the K+ equilibrium potential and could be blocked by submillimolar concentrations of Cs+ or Ba2+, and partially by physiological concentrations of Na+. Corresponding single channels with a conductance of 17 pS could also be recorded in the cell-attached configuration. A small voltage-independent current was also observed which was resistant to classic K+ and Cl- channel blockers but which could be abolished by replacement of Na+ with the impermeant cation N-methyl-D-glucamine (NMDG+). Corresponding non-selective single channels of 20 pS could be seen in inside-out mode. These results demonstrate that ASM from small bronchioles differs in terms of ion currents and channels from ASM derived from large airways, with possible implications for function.

A Current Activated on Depletion of Intracellular Ca2+Stores Can Regulate Exocytosis in Adrenal Chromaffin Cells

Exocytosis in excitable cells is strongly coupled to Ca2+ entry through voltage-gated channels but can be evoked by activation of membrane receptors that release Ca2+ from inositol 1,4, 5-trisphosphate-sensitive internal stores. In many cell types, depletion of Ca2+ stores activates Ca2+ influx across the plasma membrane, a process known as capacitative or store-operated Ca2+ entry. This influx is mediated by a number of voltage-independent, Ca2+-selective currents. In addition to replenishing Ca2+ stores, these currents are hypothesized to play an important role in agonist-evoked secretion in nonexcitable cells, although this has not been confirmed experimentally. The existence and physiological function of such currents in excitable cells is not known. Using the capacitance detection technique to monitor exocytosis, we provide direct experimental evidence that a similar mechanism exists in bovine adrenal chromaffin cells. Depletion of intracellular Ca2+ stores with thapsigargin, a SERCA pump inhibitor, or with BAPTA, an exogenous Ca2+ chelator, activates a small-amplitude, voltage-independent current that is carried by Ca2+ and Na+ ions. Ca2+ entry through this pathway is sufficient to stimulate exocytosis at negative membrane potentials. In addition, depolarization-evoked exocytosis is markedly facilitated on activation of the current. These data suggest that excitable cells possess a store-operated Ca2+ influx mechanism that may both directly trigger exocytosis and modulate excitation-secretion coupling.

Structure, chromosome localization, and tissue distribution of the mouse twik K + channel gene

We have recently discovered a new class of potassium channels with two pore-forming domains and four membrane-spanning domains. When heterologously expressed, these channels produce time- and voltage-independent currents that classify them as background or leak channels. TWIK (for t andem of P domains in a w eak i nwardly rectifying K + channel) was the first member of this family to be cloned. Here, we describe the genomic organization of TWIK in the mouse. The coding sequence as well as the untranslated sequences are contained in three exons. The twik gene (or KCNK1) has been mapped to chromosome 8, consistent with its localization to 1q42–43 in human. The twik gene is expressed in virtually all mouse tissues. It is most abundantly expressed in brain and moderately in other organs such as kidney. The level of expression is increased in brain and kidney from neonate to adult animals, but the TWIK message is also detected during embryogenesis, as early as day 7 post conception.