scholarly journals A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores

2017 ◽  
Vol 216 (6) ◽  
pp. 1715-1730 ◽  
Author(s):  
Wuyang Wang ◽  
Xiaoli Zhang ◽  
Qiong Gao ◽  
Maria Lawas ◽  
Lu Yu ◽  
...  
Keyword(s):  
Bk Channels ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Genetic Ablation ◽  
Membrane Contact Sites ◽  
Selective Permeability ◽  
Voltage Dependent ◽  
Cytosolic Side ◽  
Membrane Permeabilities ◽  
Membrane Contact

The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane’s selective permeability to K+. We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na+ and H+, but not K+, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca2+ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K+-selective conductance (LysoKVCa). LysoKVCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoKVCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca2+ stores may be refilled from endoplasmic reticulum (ER) Ca2+ via ER–lysosome membrane contact sites. We propose that LysoKVCa serves as the perilysosomal Ca2+ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoKVCa, or abolition of its Ca2+ sensitivity, blocks refilling and maintenance of lysosomal Ca2+ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.

Functional and molecular characterization of the fluid secretion mechanism in human parotid acinar cells

2007 ◽  
Vol 292 (6) ◽  
pp. R2380-R2390 ◽  
Author(s):  
Tetsuji Nakamoto ◽  
Alaka Srivastava ◽  
Victor G. Romanenko ◽  
Catherine E. Ovitt ◽  
Patricia Perez-Cornejo ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Ion Transport ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Specific Cell ◽  
Parotid Glands ◽  
Voltage Dependent ◽  
Intracellular Ca

The strategies available for treating salivary gland hypofunction are limited because relatively little is known about the secretion process in humans. An initial microarray screen detected ion transport proteins generally accepted to be critically involved in salivation. We tested for the activity of some of these proteins, as well as for specific cell properties required to support fluid secretion. The resting membrane potential of human acinar cells was near −51 mV, while the intracellular [Cl−] was ∼62 mM, about fourfold higher than expected if Cl ions were passively distributed. Active Cl− uptake mechanisms included a bumetanide-sensitive Na+-K+-2Cl− cotransporter and paired DIDS-sensitive Cl−/HCO3− and EIPA-sensitive Na+/H+ exchangers that correlated with expression of NKCC1, AE2, and NHE1 transcripts, respectively. Intracellular Ca2+ stimulated a niflumic acid-sensitive Cl− current with properties similar to the Ca2+-gated Cl channel BEST2. In addition, intracellular Ca2+ stimulated a paxilline-sensitive and voltage-dependent, large-conductance K channel and a clotrimazole-sensitive, intermediate-conductance K channel, consistent with the detection of transcripts for KCNMA1 and KCNN4, respectively. Our results demonstrate that the ion transport mechanisms in human parotid glands are equivalent to those in the mouse, confirming that animal models provide valuable systems for testing therapies to prevent salivary gland dysfunction.

Maxi K+ channel in apical membrane of vestibular dark cells

1992 ◽  
Vol 262 (6) ◽  
pp. C1430-C1436 ◽  
Author(s):  
S. Takeuchi ◽  
D. C. Marcus ◽  
P. Wangemann
Keyword(s):  
Apical Membrane ◽  
K Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Dark Cells ◽  
Voltage Dependent ◽  
Cytosolic Side ◽  
Selective Channel ◽  
Extracellular Side ◽  
Vestibular Dark Cells

Recordings were made on excised apical membrane patches from vestibular dark cells from the semicircular canal of gerbils to determine if ion channels could be involved in the process of K+ secretion. Both nonselective cation channels [Am. J. Physiol. 262 (Cell Physiol. 31): C1430-C1436, 1992] and K(+)-selective channels were found. The K+ channels occurred in only 0.7% of the patches. In symmetrical 145 mM KCl solutions, the current-voltage (I-V) relation of the K(+)-selective channel was linear, indicating the absence of rectification, and the conductance was 240 +/- 8 pS (n = 8). The Goldman-Hodgkin-Katz equation for current carried solely by K+ could be fitted to the I-V relation in asymmetrical K+ and Na+ solutions and yielded a K+ permeability of 5.78 x 10(-13) cm3/s (n = 12). The channel was shown to be impermeable to Li+, NH4+, N-methyl-D-glucamine, and Cl-. Channel activity increased with depolarization and with increasing free [Ca2+]; for voltages between +40 and -60 mV, the strongest regulation occurred in the range 10(-6) to 10(-5) M Ca2+. Tetraethylammonium (2 x 10(-2) M) had from the cytosolic side no effect on the open probability (Po) but completely inhibited activity from the extracellular side. Po was reduced by Ba2+ (5 x 10(-3) M), verapamil (10(-4) M), quinine (10(-4) M), and quinidine (10(-4) and 10(-3) M), while lidocaine (5 x 10(-3) M) had no measurable effect on Po but decreased the amplitude. Rb+ and Cs+ were either poorly permeable or partially blocked the channel in a voltage-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells

1997 ◽  
Vol 272 (4) ◽  
pp. C1271-C1278 ◽  
Author(s):  
W. Peng ◽  
J. R. Hoidal ◽  
S. V. Karwande ◽  
I. S. Farrukh
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Chronic Hypoxia ◽  
Muscle Cells ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Delayed Rectifier ◽  
Voltage Dependent ◽  
Dependent Protein ◽  
Pulmonary Arterial

We investigated the effects of chronic hypoxia on the major outward K+ currents in early cultured human main pulmonary arterial smooth muscle cells (HPSMC). Unitary currents were measured from inside-out, outside-out, and cell-attached patches of HPSMC. Chronic hypoxia depolarized resting membrane potential (Em) and reduced the activity of a charybdotoxin (CTX)- and iberiotoxin-sensitive, Ca2+-dependent K+ channel (KCa). The 4-aminopyridine-sensitive and CTX-insensitive channel or the delayed rectifier K+ channel was unaffected by chronic hypoxia. Chronic hypoxia caused a +33- to +53-mV right shift in voltage-dependent activation of K(Ca) and a decrease in K(Ca) activity at all cytosolic Ca2+ concentrations ([Ca2+]i) in the range of 0.1-10 microM. Thus the hypoxia-induced decrease in K(Ca) activity was most likely due to a decrease in K(Ca) sensitivity to Em and [Ca2+]i. Chronic hypoxia reduced the ability of nitric oxide (NO.) and guanosine 3',5'-cyclic monophosphate (cGMP) to activate K(Ca). The cGMP-dependent protein kinase-induced activation of K(Ca) was also significantly inhibited by chronic hypoxia. In addition, inhibiting channel dephosphorylation with calyculin A caused significantly less increase in K(Ca) activity in membrane patches excised from chronically hypoxic HPSMC compared with normoxic controls. This suggests that the mechanism by which hypoxia modulates NO.-induced K(Ca) activation is by decreasing the NO./cGMP-mediated phosphorylation of the channel.

scholarly journals Novel Gating Mechanism of Polyamine Block in the Strong Inward Rectifier K Channel Kir2.1

1999 ◽  
Vol 113 (4) ◽  
pp. 555-564 ◽  
Author(s):  
Jong-Kook Lee ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Open Channel ◽  
Cell Types ◽  
Inward Rectification ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Channel Block ◽  
Open Channel Block ◽  
Gating Mechanism ◽  
Voltage Dependent ◽  
Polyamine Toxins

Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.

scholarly journals Allosteric Gating of a Large Conductance Ca-activated K+ Channel

1997 ◽  
Vol 110 (3) ◽  
pp. 257-281 ◽  
Author(s):  
D.H. Cox ◽  
J. Cui ◽  
R.W. Aldrich
Keyword(s):  
Steady State ◽  
General Scheme ◽  
Kinetic Scheme ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Kinetic Properties ◽  
Allosteric Proteins ◽  
Channel Opening ◽  
Voltage Dependent

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca2+. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca2+ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca2+ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca2+ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.

scholarly journals The topology of the ER-resident phospholipid methyltransferase Opi3 of Saccharomyces cerevisiae is consistent with in trans catalysis

2020 ◽  
Vol 295 (8) ◽  
pp. 2473-2482 ◽  
Author(s):  
Grzegorz Pawlik ◽  
Mike F. Renne ◽  
Matthijs A. Kol ◽  
Anton I. P. M. de Kroon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Membrane Topology ◽  
Site Directed Mutagenesis ◽  
Membrane Contact Sites ◽  
Substituted Cysteine Accessibility ◽  
Cytosolic Side ◽  
In Trans ◽  
Membrane Contact ◽  
Topology Prediction

Phospholipid N-methyltransferases (PLMTs) synthesize phosphatidylcholine by methylating phosphatidylethanolamine using S-adenosylmethionine as a methyl donor. Eukaryotic PLMTs are integral membrane enzymes located in the endoplasmic reticulum (ER). Recently Opi3, a PLMT of the yeast Saccharomyces cerevisiae was proposed to perform in trans catalysis, i.e. while localized in the ER, Opi3 would methylate lipid substrates located in the plasma membrane at membrane contact sites. Here, we tested whether the Opi3 active site is located at the cytosolic side of the ER membrane, which is a prerequisite for in trans catalysis. The membrane topology of Opi3 (and its human counterpart, phosphatidylethanolamine N-methyltransferase, expressed in yeast) was addressed by topology prediction algorithms and by the substituted cysteine accessibility method. The results of these analyses indicated that Opi3 (as well as phosphatidylethanolamine N-methyltransferase) has an N-out C-in topology and contains four transmembrane domains, with the fourth forming a re-entrant loop. On the basis of the sequence conservation between the C-terminal half of Opi3 and isoprenyl cysteine carboxyl methyltransferases with a solved crystal structure, we identified amino acids critical for Opi3 activity by site-directed mutagenesis. Modeling of the structure of the C-terminal part of Opi3 was consistent with the topology obtained by the substituted cysteine accessibility method and revealed that the active site faces the cytosol. In conclusion, the location of the Opi3 active site identified here is consistent with the proposed mechanism of in trans catalysis, as well as with conventional catalysis in cis.

scholarly journals Gating Properties Conferred on Bk Channels by the β3b Auxiliary Subunit in the Absence of Its Nh2- and Cooh Termini

2001 ◽  
Vol 117 (6) ◽  
pp. 607-628 ◽  
Author(s):  
Xu-Hui Zeng ◽  
J.-P. Ding ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Outward Current ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Channel Current ◽  
Leftward Shift ◽  
Auxiliary Subunit ◽  
Voltage Dependent ◽  
Primary Determinant

Both β1 and β2 auxiliary subunits of the BK-type K+ channel family profoundly regulate the apparent Ca2+ sensitivity of BK-type Ca2+-activated K+ channels. Each produces a pronounced leftward shift in the voltage of half-activation (V0.5) at a given Ca2+ concentration, particularly at Ca2+ above 1 μM. In contrast, the rapidly inactivating β3b auxiliary produces a leftward shift in activation at Ca2+ below 1 μM. In the companion work (Lingle, C.J., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. J. Gen. Physiol. 117:583–605, this issue), we have shown that some of the apparent β3b-mediated shift in activation at low Ca2+ arises from rapid unblocking of inactivated channels, unlike the actions of the β1 and β2 subunits. Here, we compare effects of the β3b subunit that arise from inactivation, per se, versus those that may arise from other functional effects of the subunit. In particular, we examine gating properties of the β3b subunit and compare it to β3b constructs lacking either the NH2- or COOH terminus or both. The results demonstrate that, although the NH2 terminus appears to be the primary determinant of the β3b-mediated shift in V0.5 at low Ca2+, removal of the NH2 terminus reveals two other interesting aspects of the action of the β3b subunit. First, the conductance-voltage curves for activation of channels containing the β3b subunit are best described by a double Boltzmann shape, which is proposed to arise from two independent voltage-dependent activation steps. Second, the presence of the β3b subunit results in channels that exhibit an anomalous instantaneous outward current rectification that is correlated with a voltage dependence in the time-averaged single-channel current. The two effects appear to be unrelated, but indicative of the variety of ways that interactions between β and α subunits can affect BK channel function. The COOH terminus of the β3b subunit produces no discernible functional effects.

scholarly journals The liverwort Marchantia polymorpha operates a depolarization-activated Slowpoke (SLO) K+ channel that recognises pH changes in the environment

2021 ◽  
Author(s):  
Frances C. Sussmilch ◽  
Jennifer Boehm ◽  
Guido Gessner ◽  
Tobias Maierhofer ◽  
Thomas D. Mueller ◽  
...  
Keyword(s):  
Critical Role ◽  
Bk Channels ◽  
Bk Channel ◽  
K Channel ◽  
Marchantia Polymorpha ◽  
Terrestrial Environment ◽  
Ph Changes ◽  
Kv Channels ◽  
Voltage Dependent ◽  
Genes Encoding

Voltage-dependent ion channels are a prerequisite for cellular excitability and electrical communication - important traits for multicellular organisms to thrive in a changeable terrestrial environment. Based on their presence in extant embryophytes and closely-related green algae, the first plants to survive on land likely possessed genes encoding channels with homology to large-conductance calcium-activated K+ channels (BK channels from the Slo family) in addition to primary voltage-gated potassium channels from the plant VG-type family (Shaker or Kv channels). While the function and gating of Shaker channels has been characterised in flowering plants, so far knowledge of BK channels has been limited to animal models. In humans, BK-mediated K+ efflux has a critical role in sperm motility and membrane polarisation to enable fertilisation. In the liverwort Marchantia polymorpha, the MpBK2a channel gene is most highly expressed in male reproductive tissue, suggesting that these channels may function in sexual reproduction. We characterised MpBK2a channels and found them to be strongly K+-selective, outward-rectifying, 80-pS channels capable of repolarising the membrane after stimulus-dependent depolarisation. In contrast to its animal counterpart, MpBK2a is insensitive to cytoplasmic Ca2+ variations but effectively gated by pH changes. Given that this plant BK channel is active even in the presence of trace amounts of external K+ and at low pH, the liverwort channel could have stabilised the membrane potential under stressful pre-historic conditions including nutrient-depleted and acid environments as early plant pioneers conquered land.

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