K(+) channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K(+) channels, pH-regulated K2P channels and the ...transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K2P and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K(+) homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K2P channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K2P channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K2P and Kir K(+)-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge.
Sea louse ectoparasitosis is a major threat to fish aquaculture. Avermectins such as ivermectin and emamectin have been effectively used against sea louse infestation, but the emergence of resistance ...has limited their use. A better understanding of the molecular targets of avermectins is essential to the development of novel treatment strategies or new, more effective drugs. Avermectins are known to act by inhibiting neurotransmission through allosteric activation of glutamate-gated chloride channels (GluCls). We have investigated the GluCl subunit present in Caligus rogercresseyi, a sea louse affecting aquaculture in the Southern hemisphere. We identify four new subunits, CrGluCl-B to CrGluCl-E, and characterise them functionally. CrGluCl-A (previously reported as CrGluClα), CrGluCl-B and CrGluCl-C all function as glutamate channel receptors with different sensitivities to the agonist, but in contrast to subunit -A and -C, CrGluCl-B is not activated by ivermectin but is rather antagonised by the drug. CrGluCl-D channel appears active in the absence of any stimulation by glutamate or ivermectin and CrGluCl-E does not exhibit any activity. Notably, the expression of CrGluCl-B with either -A or -C subunits gives rise to receptors unresponsive to ivermectin and showing altered response to glutamate, suggesting that coexpression has led to the preferential formation of heteromers to which the presence of CrGluCl-B confers the property of ivermectin-activation refractoriness. Furthermore, there was evidence for heteromer formation with novel properties only when coexpressing pairs E/C and D/B CrGluCl subtypes. Site-directed mutagenesis shows that three transmembrane domain residues contribute to the lack of activation by ivermectin, most crucially Gln 15' in M2, with mutation Q15'T (the residue present in ivermectin-activated subunits A and C) conferring ivermectin activation to CrGluCl-B. The differential response to avermectin of these Caligus rogercresseyi GluClsubunits, which are highly conserved in the Northern hemisphere sea louse Lepeophtheirus salmonis, could have an influence on the response of these parasites to treatment with macrocyclic lactones. They could serve as molecular markers to assess susceptibility to existing treatments and might be useful molecular targets in the search for novel antiparasitic drugs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Key points
Kir7.1 K+ channel expressed in retinal pigment epithelium is mutated in inherited retinal degeneration diseases.
We study Kir7.1 in heterologous expression to test the hypothesis that ...pathological R162 mutation to neutral amino acids results in loss of a crucial site that binds PI(4,5)P2.
Although R162W mutation inactivates Kir7.1, changes to smaller volume (e.g. Gln) amino acids are tolerated or even enhance function (Ala or Cys).
Chemical modification of Kir7.1‐R162C confirms that large residues of the size of Trp are incompatible with normal channel function even if positively charged.
In addition to R162, K164 (and possibly K159) forms a binding site for the phosphoinositide and is essential for channel activity.
R162 substitution with a large, neutral side chain like Trp exerts a dominant negative effect on Kir7.1 activity such that less than one fifth of the full activity is expected in a cell expressing the same amount of mutant and wild‐type channels.
Mutations in the Kir7.1 K+ channel, highly expressed in retinal pigment epithelium, have been linked to inherited retinal degeneration diseases. Examples are mutations changing Arg 162 to Trp in snowflake vitreoretinal degeneration (SVD) and Gln in retinitis pigmentosa. R162 is believed to be part of a site that binds PI(4,5)P2 and stabilises the open state. We have tested the hypothesis that R162 mutation to neutral amino acids will result in the loss of this crucial interaction to the detriment of channel function. Our findings indicate that although R612W mutation inactivates Kir7.1, changes to smaller volume (e.g. Gln) amino acids are tolerated or even enhance function (Ala or Cys). Cys chemical modification of Kir7.1‐R162C confirms that large residues of the size of Trp are incompatible with normal channel function even if positively charged. Experiments titrating the levels of plasma membrane PI(4,5)P2 with voltage‐dependent phosphatase DrVSP reveal that, in addition to R162, K164 (and possibly K159) forms a binding site for the phosphoinositide and ensures channel activity. Finally, the use of a concatemeric approach shows that substitution of R162 with a large, neutral side chain mimicking a Trp residue exerts a dominant negative effect on Kir7.1 activity such that less than one fifth of the full activity is expected in heterozygous cells carrying the SVD mutation. Our results suggest that if mutations in the human KCNJ13 gene resulting in the neutralisation of R162 and Kir7.1 malfunction led to retinal degeneration diseases, their severity might depend on the nature of the side chain of the replacing amino acid.
Key points
Kir7.1 K+ channel expressed in retinal pigment epithelium is mutated in inherited retinal degeneration diseases.
We study Kir7.1 in heterologous expression to test the hypothesis that pathological R162 mutation to neutral amino acids results in loss of a crucial site that binds PI(4,5)P2.
Although R162W mutation inactivates Kir7.1, changes to smaller volume (e.g. Gln) amino acids are tolerated or even enhance function (Ala or Cys).
Chemical modification of Kir7.1‐R162C confirms that large residues of the size of Trp are incompatible with normal channel function even if positively charged.
In addition to R162, K164 (and possibly K159) forms a binding site for the phosphoinositide and is essential for channel activity.
R162 substitution with a large, neutral side chain like Trp exerts a dominant negative effect on Kir7.1 activity such that less than one fifth of the full activity is expected in a cell expressing the same amount of mutant and wild‐type channels.
K2P K(+) channels with two pore domains in tandem associate as dimers to produce so-called background conductances that are regulated by a variety of stimuli. Whereas gating in K2P channels has been ...poorly understood, recent developments have provided important clues regarding the gating mechanism for this family of proteins. Two modes of gating present in other K(+) channels have been considered. The first is the so-called activation gating that occurs by bundle crossing and the splaying apart of pore-lining helices commanding ion passage. The second mode involves a change in conformation at the selectivity filter (SF), which impedes ion flow at this narrow portion of the conduction pathway and accounts for extracellular pH modulation of several K2P channels. Although some evidence supports the existence of an activation gate in K2P channels, recent results suggest that perhaps all stimuli, even those sensed at a distant location in the protein, are also mediated by SF gating. Recently resolved crystal structures of K2P channels in conductive and nonconductive conformations revealed that the nonconductive state is reached by blockade by a lipid acyl chain that gains access to the channel cavity through intramembrane fenestrations. Here we discuss whether this novel type of gating, proposed so far only for membrane tension gating, might mediate gating in response to other stimuli or whether SF gating is the only type of opening/closing mechanism present in K2P channels.
K
+
channels fulfill roles spanning from the control of excitability to the regulation of transepithelial transport. Here we review two groups of K
+
channels, pH-regulated K
2P
channels and the ...transport group of Kir channels. After considering advances in the molecular aspects of their gating based on structural and functional studies, we examine their participation in certain chosen physiological and pathophysiological scenarios. Crystal structures of K
2P
and Kir channels reveal rather unique features with important consequences for the gating mechanisms. Important tasks of these channels are discussed in kidney physiology and disease, K
+
homeostasis in the brain by Kir channel-equipped glia, and central functions in the hearing mechanism in the inner ear and in acid secretion by parietal cells in the stomach. K
2P
channels fulfill a crucial part in central chemoreception probably by virtue of their pH sensitivity and are central to adrenal secretion of aldosterone. Finally, some unorthodox behaviors of the selectivity filters of K
2P
channels might explain their normal and pathological functions. Although a great deal has been learned about structure, molecular details of gating, and physiological functions of K
2P
and Kir K
+
-transport channels, this has been only scratching at the surface. More molecular and animal studies are clearly needed to deepen our knowledge.
The present study explored the mediation role of past exercise adherence, self-reported frequency and intentions in the association between past experience and future exercise adherence. In total, ...431 exercisers (female = 216; male = 215) aged 18 and 64 years, engaged in fitness activities such as group fitness classes and resistance training, were included in the analysis. Serial mediation procedures were employed to examine the direct, indirect, and total indirect effects among variables. The predictor variable and all mediators displayed a positive and significant association with future six-month adherence. Past six-month exercise adherence displayed the most significant association with future six-month adherence. The sequential indirect path from exercise experience → past six-months adherence → self-reported frequency → intentions future six-months adherence displayed a positive and significant effect (β = .19 CI95% = .09, .31), presenting a partial mediation effect. Past behaviour is the most significant predictor of future adherence, and thus interventions should be based on promoting consistent exercise frequency. Professionals working in the fitness centre context can identify possible dropouts based on their past behaviour and intentions to be physically active in the future.
Neurodegenerative disorders such as Parkinson and Alzheimer disease cause motor and cognitive dysfunction and belong to a heterogeneous group of common and disabling disorders. Although the complex ...molecular pathophysiology of neurodegeneration is largely unknown, major advances have been achieved by elucidating the genetic defects underlying mendelian forms of these diseases. This has led to the discovery of common pathophysiological pathways such as enhanced oxidative stress, protein misfolding and aggregation and dysfunction of the ubiquitin-proteasome system. Here, we describe loss-of-function mutations in a previously uncharacterized, predominantly neuronal P-type ATPase gene, ATP13A2, underlying an autosomal recessive form of early-onset parkinsonism with pyramidal degeneration and dementia (PARK9, Kufor-Rakeb syndrome). Whereas the wild-type protein was located in the lysosome of transiently transfected cells, the unstable truncated mutants were retained in the endoplasmic reticulum and degraded by the proteasome. Our findings link a class of proteins with unknown function and substrate specificity to the protein networks implicated in neurodegeneration and parkinsonism.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
K
channel Kir7.1 expressed at the apical membrane of the retinal pigment epithelium (RPE) plays an essential role in retinal function. An isoleucine-to-threonine mutation at position 120 of the ...protein is responsible for blindness-causing vitreo-retinal dystrophy. We have studied the molecular mechanism of action of Kir7.1-I120T in vitro by heterologous expression and in vivo in CRISPR-generated knockin mice. Full-size Kir7.1-I120T reaches the plasma membrane but lacks any activity. Analysis of Kir7.1 and the I120T mutant in mixed transfection experiments, and that of tandem tetrameric constructs made by combining wild type (WT) and mutant protomers, leads us to conclude that they do not form heterotetramers in vitro. Homozygous I120T/I120T mice show cleft palate and tracheomalacia and do not survive beyond P0, whereas heterozygous WT/I120T develop normally. Membrane conductance of RPE cells isolated from WT/WT and heterozygous WT/I120T mice is dominated by Kir7.1 current. Using Rb
as a charge carrier, we demonstrate that the Kir7.1 current of WT/I120T RPE cells corresponds to approximately 50% of that in cells from WT/WT animals, in direct proportion to WT gene dosage. This suggests a lack of compensatory effects or interference from the mutated allele product, an interpretation consistent with results obtained using WT/- hemizygous mouse. Electroretinography and behavioral tests also show normal vision in WT/I120T animals. The hypomorphic ion channel phenotype of heterozygous Kir7.1-I120T mutants is therefore compatible with normal development and retinal function. The lack of detrimental effect of this degree of functional deficit might explain the recessive nature of Kir7.1 mutations causing human eye disease.
Human retinal pigment epithelium K
channel Kir7.1 is affected by generally recessive mutations leading to blindness. We investigate one such mutation, isoleucine-to-threonine at position 120, both in vitro and in vivo in knockin mice. The mutated channel is inactive and in heterozygosis gives a hypomorphic phenotype with normal retinal function. Mutant channels do not interfere with wild-type Kir7.1 channels which are expressed concomitantly without hindrance, providing an explanation for the recessive nature of the disease.
K
channels are important in intestinal epithelium as they ensure the ionic homeostasis and electrical potential of epithelial cells during anion and fluid secretion. Intestinal epithelium ...cAMP-activated anion secretion depends on the activity of the (also cAMP dependent) KCNQ1-KCNE3 K
channel, but the secretory process survives after genetic inactivation of the K
channel in the mouse. Here we use double mutant mice to investigate which alternative K
channels come into action to compensate for the absence of KCNQ1-KCNE3 K
channels. Our data establish that whilst Ca
-activated K
3.1 channels are not involved, K
two-pore domain TASK-2 K
channels are major players providing an alternative conductance to sustain the intestinal secretory process. Work with double mutant mice lacking both TASK-2 and KCNQ1-KCNE3 channels nevertheless points to yet-unidentified K
channels that contribute to the robustness of the cAMP-activated anion secretion process.
Anion and fluid secretion across the intestinal epithelium, a process altered in cystic fibrosis and secretory diarrhoea, is mediated by cAMP-activated CFTR Cl
channels and requires the simultaneous activity of basolateral K
channels to maintain cellular ionic homeostasis and membrane potential. This function is fulfilled by the cAMP-activated K
channel formed by the association of pore-forming KCNQ1 with its obligatory KCNE3 β-subunit. Studies using mice show sizeable cAMP-activated intestinal anion secretion in the absence of either KCNQ1 or KCNE3 suggesting that an alternative K
conductance must compensate for the loss of KCNQ1-KCNE3 activity. We used double mutant mouse and pharmacological approaches to identify such a conductance. Ca
-dependent anion secretion can also be supported by Ca
-dependent K
3.1 channels after independent CFTR activation, but cAMP-dependent anion secretion is not further decreased in the combined absence of K
3.1 and KCNQ1-KCNE3 K
channel activity. We show that the K
K
channel TASK-2 is expressed in the epithelium of the small and large intestine. Tetrapentylammonium, a TASK-2 inhibitor, abolishes anion secretory current remaining in the absence of KCNQ1-KCNE3 activity. A double mutant mouse lacking both KCNQ1-KCNE3 and TASK-2 showed a much reduced cAMP-mediated anion secretion compared to that observed in the single KCNQ1-KCNE3 deficient mouse. We conclude that KCNQ1-KCNE3 and TASK-2 play major roles in the intestinal anion and fluid secretory phenotype. The persistence of an, admittedly reduced, secretory activity in the absence of these two conductances suggests that further additional K
channel(s) as yet unidentified contribute to the robustness of the intestinal anion secretory process.
Kir7.1 is an inwardly rectifying K+ channel present in epithelia where it shares membrane localization with the Na+/K+-pump. In the present communication we report the presence of a novel splice ...variant of Kir7.1 in mouse tissues including kidney, lung, choroid plexus and retinal pigment epithelium (RPE). The variant named mKir7.1-SV2 lacks most of the C-terminus domain but is predicted to have the two transmembrane domains and permeation pathway unaffected. Similarly truncated predicted proteins, Kir7.1-R166X and Kir7.1-Q219X, would arise from mutations associated with Leber Congenital Amaurosis, a rare recessive hereditary retinal disease that results in vision loss at early age. We found that mKir7.1-SV2 and the pathological variants do not produce any channel activity when expressed alone in HEK-293 cells due to their scarce presence in the plasma membrane. Simultaneous expression with the full length Kir7.1 however leads to a reduction in activity of the wild-type channel that might be due to partial proteasome degradation of WT-mutant channel heteromers.
•A novel splice variant SV2 of Kir7.1 potassium channel is described.•SV2 codes a truncated peptide as do nonsense mutants causing human retinal disease.•Neither SV2 nor the pathological mutations are functional.•Truncated peptides appear to promote proteasomal degradation of wildtype channels.
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