Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.     

SK channels in excitability, pacemaking and synaptic integration

Small conductance calcium-activated potassium channels link elevations of intracellular calcium ions to membrane potential, exerting a hyperpolarizing influence when activated. The consequences of SK channel activity have been revealed by the specific blocker apamin, a peptide toxin from honeybee venom. Recent studies have revealed unexpected roles for SK channels in fine-tuning intrinsic cell firing properties and in responsiveness to synaptic input. They have also identified specific roles for different SK channel subtypes. A host of Ca2+ sources, including distinct subtypes of voltage-dependent calcium channels, intracellular Ca2+ stores and Ca2+-permeable ionotropic neurotransmitter receptors, activate SK channels. The macromolecular complex in which the Ca2+ source, SK channels and various modulators are assembled determines the kinetics and consequences of SK channel activation.     

Inwardly rectifying current-voltage relationship of small-conductance Ca2+-activated K+ channels rendered by intracellular divalent cation blockade

Small conductance Ca2+-activated K+ channels (SK(Ca) channels) are a group of K+-selective ion channels activated by submicromolar concentrations of intracellular Ca2+ independent of membrane voltages. We expressed a cloned SK(Ca) channel, rSK2, in Xenopus oocytes and investigated the effects of intracellular divalent cations on the current-voltage (I-V) relationship of the channels. Both Mg2+ and Ca2+ reduced the rSK2 channel currents in voltage-dependent manners from the intracellular side and thus rectified the I-V relationship at physiological concentration ranges. The apparent affinity of Mg2+ was changed as a function of both transmembrane voltage and intracellular Ca2+ concentration. Extracellular K+ altered the voltage dependence as well as the apparent affinities of Mg2+ binding from intracellular side. Thus, the inwardly rectifying I-V relationship of SK(Ca) channels is likely due to the voltage-dependent blockade of intracellular divalent cations and that the binding site is located within the ion-conducting pathway. Therefore, intracellular Ca2+ modulates the permeation characteristics of SK(Ca) channels by altering the I-V relationship as well as activates the channel by interacting with the gating machinery, calmodulin, and SK(Ca) channels can be considered as Ca2+-activated inward rectifier K+ channels.     

BKCa channels activating at resting potential without calcium in LNCaP prostate cancer cells

Large-conductance Ca2+-dependent K+ (BK(Ca)) channels are activated by intracellular Ca2+ and membrane depolarization in an allosteric manner. We investigated the pharmacological and biophysical characteristics of a BK(Ca)-type K+ channel in androgen-dependent LNCaP (lymph node carcinoma of the prostate) cells with novel functional properties, here termed BK(L). K+ selectivity, high conductance, activation by Mg2+ or NS1619, and inhibition by paxilline and penitrem A largely resembled the properties of recombinant BK(Ca) channels. However, unlike conventional BK(Ca) channels, BK(L) channels activated in the absence of free cytosolic Ca2+ at physiological membrane potentials; the half-maximal activation voltage was shifted by about -100 mV compared with BK(Ca) channels. Half-maximal Ca2+-dependent activation was observed at 0.4 microM: for BK(L) (at -20 mV) and at 4.1 microM: for BK(Ca) channels (at +50 mV). Heterologous expression of hSlo1 in LNCaP cells increased the BK(L) conductance. Expression of hSlo-beta1 in LNCaP cells shifted voltage-dependent activation to values between that of BK(L) and BK(Ca) channels and reduced the slope of the P (open) (open probability)-voltage curve. We propose that LNCaP cells harbor a so far unknown type of BK(Ca) subunit, which is responsible for the BK(L) phenotype in a dominant manner. BK(L)-like channels are also expressed in the human breast cancer cell line T47D. In addition, functional expression of BK(L) in LNCaP cells is regulated by serum-derived factors, however not by androgens.     

Intracellular calcium and the control of neuronal pacemaker activity

Pacemaker activity of the Aplysia bursting pacemaker neuron R-15 was analyzed. It was shown that the free intracellular Ca2+ concentration, as measured by arsenazo III, increases during the depolarizing phase of the pacemaker cycle and declines throughout the hyperpolarizing phase that follows. This increase in Ca2+ results from the activation of voltage-dependent Ca2+ channels that open during the depolarizing phase of the cycle. The extracellular K+ concentration also increases during the depolarizing phase of the cycle and is correlated with an outward K+ current that opposes the inward current carried by Ca2+ ions. The increase in internal Ca2+ is sufficient to activate a K+ conductance that depends on the magnitude of the change in internal Ca2+ and on membrane potential, which is responsible for the hyperpolarizing phase of the cycle. It is proposed that the membrane oscillation depends on three separate but linked systems, which include a voltage-dependent Ca2+ channel, the internal Ca2+ concentration, and a Ca2+-activated K+ channel.     

Maxi K+ channels and their relationship to the apical membrane conductance in Necturus gallbladder epithelium

Using the patch-clamp technique, we have identified large-conductance (maxi) K+ channels in the apical membrane of Necturus gallbladder epithelium, and in dissociated gallbladder epithelial cells. These channels are more than tenfold selective for K+ over Na+, and exhibit unitary conductance of approximately 200 pS in symmetric 100 mM KCl. They are activated by elevation of internal Ca2+ levels and membrane depolarization. The properties of these channels could account for the previously observed voltage and Ca2+ sensitivities of the macroscopic apical membrane conductance (Ga). Ga was determined as a function of apical membrane voltage, using intracellular microelectrode techniques. Its value was 180 microS/cm2 at the control membrane voltage of -68 mV, and increased steeply with membrane depolarization, reaching 650 microS/cm2 at -25 mV. We have related maxi K+ channel properties and Ga quantitatively, relying on the premise that at any apical membrane voltage Ga comprises a leakage conductance and a conductance due to maxi K+ channels. Comparison between Ga and maxi K+ channels reveals that the latter are present at a surface density of 0.09/microns 2, are open approximately 15% of the time under control conditions, and account for 17% of control Ga. Depolarizing the apical membrane voltage leads to a steep increase in channel steady-state open probability. When correlated with patch-clamp studies examining the Ca2+ and voltage dependencies of single maxi K+ channels, results from intracellular microelectrode experiments indicate that maxi K+ channel activity in situ is higher than predicted from the measured apical membrane voltage and estimated bulk cytosolic Ca2+ activity. Mechanisms that could account for this finding are proposed.     

Ion channels in smooth muscle: regulators of intracellular calcium and contractility

Smooth muscle (SM) is essential to all aspects of human physiology and, therefore, key to the maintenance of life. Ion channels expressed within SM cells regulate the membrane potential, intracellular Ca2+ concentration, and contractility of SM. Excitatory ion channels function to depolarize the membrane potential. These include nonselective cation channels that allow Na+ and Ca2+ to permeate into SM cells. The nonselective cation channel family includes tonically active channels (Icat), as well as channels activated by agonists, pressure-stretch, and intracellular Ca2+ store depletion. Cl--selective channels, activated by intracellular Ca2+ or stretch, also mediate SM depolarization. Plasma membrane depolarization in SM activates voltage-dependent Ca2+ channels that demonstrate a high Ca2+ selectivity and provide influx of contractile Ca2+. Ca2+ is also released from SM intracellular Ca2+ stores of the sarcoplasmic reticulum (SR) through ryanodine and inositol trisphosphate receptor Ca2+ channels. This is part of a negative feedback mechanism limiting contraction that occurs by the Ca2+-dependent activation of large-conductance K+ channels, which hyper polarize the plasma membrane. Unlike the well-defined contractile role of SR-released Ca2+ in skeletal and cardiac muscle, the literature suggests that in SM Ca2+ released from the SR functions to limit contractility. Depolarization-activated K+ chan nels, ATP-sensitive K+ channels, and inward rectifier K+ channels also hyperpolarize SM, favouring relaxation. The expression pattern, density, and biophysical properties of ion channels vary among SM types and are key determinants of electrical activity, contractility, and SM function.     

Activation of the BK (SLO1) potassium channel by mallotoxin

Pharmacologic approaches to activate K+ channels represent an emerging strategy to regulate membrane excitability. Here we report the identification and characterization of a lipid soluble toxin, mallotoxin (rottlerin), which potently activates the large conductance voltage and Ca2+-activated K+ channel (BK) expressed in a heterologous expression system and human vascular smooth muscle cells, shifting the conductance/voltage relationship by >100 mV. Probing the mechanism of action, we discover that the BK channel can be activated in the absence of divalent cations (Ca2+, Mg2+), suggesting that the mallotoxin mechanism of action involves the voltage-dependent gating of the channel. Mallotoxin-activated channels remain incrementally sensitive to Ca2+ and beta subunits. In comparison to other small hydrophobic poisons, anesthetic agents, and protein toxins that inhibit ion channel activity, mallotoxin potently activates channel activity. In certain respects, mallotoxin acts as a BK channel beta1 subunit mimetic, preserving BK channel Ca2+ sensitivity yet adjusting the set-point for BK channel activation to a more hyperpolarized membrane potential.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

Ion channels in opossum kidney cells.

This review discusses the activation of ion transport pathways during regulatory volume decrease in opossum kidney (OK) cells. OK cells regulate their volume when exposed to a hypotonic medium. The changes in cell volume are caused by activation of ion transport pathways and the accompanying osmotically driven water movement so that the increased cell volume returns toward physiological levels. The reshrinking of hypotonically swollen cells is termed regulatory volume decrease. In OK cells separate K+ and Cl- conductances are activated. The Na+/H+ cotransport system seems not to be involved. The potassium pathway is mediated by a K+ channel with a slope conductance of about 12 pS. The occasionally observed widely distributed Ca2(+)- and voltage-dependent K+ channel of large unit conductance (120 pS) seems not to be involved. The volume regulatory decrease is accompanied by a cell depolarization from a resting potential of about -60 mV to about -20 mV followed by a repolarization. It will be discussed whether the depolarization is caused by the observed activation of stretch-sensitive ion channels of about 30 and 40 pS, respectively. The transient behavior of the cell volume parallels the time-dependent change of the total membrane current. For both recording techniques the volume regulatory decrease can be blocked by quinine. In addition an inward rectifying K+ channel of about 80 pS has been observed in high KCl solution.     

A marriage of convenience: beta-subunits and voltage-dependent K+ channels

The movement of ions across cell membranes is essential for a wide variety of fundamental physiological processes, including secretion, muscle contraction, and neuronal excitation. This movement is possible because of the presence in the cell membrane of a class of integral membrane proteins dubbed ion channels. Ion channels, thanks to the presence of aqueous pores in their structure, catalyze the passage of ions across the otherwise ion-impermeable lipid bilayer. Ion conduction across ion channels is highly regulated, and in the case of voltage-dependent K(+) channels, the molecular foundations of the voltage-dependent conformational changes leading to the their open (conducting) configuration have provided most of the driving force for research in ion channel biophysics since the pioneering work of Hodgkin and Huxley (Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. 117, 500-544). The voltage-dependent K(+) channels are the prototypical voltage-gated channels and govern the resting membrane potential. They are responsible for returning the membrane potential to its resting state at the termination of each action potential in excitable membranes. The pore-forming subunits (alpha) of many voltage-dependent K(+) channels and modulatory beta-subunits exist in the membrane as one component of macromolecular complexes, able to integrate a myriad of cellular signals that regulate ion channel behavior. In this review, we have focused on the modulatory effects of beta-subunits on the voltage-dependent K(+) (Kv) channel and on the large conductance Ca(2+)- and voltage-dependent (BK(Ca)) channel.     

Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell.

Numerous biological assays and pharmacological studies on various higher plant tissues have led to the suggestion that voltage-dependent plasma membrane Ca2+ channels play prominent roles in initiating signal transduction processes during plant growth and development. However, to date no direct evidence has been obtained for the existence of such depolarization-activated Ca2+ channels in the plasma membrane of higher plant cells. Carrot suspension cells (Daucus carota L.) provide a well-suited system to determine whether voltage-dependent Ca2+ channels are present in the plasma membrane of higher plants and to characterize the properties of putative Ca2+ channels. It is known that both depolarization, caused by raising extracellular K+, and exposure to fungal toxins or oligogalacturonides induce Ca2+ influx into carrot cells. By direct application of patch-clamp techniques to isolated carrot protoplasts, we show here that depolarization of the plasma membrane positive to -135 mV activates Ca(2+)-permeable channels. These voltage-dependent ion channels were more permeable to Ca2+ than K+, while displaying large permeabilities to Ba2+ and Mg2+ ions. Ca(2+)-permeable channels showed slow and reversible inactivation. The single-channel conductance was 13 pS in 40 mM CaCl2. These data provide direct evidence for the existence of voltage-dependent Ca2+ channels in the plasma membrane of a higher plant cell and point to physiological mechanisms for plant Ca2+ channel regulation. The depolarization-activated Ca(2+)-permeable channels identified here could constitute a regulated pathway for Ca2+ influx in response to physiologically occurring stimulus-induced depolarizations in higher plant cells.     

Electroporative fast pore-flickering of the annexin V-lipid surface complex, a novel gating concept for ion transport

In contact with lipid bilayers and Ca2+-ions, the intracellular protein human annexin V (wild-type), Mr = 35,800, forms two types of cation-selective channels for the transport of Ca2+-, K+-, Na+- and Mg2+-ions, depending on the protein concentration [AN]. Type (I) channel events are large and predominant at high values [AN] > or = K = 5 nM at 296 K. At 50 mM Ca2+, symmetrical on both membrane sides, AN added at the cis side, the conductance is gCa(I) = 22 +/- 2 pS and at symmetrical 0.1 M K+-conditions: gK(I) = 32 +/- 3 pS, associated with two mean open-times tau1(I) = 0.68 +/- 0.2 ms and tau2(I) = 31 +/- 2 ms. Monoclonal anti-AN antibodies added to the trans-side first increase the mean open-times and then abolish the channel activity, suggesting that type (I) channels refer to a membrane spanning protein complex, probably a trimer T, which at [AN] > K changes its membrane organization to a higher oligomer, probably to the side-by-side double-trimer T2. The smaller type (II) channel events are predominant at low [AN] < or = K and refer to the (electroporative) adsorption complex of the monomer. The conductances g(i)(II) for symmetrical concentrations depend non-linearly on the voltage Um = Uext + U(AN), where U(AN) = 0.02 +/- 0.002 V is the electrostatic contribution of the Ca2+-AN complex and Uext the externally applied voltage. There is only one mean open-time tau(o)(II) which is voltage-dependent according to a functional of b x Um2 where b = 113.9 +/- 15 V(-2), yielding an activation Gibbs free energy of Ga = RT x b x Um2. The conformational flicker probability f(i)(II) in g(i)(II) = g(i)0(II) x gamma(i) x f(i)(II) is non-linearly voltage-dependent according to a functional of a x Um2. The Nernst term gamma(i) refers to asymmetrical ion concentrations. From a = 50 V(-2), independent of the ion type, we obtain f(i)0(II) = 0.03 +/- 0.002 and the conductances for the fully open-channel states: gCa0(II) = 69 +/- 3 pS (0.05 M Ca2+) and gK0(II) = 131 +/- 5 pS (1.2 M K+). From the electroporation term a = pi[r(p)2]epsilon0(epsilon(w) - epsilon(m))/(2 kTd) we determine the mean pore radius of the complex in its fully open state as r(p)= 0.86 +/- 0.05 nm. The adsorbed annexin V (Ca2+) monomer appears to electrostatically facilitate the electric pore formation at the contact interface between the protein and the lipid phase. The complex rapidly flickers and thus limits the ion transport in a voltage-dependent manner.     

Electrical pacemaker mechanisms of pancreatic islet cells

Glucose, the major physiological stimulus for insulin secretion, induces a periodic bursting pattern of Ca2+ action potentials that are thought to mediate the uptake of Ca2+ into the intracellular pool of free Ca2+, which controls the rate of insulin release. Evidence is reviewed that shows that the voltage-dependent Ca2+ spikes are driven by a slow, voltage-dependent plateau depolarization that may also be caused by Ca2+ influx. Current evidence suggests that this plateau conductance is periodically terminated in turn by a pacemaker current through membrane K+ channels that are activated by intracellular free Ca2+. The control of electrical activity by different modulators of insulin release may involve interactions with this system at several points, including changes of the sensitivity of K+ channels to intracellular Ca2+ and to changes of intracellular Ca2+ buffering capacity.     

Stable and functional expression of the calcium channel alpha 1 subunit from smooth muscle in somatic cell lines.

Voltage-activated calcium channels are membrane spanning proteins that allow the controlled entry of Ca2+ into the cytoplasm of cells. The principal channel forming subunit of an L-type calcium channel is the alpha 1 subunit. Transfection of Chinese hamster ovary (CHO) cells with complementary DNA encoding the calcium channel alpha 1 subunit from smooth muscle led to the expression of functional calcium channels which bind calcium channel blockers and show the voltage-dependent activation and slow inactivation and unitary current conductance characteristic of calcium channels in smooth muscle. The currents mediated by these channels are sensitive towards dihydropyridine-type blockers and agonists indicating that the calcium channel blocker receptor sites were present in functional form. The smooth muscle alpha 1 subunit cDNA alone is sufficient for stable expression of functional calcium channels with the expected kinetic and pharmacological properties in mammalian somatic cells.     

Multiple types of voltage-dependent Ca2+-activated K+ channels of large conductance in rat brain synaptosomal membranes

K+-selective ion channels from a mammalian brain synaptosomal membrane preparation were inserted into planar phospholipid bilayers on the tips of patch-clamp pipettes, and single-channel currents were measured. Multiple distinct classes of K+ channels were observed. We have characterized and described the properties of several types of voltage-dependent, Ca2+-activated K+ channels of large single-channel conductance (greater than 50 pS in symmetrical KCl solutions). One class of channels (Type I) has a 200-250-pS single-channel conductance. It is activated by internal calcium concentrations greater than 10(-7) M, and its probability of opening is increased by membrane depolarization. This channel is blocked by 1-3 mM internal concentrations of tetraethylammonium (TEA). These channels are similar to the BK channel described in a variety of tissues. A second novel group of voltage-dependent, Ca2+-activated K+ channels was also studied. These channels were more sensitive to internal calcium, but less sensitive to voltage than the large (Type I) channel. These channels were minimally affected by internal TEA concentrations of 10 mM, but were blocked by a 50 mM concentration. In this class of channels we found a wide range of relatively large unitary channel conductances (65-140 pS). Within this group we have characterized two types (75-80 pS and 120-125 pS) that also differ in gating kinetics. The various types of voltage-dependent, Ca2+-activated K+ channels described here were blocked by charybdotoxin added to the external side of the channel. The activity of these channels was increased by exposure to nanomolar concentrations of the catalytic subunit of cAMP-dependent protein kinase. These results indicate that voltage-dependent, charybdotoxin-sensitive Ca2+-activated K+ channels comprise a class of related, but distinguishable channel types. Although the Ca2+-activated (Type I and II) K+ channels can be distinguished by their single-channel properties, both could contribute to the voltage-dependent Ca2+-activated macroscopic K+ current (IC) that has been observed in several neuronal somata preparations, as well as in other cells. Some of the properties reported here may serve to distinguish which type contributes in each case. A third class of smaller (40-50 pS) channels was also studied. These channels were independent of calcium over the concentration range examined (10(-7)-10(-3) M), and were also independent of voltage over the range of pipette potentials of -60 to +60 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent ionic conductances in the human malignant astrocytoma cell line U87-MG

Although the human malignant astrocytoma cell line U87-MG has been used in numerous studies, few findings are available on the properties of its membrane ion conductances. Characterization of the ion channels expressed in these cells will make it possible to study membrane ion conductance changes when a receptor is activated by its ligand. This will help to elucidate the functional properties of these receptors and their signal-transduction pathways in pathophysiological events. This work studied the voltage-dependent ionic conductances of U87-MG cells using the Whole-Cell Recording patch-clamp technique. Six types of voltage-dependent ionic currents were identified: (i) a TEA-, 4-AP- and CTX-sensitive Ca2+-dependent K+ current, (ii) a transient K+ current inhibited by 4-AP, (iii) an inwardly rectifying K+ current blocked by Ba2+ and 4-AP, (iv) a DIDS- and SITS-sensitive Cl- current, (v) a TTX-sensitive Na+ conductance and (vi) a L-type Ca2+ conductance activated by BayK-8644 and inhibited by Ni and the L-type Ca2+ channel inhibitor, nifedipine. In addition, electrical depolarizations elicited inward currents due to voltage-independent, Ca2+-dependent K+ influx against the electrochemical gradient, probably via an ouabain-sensitive Na+-K+ pump.     

Vacuolar calcium channels

The central vacuole is the largest Ca2+ store in a mature plant cell. Ca2+ release from this store contributes to Ca2+-mediated intracellular signalling in a variety of physiological responses. However, the routes for vacuolar Ca2+ release are not well characterized. To date, at least two voltage-dependent and two ligand-gated Ca2+-permeable channels have been reported in plant vacuoles. However, the so-called VVCa (vacuolar voltage-gated Ca2+) channel most probably is not a separate channel but is identical to another voltage-dependent channel-the so-called SV (slow vacuolar) channel. Studies in the last few years have added a new dimension to our knowledge of SV channel-mediated ion transport and the mechanisms of its regulation by multiple natural factors. Recently, the SV channel was identified as the product of the TPC1 gene in Arabidopsis. In contrast, the TPC1 channel from other species was thought to be localized in the plasma membrane. A re-evaluation of this work under the assumption that the TPC1 channel is generally a vacuolar channel provides interesting insights into the physiological function of the TPC1/SV channel. Considerably less is known about vacuolar Ca2+ channels that are supposed to be activated by inositol 1,4,5-trisphosphate or cADP ribose. The major problems are controversial reports about functional characteristics, and a remarkable lack of homologues of animal ligand-gated Ca2+ channels in higher plants. To help understand Ca2+-mediated intracellular signalling in plant cells, a critical update of existing experimental evidence for vacuolar Ca2+ channels is presented.     

Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume

Large conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca(2+) are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K(+) channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K(+) flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl(-) efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.