Divalent Cation Permeability Research Articles

Claudins and nephrolithiasis.

The greatest risk factor for kidney stone formation is increased urinary calcium excretion. Most filtered calcium is reabsorbed from the proximal tubule and the thick ascending limb (TAL) of Henle's loop via a paracellular pathway. Claudins are tight junction proteins that confer the permeability properties of an epithelium. We review the contribution of renal claudins to nephron calcium permeability and how perturbations in these pathways cause alterations in tubular calcium transport, hypercalciuria, nephrocalcinosis, or nephrolithiasis. Claudin-16 and Claudin-19 form a complex with claudin-3 enabling divalent cation permeability in the TAL. Claudin-14 interacts with claudin-16 to attenuate calcium permeability through this pore. Intronic mutations in claudin-14 increase expression causing hypercalciuria and kidney stones. A different type of TAL tight junction pore is composed of claudin-10b, which does not preferentially permeate calcium. Deletion of claudin-10b results in increased expression of the claudin-16/claudin-19 complex expressed in the medullary TAL and nephrocalcinosis. Alterations to claudins expressed in the TAL tight junction greatly affects calcium homeostasis as highlighted by point mutations in claudin-16 or claudin-19 causing FHHNC or gain of function mutations in claudin-14 causing kidney stones.

Axial and cellular heterogeneity in electrolyte transport pathways along the thick ascending limb.

The thick ascending limb (TAL) extends from the border of the inner medulla to the renal cortex, thus ascending through regions with wide differences in tissue solute and electrolyte concentrations. Structural and functional differences between TAL cells in the medulla (mTAL) and the cortex (cTAL) would therefore be useful to adapt TAL transport function to a changing external fluid composition. While mechanisms common to all TAL cells play a central role in the reclamation of about 25% of the NaCl filtered by the kidney, morphological features, Na+ / K+ -ATPase activity, NKCC2 splicing and phosphorylation do vary between segments and cells. The TAL contributes to K+ homeostasis and TAL cells with high or low basolateral K+ conductances have been identified which may be involved in K+ reabsorption and secretion respectively. Although transport rates for HCO3- do not differ between mTAL and cTAL, divergent axial and cellular expression of H+ transport proteins in TAL have been documented. The reabsorption of the divalent cations Ca2+ and Mg2+ is highest in cTAL and paralleled by differences in divalent cation permeability and the expression of select claudins. Morphologically, two cell types with different cell surface phenotypes have been described that still need to be linked to specific functional characteristics. The unique external environment and its change along the longitudinal axis require an axial functional heterogeneity for the TAL to optimally participate in conserving electrolyte homeostasis. Despite substantial progress in understanding TAL function, there are still considerable knowledge gaps that are just beginning to become bridged.

Induction of divalent cation permeability by heterologous expression of a voltage sensor domain

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K+ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.

All atom NMDA receptor transmembrane domain model development and simulations in lipid bilayers and water.

N-methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family that mediate excitatory synaptic transmission in the central nervous system. The channels of NMDARs are permeable to Ca2+ but blocked by Mg2+, distinctive properties that underlie essential brain processes such as induction of synaptic plasticity. However, due to limited structural information about the NMDAR transmembrane ion channel forming domain, the mechanism of divalent cation permeation and block is understood poorly. In this paper we developed an atomistic model of the transmembrane domain (TMD) of NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors). The model was generated using (a) a homology model based on the structure of the NaK channel and a partially resolved structure of an AMPA receptor (AMPAR), and (b) a partially resolved X-ray structure of GluN1/2B NMDARs. Refinement and extensive Molecular Dynamics (MD) simulations of the NMDAR TMD model were performed in explicit lipid bilayer membrane and water. Targeted MD with simulated annealing was introduced to promote structure refinement. Putative positions of the Mg2+ and Ca2+ ions in the ion channel divalent cation binding site are proposed. Differences in the structural and dynamic behavior of the channel protein in the presence of Mg2+ or Ca2+ are analyzed. NMDAR protein conformational flexibility was similar with no ion bound to the divalent cation binding site and with Ca2+ bound, whereas Mg2+ binding reduced protein fluctuations. While bound at the binding site both ions retained their preferred ligand coordination numbers: 6 for Mg2+, and 7–8 for Ca2+. Four asparagine side chain oxygens, a back-bone oxygen, and a water molecule participated in binding a Mg2+ ion. The Ca2+ ion first coordination shell ligands typically included four to five side-chain oxygen atoms of the binding site asparagine residues, two water molecules and zero to two backbone oxygens of the GluN2B subunits. These results demonstrate the importance of high-resolution channel structures for elucidation of mechanisms of NMDAR permeation and block.

Will it Permeate? Predicting Ion Channel Ion Selectivity, Permittivity and Block Mechanisms

Modeling permeation of ions through an open transmembrane channel remains a difficult problem in theoretical biophysics. In most general terms a successful approach requires a faithful representation of an ion interaction with water, the channel protein, and other ions in solution, as well as a transport model for ion diffusion through the channel at a variety of applied voltages and salt concentrations. The process of ion permeation occurs over distances of hundred angstroms, and involves time-scale spanning from picoseconds to micro- or, even, milliseconds. In the last two decades a significant progress was achieved in both aspects of the problem: i) improving modeling of ion-protein interactions at short time-scales via empirical force-field employing molecular dynamics simulations, as well as ii) developing an ion-diffusion formalisms for long-time-scale ion motion at various voltages. In this talk I will describe a general hierarchical approach we developed for modeling ion permeation, in which information about ion - protein interactions can be supplied to a generalized electro-diffusion model (PNP) in the form of a Potential of Mean Force (PMF) of an ion in the channel (Mamonov et al. 2003). A generalized PNP to include a soft channel wall (SIP-PNP) (Simakov and Kurnikova, JPC2010) is a simplified model suitable for wider ion channels. Example applications, and limitations of the methods at both atomistic and continuum level of the modeling hierarchies will also be provided. I will also discuss our recent progress in modeling divalent cation permeation and blocking of a model pore of the NMDA receptor, which presents additional challenges to the theory of ion channel permeation (Kurnikov and Kurnikova, JPC 2015; Mesbahi et al. 2016).

Single channel measurements demonstrate the voltage dependence of permeation through N-type and L-type CaV channels

The delivery of Ca2+ into cells by CaV channels provides the trigger for many cellular actions, such as cardiac muscle contraction and neurotransmitter release. Thus, a full understanding of Ca2+ permeation through these channels is critical. Using whole-cell voltage-clamp recordings, we recently demonstrated that voltage modulates the apparent affinity of N-type (CaV2.2) channels for permeating Ca2+ and Ba2+ ions. While we took many steps to ensure the high fidelity of our recordings, problems can occur when CaV currents become large and fast, or when currents run down. Thus, we use here single channel recordings to further test the hypothesis that permeating ions interact with N-type channels in a voltage-dependent manner. We also examined L-type (CaV1.2) channels to determine if these channels also exhibit voltage-dependent permeation. Like our whole-cell data, we find that voltage modulates N-channel affinity for Ba2+ at voltages > 0 mV, but has little or no effect at voltages < 0 mV. Furthermore, we demonstrate that permeation through L-channel is also modulated by voltage. Thus, voltage-dependence may be a common feature of divalent cation permeation through CaV1 and CaV2 channels (i.e. high-voltage activated CaV channels). The voltage dependence of CaV1 channel permeation is likely a mechanism mediating sustained Ca2+ influx during the plateau phase of the cardiac action potential.

A Mechanosensitive Channel (MscS) with Multiple Conducting States Shows Stronger Selectivity for Anions in the Presence of Divalent Cations

The mechanosensitive channel of small conductance (MscS) in E. coli plays an integral role in osmoprotection[1]. The channel gates in response to an increase in membrane tension as a result of hypo-osmotic shock and acts as an ‘emergency release valve’ preventing cell lysis. Very little is known about the permeation of divalent cations through MscS. This was addressed by reconstituting MscS into azolectin liposomes (protein:lipid - 1:10,000) using the dehydration-rehydration method as previously described[2]. Single-channel recordings were performed in symmetrical 100 mM BaCl2, CaCl2, and MgCl2. At least 5 distinct conducting states were present at +50 mV pipette potential. A further increase in pipette potential to +70 mV resulted in 8 distinct conducting states which were conserved up to the highest voltage tested (+150 mV). It is not known whether these sub-conducting states are physiologically relevant. In addition to these sub-conducting states the substitution of K+ by Ba2+, Ca2+ and Mg2+ increased MscS rectification at negative pipette potentials. The rectification increased as the hydrated ionic radius of the major permeating cation increased (Mg2+ = Ca2+ = Ba2+ > Li+>Na+>K+>Rb+>Cs+). It was hypothesised that divalent cations interact and screen electronegative charges surrounding the portals of the cytoplasmic domain of MscS. If this were the case then divalent cations would increase the anion selectivity of MscS. In line with this prediction in asymmetric BaCl2 the calculated permeability ratio was PCl/PBa = ∼11.5 as opposed to PCl/PK = ∼3.5 in asymmetric KCl. The significance of the effects of divalent ions on MscS gating and ion selectivity is unknown at present, however they may give us a new insight into the global gating mechanism of MscS and importance of calcium in osmoregulation of bacterial cells.

The Permeation of Divalent Cations Through CNGA1 and CNGB1 Channels

We have analysed the permeation of divalent cations through homomeric CNGA1 and heteromeric CNGA1 and CNGB1 channels when 110 mM NaCl were in the patch pipette and 76.6 mM of CaCl2, MgCl2, SrCl2 and BaCl in the medium bathing the intracellular side of the patch. For homomeric CNGA1 channels the reversal potential Vrev was −6.5±2.5, −5.5±3.2, 21.3±7.2 and 48.6±8.5 mV and for heteromeric CNGA1 and CNGB1 channels Vrev was −1.5±0.9, 5.1±2.4, 7±3.2 and 2.3±1.2 mV for Ca2+, Mg2+, Sr2+ and Ba2+ respectively. For homomeric CNGA1 channels, the ratio of the current at +200 mV carried by Na+ and Ca2+, Mg2+, Sr2+ and Ba2+ was 0.05±0.03, 0.045±0.02,0.1±0.02 and 0.08±0.035 respectively. For heteromeric CNGA1 and CNGB1 these ratios were 0.18±0.04, 0.08±0.03, 0.21±0.05 and 0.13±0.03 respectively. At negative voltages divalent cations blocked the inward Na+ current more powerfully in homomeric than in heteromeric channels. These results show that divalent cations permeate more easily through heteromeric than homomeric CNG channels and that they block less powerfully the current carried by monovalent cations in hetoromeric channels.

Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand, and the distal C-terminal domain

L-type Ca2+ currents conducted by Cav1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg2+ (Mgi) inhibits the ionic current of Cav1.2 channels. Because Mgi is altered in ischemia and heart failure, its regulation of Cav1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mgi on voltage-dependent inactivation (VDI) of Cav1.2 channels using Na+ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca2+-dependent inactivation process. We confirmed that increased Mgi reduces peak ionic currents and increases VDI of Cav1.2 channels in ventricular myocytes and in transfected cells when measured with Na+ as permeant ion. The increased rate and extent of VDI caused by increased Mgi were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mgi to the EF-hand (KD ≈ 0.9 mM) near the resting level of Mgi in ventricular myocytes. VDI was more rapid for L-type Ca2+ currents in ventricular myocytes than for Cav1.2 channels in transfected cells. Coexpression of Cavβ2b subunits and formation of an autoinhibitory complex of truncated Cav1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Cav1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mgi binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Cav1.2 channels by Mgi, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.

A light switch controlling Ca2+‐permeable AMPA receptors in the retina

At most excitatory, glutamatergic synapses in the brain, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs) make distinct contributions to the postsynaptic response: AMPARs mediate transient conductances that are well suited to fast information transfer; NMDARs associate glutamate binding with postsynaptic depolarization to mediate a slower conductance that, unlike that of most AMPARs, includes Ca2+ influx that can trigger long-lasting changes in synaptic strength. This delegation of postsynaptic roles has been blurred recently by evidence implicating Ca2+-permeable AMPARs (CP-AMPARs) in different forms of synaptic transmission and plasticity. AMPARs are heterotetramers comprising various combinations of subunits GluR1–4, each of which is coded to express an uncharged glutamine in the pore region that permits permeation of divalent cations. In GluR2, RNA editing recodes this glutamine to a positively charged arginine, rendering GluR2-containing AMPARs Ca2+ impermeable (Dingledine et al. 1992). Many neurons, however, express AMPARs that lack GluR2 and are, consequently, Ca2+ permeable. Particularly in interneurons, CP-AMPARs often replace NMDARs as the primary source of postsynaptic Ca2+ influx. For example, Ca2+ influx through CP-AMPARs induces long-term potentiation (LTP) of excitatory synapses onto interneurons in the amygdala (Mahanty & Sah, 1998) and triggers GABA release from A17 amacrine cells in the retina (Chavez et al. 2006). CP-AMPAR channels are distinguished electrophysiolgically via voltage-dependent block by (and eventual permeation of) endogenous intracellular polyamines that confers biphasic rectification on an otherwise linear I–V relationship (Bahring et al. 1997). CP-AMPARs also are blocked by exogenous extracellular polyamines such as philanthotoxin (Washburn et al. 1997). Ca2+ permeability and polyamine sensitivity typically go hand in hand, but a paper in this issue of The Journal of Physiology reports CP-AMPARs in rat retinal interneurons that are insensitive to philanthotoxin (Osswald et al. 2007). The expression of these receptors coincides with eye opening and is delayed by rearing animals in darkness, suggesting that they may participate in a critical phase of retinal development. Functional CP-AMPARs are detected using cobalt staining and electrophysiology. Cobalt permeates CP-AMPARs and so fills cells expressing activated receptors. Following exposure to cobalt and glutamate, retinas were fixed according to a protocol that rendered cobalt-positive horizontal and amacrine cells a deep orange. Particularly strong staining was observed in AII amacrine cells, glycinergic interneurons known to express CP-AMPARs. Cobalt staining was not induced by membrane depolarization in the absence of glutamate and was unaffected by NMDAR and metabotropic glutamate receptor (mGluR) antagonists but was abolished by AMPAR antagonists, including philanthotoxin. During the third postnatal week, around the time of eye opening (∼P14), cobalt staining persisted, but philanthotoxin sensitivity disappeared in horizontal cells and was reduced in AII cells, particularly in processes ramifying in the outer portion of the inner plexiform layer (IPL), where synapses transmitting ‘OFF’ visual signals are made. AII cells, but not horizontal cells, recovered philanthotoxin sensitivity 2 weeks later. Decreased philanthotoxin sensitivity did not occur in dark-reared animals, suggesting that light-induced activity may trigger the change. Interestingly, voltage clamp recordings of spontaneous synaptic currents in AII cells also reflected the transient disappearance of philanthotoxin sensitivity. This, together with the morphological data described above, would suggest that most spontaneous activity in AIIs arises at synapses in the ‘OFF’ region of the IPL. This is a bit surprising, as most synapses onto AIIs are made by rod bipolar cells (RBCs) in the ON region, and spontaneous events in AIIs are indistinguishable from evoked quantal events from RBCs (Singer et al. 2004). The molecular mechanism underlying this peculiar developmental switch remains unknown, but the authors propose the possibility of a novel AMPAR subtype (or novel RNA editing of known subunits), a reasonable suggestion given that other glutamate receptors (e.g. mGluR6) and transporters (e.g. EAAT5) appear unique to the retina. The functional impact of these philanthotoxin-insensitive CP-AMPARs also remains to be determined. Relief of polyamine sensitivity could change postsynaptic responsivity to patterns of synaptic input (Rozov & Burnashev, 1999) during a time when excitatory synaptic connections with bipolar cells are first being established (Horsburgh & Sefton, 1987). Alternatively, the authors suggest that these receptors may act as temporary molecular cues to direct formation of synaptic connections within specific sublaminae of the IPL, a critical step in the proper wiring of ON and OFF visual pathways.

Hypomagnesemia with Secondary Hypocalcemia due to a Missense Mutation in the Putative Pore-forming Region of TRPM6

Hypomagnesemia with secondary hypocalcemia is an autosomal recessive disorder caused by mutations in the TRPM6 gene. Current experimental evidence suggests that TRPM6 may function in a specific association with TRPM7 by means of heterooligomeric channel complex formation. Here, we report the identification and functional characterization of a new hypomagnesemia with secondary hypocalcemia missense mutation in TRPM6. The affected subject presented with profound hypomagnesemia and hypocalcemia caused by compound heterozygous mutation in the TRPM6 gene: 1208(-1)G > A affecting the acceptor splice site preceding exon 11, and 3050C > G resulting in the amino acid change (P1017R) in the putative pore-forming region of TRPM6. To assess the functional consequences of the P1017R mutation, TRPM6(P1017R) and wild-type TRPM6 were co-expressed with TRPM7 in Xenopus oocytes and HEK 293 cells, and currents were assessed by two-electrode voltage clamp and whole cell patch clamp measurements, respectively. Co-expression of wild-type TRPM6 and TRPM7 resulted in a significant increase in the amplitude of TRPM7-like currents. In contrast, TRPM6(P1017R) suppressed TRPM7 channel activity. In line with these observations, TRPM7, containing the corresponding mutation P1040R, displayed a dominant-negative effect upon co-expression with wild-type TRPM7. Confocal microscopy and fluorescence resonance energy transfer recordings demonstrated that the P1017R mutation neither affects assembly of TRPM6 with TRPM7, nor co-trafficking of heteromultimeric channel complexes to the cell surface. We conclude that a functional defect in the putative pore of TRPM6/7 channel complexes is sufficient to impair body magnesium homeostasis.

Ion pore properties of ionotropic glutamate receptors are modulated by a transplanted potassium channel selectivity filter

The canonical potassium channel selectivity filter motif TVGYG was transplanted into ionotropic glutamate receptors (iGluRs) of the AMPA and NMDA subtype to test whether it renders the iGluRs K + selective. The TVGYG motif modulated several ion pore properties of AMPA receptor as well as NMDA receptor mutants, e.g., the intra- and extracellular polyamine block, current/voltage relationships, open channel block by MK801 and Mg 2+, and permeability for divalent cations. However, introduction of the selectivity filter failed to increase the K + selectivity of homomeric AMPA and heteromeric NMDA receptor complexes, which may be due to absence of selectivity filter-stabilizing interaction sites in the iGluR pore domain. Our findings indicate that even if glutamate receptors appear to have the intrinsic capacity for K + permeability, as is demonstrated by the prokaryotic, glutamate-gated, K + selective GluR0, the isolated selectivity filter is not able to confer K + permeability to the relatively unselective iGluR cation pore.

Functional Characterization of Homo- and Heteromeric Channel Kinases TRPM6 and TRPM7

TRPM6 and TRPM7 are two known channel kinases that play important roles in various physiological processes, including Mg2+ homeostasis. Mutations in TRPM6 cause hereditary hypomagnesemia and secondary hypocalcemia (HSH). However, whether TRPM6 encodes functional channels is controversial. Here we demonstrate several signature features of TRPM6 that distinguish TRPM6 from TRPM7 and TRPM6/7 channels. We show that heterologous expression of TRPM6 but not the mutant TRPM6S141L produces functional channels with divalent cation permeability profile and pH sensitivity distinctive from those of TRPM7 channels and TRPM6/7 complexes. TRPM6 exhibits unique unitary conductance that is 2- and 1.5-fold bigger than that of TRPM7 and TRPM6/7. Moreover, micromolar levels of 2-aminoethoxydiphenyl borate (2-APB) maximally increase TRPM6 but significantly inhibit TRPM7 channel activities; whereas millimolar concentrations of 2-APB potentiate TRPM6/7 and TRPM7 channel activities. Furthermore, Mg2+ and Ca2+ entry through TRPM6 is enhanced three- to fourfold by 2-APB. Collectively, these results indicate that TRPM6 forms functional homomeric channels as well as heteromeric TRPM6/7 complexes. The unique characteristics of these three channel types, TRPM6, TRPM7, and TRPM6/7, suggest that they may play different roles in vivo.

Characterization of functional P2X 1 receptors in mouse megakaryocytes

Introduction Although accumulating evidence within the past 5 years strongly supports the importance of platelet P2X 1 receptors in hemostasis and thrombosis, P2X 1 receptors of platelet and/or its progenitor cell, megakaryocyte, have not been fully characterized. The aim of this study was to electrophysiologically and pharmacologically characterize the functional P2X 1 receptors on mouse megakaryocytes. Materials and methods The currents in response to nucleotides were examined using the patch-clamp whole-cell recording. Results The agonist profile of megakaryocyte P2X 1 receptors was ATP > α,β-methylene ATP > β,γ-methylene ATP. The P2X 1 receptors exhibited substantial monovalent as well as divalent cation permeability and the ratios of Na + to Cs + and Ca 2+ to Cs + permeability were 1 and 2.5, respectively. P2X receptor antagonists except suramin significantly inhibited the P2X 1 responses with an IC 50 values of 0.4 μM for pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS), 0.3 μM for 2′,3′- O-(2,4,6-trinitophenyl)-adenosine 5′-triphosphate (TNP-ATP), 20 μM for reactive blue 2 (RB2), or 160 μM for 8,8′-(carbonylbis(imino-3,1-phenylene carbonylimino)bis(1,3,5-naphthalenetrisulfonic acid) (NF023), respectively. Suramin had no significant effect on the P2X 1 responses. In rat megakaryocytes, suramin similarly had no significant effect on the P2X 1 responses, but abolished the P2Y receptor-mediated responses, indicating that the suramin was active under present experimental condition. Conclusions These results provide the basic properties of mouse megakaryocyte P2X 1 receptors and would be helpful to examine the P2 receptor signaling in platelets and megakaryocytes.

Association of Paracellin-1 with ZO-1 Augments the Reabsorption of Divalent Cations in Renal Epithelial Cells

Paracellin-1 (PCLN-1) belongs to the claudin family of tight junction proteins and possibly plays a critical role in the reabsorption of magnesium and calcium. So far, the physiological properties of PCLN-1 have not been clarified. In the present study, we investigated whether PCLN-1 is associated with ZO-1. We also investigated whether (45)Ca(2+) transport across the paracellular barrier is affected by this association. In vitro binding analysis using glutathione S-transferase fusion protein showed that the C-terminal TRV sequence, especially Thr and Val residues, of PCLN-1 interacts with ZO-1. Next, PCLN-1 was stably expressed in Madin-Darby canine kidney cells using a FLAG tagging vector. ZO-1 was co-immunoprecipitated with the wild-type PCLN-1 and the alanine substitution (TAV) mutant. However, mutants of the deletion (Delta TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the Delta TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of (45)Ca(2+) transport from apical to basal compartments, compared with those expressing the Delta TRV, ARV, and TRA mutants and the mock cells. (45)Ca(2+) transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced (45)Ca(2+) transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1.ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells.

Mitochondrial Uniporter Identified

Kirichok et al . have identified a novel calcium (Ca 2+ )-selective ion channel as the elusive mitochondrial Ca 2+ uniporter. Mitochondrial accumulation of Ca 2+ shapes intracellular Ca 2+ signals, regulates energy production, and has been implicated in cell death. The nature of the mitochondrial Ca 2+ uniporter that enables this uptake, however, has been unclear. Kirichok et al . patch-clamped "mitoplasts" (vesicles derived from the inner mitochondrial membranes) from COS-7 cells and measured an inwardly rectifying Ca 2+ current in whole mitoplasts that, like the mitochondrial Ca 2+ uniporter, was inhibited by Ruthenium 360 and Ruthenium Red. This current, which the authors termed I MiCa , also showed the same cation selectivity and order of divalent cation permeability as the mitochondrial Ca 2+ uniporter. The behavior of single MiCa channels analyzed by recording from inside-out mitoplast patches could account for the properties of the macroscopic I MiCa . MiCa was voltage-dependent, was highly selective for Ca 2+ , and carried sufficient Ca 2+ current to account for mitochondrial Ca 2+ uptake. Thus, the authors concluded that the MiCa channel functioned as the mitochondrial Ca 2+ uniporter. Y. Kirichok, G. Krapivinsky, D. E. Clapham, The mitochondrial calcium uniporter is a highly selective ion channel. Nature 427 , 360-364 (2004). [Online Journal]

Channelrhodopsin-2, a directly light-gated cation-selective membrane channel.

Microbial-type rhodopsins are found in archaea, prokaryotes, and eukaryotes. Some of them represent membrane ion transport proteins such as bacteriorhodopsin, a light-driven proton pump, or channelrhodopsin-1 (ChR1), a recently identified light-gated proton channel from the green alga Chlamydomonas reinhardtii. ChR1 and ChR2, a related microbial-type rhodopsin from C. reinhardtii, were shown to be involved in generation of photocurrents of this green alga. We demonstrate by functional expression, both in oocytes of Xenopus laevis and mammalian cells, that ChR2 is a directly light-switched cation-selective ion channel. This channel opens rapidly after absorption of a photon to generate a large permeability for monovalent and divalent cations. ChR2 desensitizes in continuous light to a smaller steady-state conductance. Recovery from desensitization is accelerated by extracellular H+ and negative membrane potential, whereas closing of the ChR2 ion channel is decelerated by intracellular H+. ChR2 is expressed mainly in C. reinhardtii under low-light conditions, suggesting involvement in photoreception in dark-adapted cells. The predicted seven-transmembrane alpha helices of ChR2 are characteristic for G protein-coupled receptors but reflect a different motif for a cation-selective ion channel. Finally, we demonstrate that ChR2 may be used to depolarize small or large cells, simply by illumination.

Ca2+-dependent Potentiation of the Nonselective Cation Channel TRPV4 Is Mediated by a C-terminal Calmodulin Binding Site

Most Ca2+-permeable ion channels are inhibited by increases in the intracellular Ca2+ concentration ([Ca2+]i), thus preventing potentially deleterious rises in [Ca2+]i. In this study, we demonstrate that currents through the osmo-, heat- and phorbol ester-sensitive, Ca2+-permeable nonselective cation channel TRPV4 are potentiated by intracellular Ca2+. Spontaneous TRPV4 currents and currents stimulated by hypotonic solutions or phorbol esters were reduced strongly at all potentials in the absence of extracellular Ca2+. The other permeant divalent cations Ba2+ and Sr2+ were less effective than Ca2+ in supporting channel activity. An intracellular site of Ca2+ action was supported by the parallel decrease in spontaneous currents and [Ca2+]i on removal of extracellular Ca2+ and the ability of Ca2+ release from intracellular stores to restore TRPV4 activity in the absence of extracellular Ca2+. During TRPV4 activation by hypotonic solutions or phorbol esters, Ca2+ entry through the channel increased the rate and extent of channel activation. Currents were also potentiated by ionomycin in the presence of extracellular Ca2+. Ca2+-dependent potentiation of TRPV4 was often followed by inhibition. By mutagenesis, we localized the structural determinant of Ca2+-dependent potentiation to an intracellular, C-terminal calmodulin binding domain. This domain binds calmodulin in a Ca2+-dependent manner. TRPV4 mutants that did not bind calmodulin lacked Ca2+-dependent potentiation. We conclude that TRPV4 activity is tightly controlled by intracellular Ca2+. Ca2+ entry increases both the rate and extent of channel activation by a calmodulin-dependent mechanism. Excessive increases in [Ca2+]i via TRPV4 are prevented by a Ca2+-dependent negative feedback mechanism.

Sphingosine 1-Phosphate, a Diffusible Calcium Influx Factor Mediating Store-operated Calcium Entry

Store-operated calcium entry (SOCE) is a fundamental mechanism of calcium signaling. The mechanisms linking store depletion to SOCE remain controversial, hypothetically involving both diffusible messengers and conformational coupling of stores to channels. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that can signal via cell surface G-protein-coupled receptors, but S1P can also act as a second messenger, mobilizing calcium directly via unknown mechanisms. We show here that S1P opens calcium entry channels in human neutrophils (PMNs) and HL60 cells without prior store depletion, independent of G-proteins and of phospholipase C. S1P-mediated entry has the typical divalent cation permeability profile and inhibitor profile of SOCE in PMNs, is fully inhibited by 1 microm Gd3+, and is independent of [Ca2+]i. Depletion of PMN calcium stores by thapsigargin induces S1P synthesis. Inhibition of S1P synthesis by dimethylsphingosine blocks thapsigargin-, ionomycin-, and platelet-activating factor-mediated SOCE despite normal store depletion. We propose that S1P is a "calcium influx factor," linking calcium store depletion to downstream SOCE.

AMPA-evoked ion influx is strongest in tangential neurons of the rat neocortical intermediate zone close to the front of the migratory stream.

In addition to the classically described radially migrating neurons, embryonic cortical areas receive neurons originating from the basal ganglia. One of the migration routes is in the intermediate zone. The front of this migration moves toward the hippocampus synchronously with the edge of the dorsally extending cortical plate. We investigated whether cells close to the front have specific properties compared with those at less advanced positions. Activation of AMPA receptors in the presence of cobalt showed that a strong influx of divalent cations could be triggered in front cells by low agonist concentration, whereas the less advanced cells needed a higher concentration to incorporate detectable amounts of cobalt. As shown by in situ hybridization, this discrepancy was not due to differential expression of GluR-2 (known to reduce permeability for divalent cations). In vivo, release of an endogenous agonist presumably affects more, or differently, the tangential cells close to the front.