Selective activation of Ca2+-activated K+ channels by co-localized Ca2+ channels in hippocampal neurons

Calcium entry through voltage-gated calcium channels can activate either large- (BK) or small- (SK) conductance calcium-activated potassium channels. In hippocampal neurons, activation of BK channels underlies the falling phase of an action potential and generation of the fast afterhyperpolarization (AHP). In contrast, SK channel activation underlies generation of the slow AHP after a burst of action potentials. The source of calcium for BK channel activation is unknown, but the slow AHP is blocked by dihydropyridine antagonists, indicating that L-type calcium channels provide the calcium for activation of SK channels. It is not understood how this specialized coupling between calcium and potassium channels is achieved. Here we study channel activity in cell-attached patches from hippocampal neurons and report a unique specificity of coupling. L-type channels activate SK channels only, without activating BK channels present in the same patch. The delay between the opening of L-type channels and SK channels indicates that these channels are 50-150 nm apart. In contrast, N-type calcium channels activate BK channels only, with opening of the two channel types being nearly coincident. This temporal association indicates that N and BK channels are very close. Finally, P/Q-type calcium channels do not couple to either SK or BK channels. These data indicate an absolute segregation of coupling between channels, and illustrate the functional importance of submembrane calcium microdomains.     

ATP inhibition and rectification of a Ca2+-activated anion channel in sarcoplasmic reticulum of skeletal muscle

We describe ATP-dependent inhibition of the 75-105-pS (in 250 mM Cl-) anion channel (SCl) from the sarcoplasmic reticulum (SR) of rabbit skeletal muscle. In addition to activation by Ca2+ and voltage, inhibition by ATP provides a further mechanism for regulating SCl channel activity in vivo. Inhibition by the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate (AMP-PNP) ruled out a phosphorylation mechanism. Cytoplasmic ATP (approximately 1 mM) inhibited only when Cl- flowed from cytoplasm to lumen, regardless of membrane voltage. Flux in the opposite direction was not inhibited by 9 mM ATP. Thus ATP causes true, current rectification in SCl channels. Inhibition by cytoplasmic ATP was also voltage dependent, having a K(I) of 0.4-1 mM at -40 mV (Hill coefficient approximately 2), which increased at more negative potentials. Luminal ATP inhibited with a K(I) of approximately 2 mM at +40 mV, and showed no block at negative voltages. Hidden Markov model analysis revealed that ATP inhibition 1) reduced mean open times without altering the maximum channel amplitude, 2) was mediated by a novel, single, voltage-independent closed state (approximately 1 ms), and 3) was much less potent on lower conductance substates than the higher conductance states. Therefore, the SCl channel is unlikely to pass Cl- from cytoplasm to SR lumen in vivo, and balance electrogenic Ca2+ uptake as previously suggested. Possible roles for the SCl channel in the transport of other anions are discussed.     

Selectivity changes during activation of mutant Shaker potassium channels

Mutations of the pore-region residue T442 in Shaker channels result in large effects on channel kinetics. We studied mutations at this position in the backgrounds of NH2-terminal-truncated Shaker H4 and a Shaker -NGK2 chimeric channel having high conductance (Lopez, G.A., Y.N. Jan, and L.Y. Jan. 1994. Nature (Lond.). 367: 179-182). While mutations of T442 to C, D, H, V, or Y resulted in undetectable expression in Xenopus oocytes, S and G mutants yielded functional channels having deactivation time constants and channel open times two to three orders of magnitude longer than those of the parental channel. Activation time courses at depolarized potentials were unaffected by the mutations, as were first-latency distributions in the T442S chimeric channel. The mutant channels show two subconductance levels, 37 and 70% of full conductance. From single-channel analysis, we concluded that channels always pass through the larger subconductance state on the way to and from the open state. The smaller subconductance state is traversed in approximately 40% of activation time courses. These states apparently represent kinetic intermediates in channel gating having voltage-dependent transitions with apparent charge movements of approximately 1.6 e0. The fully open T442S chimeric channel has the conductance sequence Rb+ > NH4+ > K+. The opposite conductance sequence, K+ > NH4+ > Rb+, is observed in each of the subconductance states, with the smaller subconductance state discriminating most strongly against Rb+.     

cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block

Elevation of intracellular cAMP levels in Necturus gallbladder epithelium (NGB) induces an apical membrane Cl- conductance (GaCl). Its characteristics (i.e., magnitude, anion selectivity, and block) were studied with intracellular microelectrode techniques. Under control conditions, the apical membrane conductance (Ga) was 0.17 mS.cm-2, primarily ascribable to GaK. With elevation of cell cAMP to maximum levels, Ga increased to 6.7 mS.cm-2 and became anion selective, with the permeability sequence SCN- > NO3- > I- > Br- > Cl- > SO4(2-) approximately gluconate approximately cyclamate. GaCl was not affected by the putative Cl- channel blockers Cu2+, DIDS, DNDS, DPC, furosemide, IAA-94, MK-196, NPPB, SITS, verapamil, and glibenclamide. To characterize the cAMP-activated Cl- channels, patch-clamp studies were conducted on the apical membrane of enzyme-treated gallbladders or on dissociated cells from tissues exposed to both theophylline and forskolin. Two kinds of Cl- channels were found. With approximately 100 mM Cl- in both bath and pipette, the most frequent channel had a linear current-voltage relationship with a slope conductance of approximately 10 pS. The less frequent channel was outward rectifying with slope conductances of approximately 10 and 20 pS at -40 and 40 mV, respectively. The Cl- channels colocalized with apical maxi-K+ channels in 70% of the patches. The open probability (Po) of both kinds of Cl- channels was variable from patch to patch (0.3 on average) and insensitive to [Ca2+], membrane voltage, and pH. The channel density (approximately 0.3/patch) was one to two orders of magnitude less than that required to account for GaCl. However, addition of 250 U/ml protein kinase A plus 1 mM ATP to the cytosolic side of excised patches increased the density of the linear 10-pS Cl- channels more than 10-fold to four per patch and the mean Po to 0.5, close to expectations from GaCl. The permeability sequence and blocker insensitivity of the PKA-activated channels were identical to those of the apical membrane. These data strongly suggest that 10-pS Cl- channels are responsible for the cAMP-induced increase in apical membrane conductance of NGB epithelium.     

Functional diversity of GABA-activated Cl- currents in Purkinje versus granule neurons in rat cerebellar slices

M2, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M2 baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25-500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25-90 pS). (3) The channels were selective for cations; tNa = 0.75 when 150 mM NaCl bathed both sides of the membrane. (4) Amantadine reduced the probablity of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs approximately Na > Li. The pH activation, amantadine block and ion selectivity of the M2 protein ion channel in bilayers are consistent with those observed on expression of the M2 protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M2 protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

Cloning and expression of a novel pH-sensitive two pore domain K+ channel from human kidney

A complementary DNA encoding a novel K+ channel, called TASK-2, was isolated from human kidney and its gene was mapped to chromosome 6p21. TASK-2 has a low sequence similarity to other two pore domain K+ channels, such as TWIK-1, TREK-1, TASK-1, and TRAAK (18-22% of amino acid identity), but a similar topology consisting of four potential membrane-spanning domains. In transfected cells, TASK-2 produces noninactivating, outwardly rectifying K+ currents with activation potential thresholds that closely follow the K+ equilibrium potential. As for the related TASK-1 and TRAAK channels, the outward rectification is lost at high external K+ concentration. The conductance of TASK-2 was estimated to be 14.5 picosiemens in physiological conditions and 59.9 picosiemens in symmetrical conditions with 155 mM K+. TASK-2 currents are blocked by quinine (IC50 = 22 microM) and quinidine (65% of inhibition at 100 microM) but not by the other classical K+ channel blockers tetraethylammonium, 4-aminopyridine, and Cs+. They are only slightly sensitive to Ba2+, with less than 17% of inhibition at 1 mM. As TASK-1, TASK-2 is highly sensitive to external pH in the physiological range. 10% of the maximum current was recorded at pH 6. 5 and 90% at pH 8.8. Unlike all other cloned channels with two pore-forming domains, TASK-2 is essentially absent in the brain. In human and mouse, TASK-2 is mainly expressed in the kidney, where in situ hybridization shows that it is localized in cortical distal tubules and collecting ducts. This localization, as well as its functional properties, suggest that TASK-2 could play an important role in renal K+ transport.     

Single-channel currents produced by the serotonin transporter and analysis of a mutation affecting ion permeation

Single-channel activities were observed in outside-out patches excised from oocytes expressing a mammalian 5-hydroxytryptamine (5-HT) transporter. Channel conductance was larger for a mutant in which asparagine177 of the third putative transmembrane domain was replaced by glycine, suggesting that this residue lies within or near the permeation pathway. The N177G mutant enables quantitative single-channel measurements; it displays two conducting states. One state, with conductance of approximately 6 pS, is induced by 5-HT and is permeable to Na+. The other state (conductance of approximately 13 pS) is associated with substrate-independent leakage current and is permeable to both Na+ and Li+. Cl- is not a major current carrier. Channel lifetimes under all conditions measured are approximately 2.5 ms. The single-channel phenomena account for previously observed macroscopic electrophysiological phenomena, including 5-HT-induced transport-associated currents and substrate-independent leakage currents. The channel openings occur several orders of magnitude less frequently than would be expected if one such opening occurred for each transport cycle and therefore do not represent an obligatory step in transport. Nevertheless, single-channel events produced by neurotransmitter transporters indicate the functional and structural similarities between transporters and ion channels and provide a new tool, at single-molecule resolution, for detailed structure-function studies of transporters.     

The S5-S6 linker of repeat I is a critical determinant of L-type Ca2+ channel conductance

The alpha1-subunits of the skeletal and cardiac L-type calcium channels (L-channels) contain nearly identical pore regions (P-regions) in each of the four internal homology repeats. In spite of this high conservation of the P-regions, native skeletal L-channels exhibit a unitary conductance that is only about half that of native cardiac L-channels. To identify structural determinants of this difference in L-channel conductance, we have characterized unitary activity in cell-attached patches of dysgenic myotubes expressing skeletal, cardiac, and chimeric L-channel alpha1-subunits. Our results demonstrate that the S5-S6 linker of repeat I (IS5-IS6 linker) is a critical determinant of the difference in skeletal and cardiac unitary conductance. The unitary conductances attributable to the wild-type skeletal (CAC6; approximately 14 pS) and cardiac (CARD1; approximately 25 pS) alpha1-subunits expressed in dysgenic myotubes are identical to those observed in native tissues. Chimeric alpha1-subunits containing skeletal sequence for the first internal repeat and all of the putative intracellular loops (SkC15), the IS5-IS6 linker and the intracellular loops (SkC51), or only the IS5-IS6 linker (SkC49) each exhibit a low, skeletal-like unitary conductance (< or = 17 pS). Constructs in which the IS5-IS6 linker is of cardiac origin (CARD1 and CSk9) display cardiac-like conductance (approximately 25 pS). Unitary conductance and the rate of channel activation are apparently independent processes, since both SkC51 and SkC49 exhibit low, skeletal-like conductance and rapid, cardiac-like rates of ensemble activation. These results demonstrate that the IS5-IS6 linker strongly influences the single channel conductance of L-channels in a manner that is independent from the rate of channel activation.     

Two types of stretch-activated channels coexist in the rabbit corneal epithelial cell

Ion channels contribute to the regulation of cellular function through control of the membrane potential and intracellular concentration of various ions. We examined stretch-activated channels in the corneal epithelial cell. Patch clamping was applied to enzymatically dissociated corneal epithelial cells to characterize their stretch-activated ion channels. The plasma membrane was stretched by applying suction to the patch pipette in cell-attached or inside-out patch configuration. The ion selectivity, voltage-dependence, and stretch-dependence were examined. Two kinds of stretch-activated channel events were observed; the previously-reported large conductance (L) channel and a novel small conductance (S) channel. The probability of recording L vs. S channels in the cell-attached configuration was about 2:1. The L channel was potassium selective with single channel conductance (gamma) of about 160 pS under the symmetrical (150 mm K+) solution. The S channel was permeable to Na+ and K+ with gamma of about 20 pS under the same conditions. Both L and S channels showed little activity in the absence of suction applied to the recording pipette. Channel activity was evoked by suction (negative pressure) stronger than -20 mmHg in both channels. The open probability (Po) and the mean current increased in proportion to further applied stretch and did not saturate for applied suction as strong as -80 mmHg, the pressure at which the gigaseal started to break. Thus, two types of stretch-activated channels coexist in corneal epithelial cells; a potassium-selective L channel and non-selective S channel. The contribution of these channels to the membrane potential is discussed.     

The large conductance, voltage-dependent, and calcium-sensitive K+ channel, Hslo, is a target of cGMP-dependent protein kinase phosphorylation in vivo

Native large conductance, voltage-dependent, and Ca2+-sensitive K+ channels are activated by cGMP-dependent protein kinase. Two possible mechanisms of kinase action have been proposed: 1) direct phosphorylation of the channel and 2) indirect via PKG-dependent activation of a phosphatase. To scrutinize the first possibility, at the molecular level, we used the human pore-forming alpha-subunit of the Ca2+-sensitive K+ channel, Hslo, and the alpha-isoform of cGMP-dependent protein kinase I. In cell-attached patches of oocytes co-expressing the Hslo channel and the kinase, 8-Br-cGMP significantly increased the macroscopic currents. This increase in current was due to an increase in the channel voltage sensitivity by approximately 20 mV and was reversed by alkaline phosphatase treatment after patch excision. In inside-out patches, however, the effect of purified kinase was negative in 12 of 13 patches. In contrast, and consistent with the intact cell experiments, purified kinase applied to the cytoplasmic side of reconstituted channels increased their open probability. This stimulatory effect was absent when heat-denatured kinase was used. Biochemical experiments show that the purified kinase incorporates gamma-33P into the immunopurified Hslo band of approximately 125 kDa. Furthermore, in vivo phosphorylation largely attenuates this labeling in back-phosphorylation experiments. These results demonstrate that the alpha-subunit of large conductance Ca2+-sensitive K+ channels is substrate for G-Ialpha kinase in vivo and support direct phosphorylation as a mechanism for PKG-Ialpha-induced activation of maxi-K channels.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Mutations causing neurodegeneration in Caenorhabditis elegans drastically alter the pH sensitivity and inactivation of the mammalian H+-gated Na+ channel MDEG1

The mammalian degenerin MDEG1 belongs to the nematode degenerin/epithelial Na+ channel superfamily. It is constitutively activated by the same mutations that cause gain-of-function of the Caenorhabditis elegans degenerins and neurodegeneration. ASIC and DRASIC, which were recently cloned, are structural homologues of MDEG1 and behave as H+-gated cation channels. MDEG1 is also a H+-activated Na+ channel, but it differs from ASIC in its lower pH sensitivity and slower kinetics. In addition to the generation of a constitutive current, mutations in MDEG1 also alter the properties of the H+-gated current. Replacement of Gly-430 in MDEG1 by bulkier amino acids, such as Val, Phe, or Thr, drastically increases the H+ sensitivity of the channel (half-maximal pH (pHm) approximately 4.4 for MDEG1, pHm approximately 6.7 for the different mutants). Furthermore, these replacements completely suppress the inactivation observed with the wild-type channel and increase the sensitivity of the H+-gated channel to blockade by amiloride by a factor of 10 without modification of its conductance and ionic selectivity. These results as well as those obtained with other mutants clearly indicate that the region surrounding Gly-430, situated just before the second transmembrane segment, is essential for pH sensitivity and gating.     

Ion channels in isolated mouse jejunal crypts

The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K+ channel (BK) (conductance, g at 0 mV = 92 +/- 6 pS), which was highly selective for K+ [PK+ (1) > PRb+ (0.6) > PCs+ (0.09) approximately PNa+ (0.07) > PLi+ (0.04)], had a low, voltage-independent open probability (Po) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 +/- 3 pS), Po was voltage dependent and it was blocked by cytoplasmic Ba2+ (5 mmol/l). An intermediate K+ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 +/- 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl- channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 +/- 2 pS), its Po was independent of voltage and unaffected by variations in cytoplasmic Ca2+ (100 nmol/l to 1 mmol/l) or ATP (0-1 mmol/l). The NSCC had a linear conductance (20 +/- 1 pS), its Po increased with depolarisation and elevation of cytoplasmic [Ca2+] (> or = 10 micromol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl- conductance was activated. This cAMP-dependent Cl- conductance was distinct from the basolateral Cl- channel and thus is most likely located in the apical membrane.     

Study of ionic currents across a model membrane channel using Brownian dynamics

Brownian dynamics simulations have been carried out to study ionic currents flowing across a model membrane channel under various conditions. The model channel we use has a cylindrical transmembrane segment that is joined to a catenary vestibule at each side. Two cylindrical reservoirs connected to the channel contain a fixed number of sodium and chloride ions. Under a driving force of 100 mV, the channel is virtually impermeable to sodium ions, owing to the repulsive dielectric force presented to ions by the vestibular wall. When two rings of dipoles, with their negative poles facing the pore lumen, are placed just above and below the constricted channel segment, sodium ions cross the channel. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength does not increase the channel conductance further. When only those ions that acquire a kinetic energy large enough to surmount a barrier are allowed to enter the narrow transmembrane segment, the channel conductance decreases monotonically with the barrier height. This barrier represents those interactions between an ion, water molecules, and the protein wall in the transmembrane segment that are not treated explicitly in the simulation. The conductance obtained from simulations closely matches that obtained from ACh channels when a step potential barrier of 2-3 kTr is placed at the channel neck. The current-voltage relationship obtained with symmetrical solutions is ohmic in the absence of a barrier. The current-voltage curve becomes nonlinear when the 3 kTr barrier is in place. With asymmetrical solutions, the relationship approximates the Goldman equation, with the reversal potential close to that predicted by the Nernst equation. The conductance first increases linearly with concentration and then begins to rise at a slower rate with higher ionic concentration. We discuss the implications of these findings for the transport of ions across the membrane and the structure of ion channels.     

On the mechanism by which 4-Aminopyridine occludes quinidine block of the cardiac K+ channel, hKv1.5

4-Aminopyridine (4-AP) binds to potassium channels at a site or sites in the inner mouth of the pore and is thought to prevent channel opening. The return of hKv1.5 off-gating charge upon repolarization is accelerated by 4-AP and it has been suggested that 4-AP blocks slow conformational rearrangements during late closed states that are necessary for channel opening. On the other hand, quinidine, an open channel blocker, slows the return or immobilizes off-gating charge only at opening potentials (>-25 mV). The aim of this study was to use quinidine as a probe of open channels to test the kinetic state of 4-AP-blocked channels. In the presence of 0.2-1 mM 4-AP, quinidine slowed charge return and caused partial charge immobilization, corresponding to an increase in the Kd of approximately 20-fold. Peak off-gating currents were reduced and decay was slowed approximately 2- to 2.5-fold at potentials negative to the threshold of channel activation and during depolarizations shorter than normally required for channel activation. This demonstrated access of quinidine to 4-AP-blocked channels, a lack of competition between the two drugs, and implied allosteric modulation of the quinidine binding site by 4-AP resident within the channel. Single channel recordings also showed that quinidine could modulate the 4-AP-induced closure of the channels, with the result that frequent channel reopenings were observed when both drugs were present. We propose that 4-AP-blocked channels exist in a partially open, nonconducting state that allows access to quinidine, even at more negative potentials and during shorter depolarizations than those required for channel activation.     

Macroscopic and microscopic properties of a cloned glutamate transporter/chloride channel

The behavior of a Cl- channel associated with a glutamate transporter was studied using intracellular and patch recording techniques in Xenopus oocytes injected with human EAAT1 cRNA. Channels could be activated by application of glutamate to either face of excised membrane patches. The channel exhibited strong selectivity for amphipathic anions and had a minimum pore diameter of approximately 5A. Glutamate flux exhibited a much greater temperature dependence than Cl- flux. Stationary and nonstationary noise analysis was consistent with a sub-femtosiemen Cl- conductance and a maximum channel Po < 1. The glutamate binding rate was similar to estimates for receptor binding. After glutamate binding, channels activated rapidly followed by a relaxation phase. Differences in the macroscopic kinetics of channels activated by concentration jumps of L-glutamate or D-aspartate were correlated with differences in uptake kinetics, indicating a close correspondence of channel gating to state transitions in the transporter cycle.     

Single-channel characteristics of wild-type IKs channels and channels formed with two minK mutants that cause long QT syndrome

IKs 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, IKs 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 IKs 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 IKs channels was approximately 16 pS (corresponding to approximately 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 IKs 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 IKs channels due to lowered single-channel conductance and altered gating.     

Regulation of cyclooxygenase-2 pathway by HER2 receptor

In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP3)-activated Ca2+ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP3 receptor (IP3R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP3, inhibition by heparin, time-dependent inactivation by high IP3 concentrations, activation by guanosine 5'o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca2+-release-activated Ca2+ channels (Icrac). These conductive properties include extremely low conductance (approximately 1 pS), very high selectivity for Ca2+ over K+ (PCa/PK>1000), inactivation by high intracellular Ca2+ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP3 after patch excision. Although the possibility cannot be completely excluded that a novel type of IP3R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains Icrac-like Ca2+ channels coupled to an IP3-responsive protein which displays properties similar to those of the IP3R expressed in the ER membrane.     

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4-5 elementary charges.