Marie Kanazirska

Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway

Mucolipin‐1 (MLN1) is a membrane protein with homology to the transient receptor potential channels and other non‐selective cation channels. It is encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), an autosomal recessive disease that is characterized by severe abnormalities in neurological development as well as by ophthalmologic defects. At the cellular level, MLIV is associated with abnormal lysosomal sorting and trafficking. Here we identify the channel function of human MLN1 and characterize its properties. MLN1 represents a novel Ca2+‐permeable channel that is transiently modulated by changes in [Ca2+]. It is also permeable to Na+ and K+. Large unitary conductances were measured in the presence of these cations. With its Ca2+ permeability and modulation by [Ca2+], MLN1 could play a major role in Ca2+ transport regulating lysosomal exocytosis and potentially other phenomena related to the trafficking of late endosomes and lysosomes.

Amyloid-? proteins activate Ca2+-permeable channels through calcium-sensing receptors

The amyloid‐beta peptides (Aβ) are produced in excess in Alzheimers disease (AD) and may contribute to neuronal dysfunction and degeneration. This study provides strong evidence for a novel cellular target for the actions of Aβ, the phospholipase C‐coupled, extracellular Ca2+‐sensing receptor (CaR). We demonstrate that Aβs produce a CaR‐mediated activation of a Ca2+‐permeable, nonselective cation channel (NCC), probably via elevation in cytosolic Ca2+ (Cai), in cultured hippocampal pyramidal neurons from normal rats and from wild type mice but not those from mice with targeted disruption of the CaR gene (CaR −/−). Aβs also activate NCC in CaR‐transfected but not in nontransfected human embryonic kidney (HEK293) cells. Thus aggregates of Aβ deposited on hippocampal neurons in AD could inappropriately activate the CaR, stimulating Ca2+‐permeable channels and causing sustained elevation of Cai with resultant neuronal dysfunction.

Assessment of frequency-dependent alterations in the level of extracellular Ca2+ in the synaptic cleft.

The synaptic cleft may be represented as a very thin disk of extracellular fluid. It is possible that at high stimulation frequencies the interval between pulses would be insufficient for diffusion of Ca2+ from the periphery of the cleft to replace extracellular Ca2+ depleted at the center of the cleft as a result of activation of postsynaptic, Ca2(+)-permeable channels. Computer modeling was employed to assess the impact of activation of glutamate receptor channels (GRCs) in the postsynaptic membrane on the level of extracellular Ca2+ within the synaptic cleft. The model includes calcium influx from the synaptic cleft into the postsynaptic compartment through GRC and calcium efflux through calcium pumps and Na/Ca exchangers. Concentrations of extracellular Ca2+ inside the cleft are estimated by using a compartmental model incorporating flux across the postsynaptic membrane and radial diffusion from the edges of the cleft. The simulations suggest that substantial extracellular Ca2+ depletion can occur in the clefts during activation of GRCs, particularly at high stimulation frequencies used to induce long-term potentiation (LTP). Only minimal transitory changes in extracellular Ca2+ are observed at low frequencies. These frequency-dependent alterations in extracellular Ca2+ dynamics are a direct reflection of the activity of GRCs and could be involved in the modulation of presynaptic function via a retrograde messenger mechanism, if there are extracellular Ca2+ sensors on the presynaptic membranes. The recently cloned extracellular Ca2(+)-sensing receptors that are known to be present in nerve terminals in hippocampus and other areas of the brain could potentially play such a role.

Cao-sensing receptor (CaR)-mediated activation of K+ channels is blunted in CaR gene-deficient mouse neurons

THE extracellular Ca2+ (Cao)-sensing receptor (CaR) is expressed in hippocampus and other brain regions, suggesting that it could mediate some of the well recognized but poorly understood direct actions of Cao on neuronal function. This study presents evidence that the CaR is functionally coupled to Ca2+-activated K+ channels. The effects of CaR agonists on these channels in neurons from wild type (WT) and CaR-deficient (CaR -/-) mice were compared. Neomycin (100 mM) and elevation of Cao from 0.5 to 3 mM significantly increased the probability of channel opening (Po) in neurons from WT but not in those from CaR -/- mice. Thus the CaR activates neuronal K+ channels and could potentially inhibit neuronal excitability and neurotransmission via membrane repolarization.

Deficient Cation Channel Regulation in Neurons From Mice With Targeted Disruption of the Extracellular Ca2+-Sensing Receptor Gene

This study presents evidence that a receptor sensitive to the concentration of extracellular Ca2+ (Ca[2+]o) (CaR) is functionally coupled to ion channels involved in modulation of neuronal excitability. This receptor is expressed in hippocampus and other brain regions, suggesting that it could mediate some of the well-recognized but poorly understood direct actions of extracellular Ca2+ (Ca[2+]o) on neuronal function. The effects of polycationic CaR agonists on the activity of a nonselective cation channel (NCC) in cultured hippocampal neurons from wild-type mice and from mice homozygous for targeted disruption of the CaR gene (CaR -/-) were compared in this study. The CaR agonists, neomycin (100 microM), spermine (300 microM), and elevation of Ca(2+)o from 0.75 to 3 mM, significantly increased the probability of channel opening (Po) in wild-type neurons. None of these agents, however, produced any effect on Po in neurons from mice lacking the CaR. The same NCC, however, could be activated by thapsigargin in neurons from both wild-type mice and CaR-deficient mice, most likely through an associated increase in the cytosolic free calcium concentration (Ca[i]). Thus the CaR regulates the activity of Ca2+-permeable NCC in hippocampal neurons and could potentially modulate key neuronal functions, including neurotransmission and neuronal excitability, via membrane depolarization.

Beneficial effects of lysosome-modulating and other pharmacological and nanocarrier agents on amyloid-beta-treated cells.

The progression of Alzheimers disease (AD) is accompanied by disturbances of the endosome/lysosome (EL) system and there is accumulation of peptides of the AD-associated amyloid beta (Abeta) type in EL vesicles of affected neurons. EL modulating agents partially ameliorate the Abeta-mediated cell abnormalities. However, no extensive studies on the potential pharmaceutical applications of combinations of such agents and their synergistic effects have been performed. This study shows the beneficial anti-amyloid effects of several combinations of lysosomal modulators and other pharmacological and new nanobiotechnological agents. Some agents potentiated each others action and some of them facilitated the anti-amyloid actions of memantine, a modifier of Ca2+-permeable channels involved in AD and one of the few drugs used for treatment of AD. Another compound used in nanobiotechnology ameliorated as a nanocarrier the beneficial effects of some of these potential pharmaceutical agents. They may be considered as additional drugs to improve the efficacy of the therapeutic approaches for AD and related neurodegenerative disorders.