Pore properties and ionic block of the rabbit epithelial calcium channel expressed in HEK 293 cells (original) (raw)
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Pfl�gers Archiv European Journal of Physiology, 1997
Transient transfection of ion channels into mammalian cells is a useful method with which to study ion channel properties. However, a general problem in transient transfection procedures is how to select cells that express the transfected cDNA. We have constructed a bicistronic vector, pCINeo/IRES-GFP, which utilises a red-shifted variant of Green Fluorescent Protein as an in vivo cell marker. Incorporation of an ion channel cDNA into the bicistronic unit allows coupled expression of the ion channel and Green Fluorescent Protein. After transient transfection of COS cells with pCINeo/IRES-GFP containing a rat delayed rectifier K + channel cDNA (RCK1, Kv1.1), all green cells (n = 32) expressed the RCK1 channel as identified by the well known kinetics, K + selectivity and pharmacology of Kv1.1. In contrast, non-fluorescent cells (n = 24) were negative with respect to RCK1 expression. It is concluded that the bicistronic pCINeo/IRES-GFP vector provides an efficient and noninvasive way of identifying cells which express ion channels after transfection. This novel method should greatly facilitate functional studies of ion channels transfected into mammalian cells.
Permeation and Gating Properties of the Novel Epithelial Ca2+ Channel
Journal of Biological Chemistry, 2000
The recently cloned epithelial Ca 2؉ channel (ECaC) constitutes the Ca 2؉ influx pathway in 1,25-dihydroxyvitamin D 3 -responsive epithelia. We have combined patchclamp analysis and fura-2 fluorescence microscopy to functionally characterize ECaC heterologously expressed in HEK293 cells. The intracellular Ca 2؉ concentration in ECaC-expressing cells was closely correlated with the applied electrochemical Ca 2؉ gradient, demonstrating the distinctive Ca 2؉ permeability and constitutive activation of ECaC. Cells dialyzed with 10 mM 1,2bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid displayed large inward currents through ECaC in response to voltage ramps. The corresponding currentvoltage relationship showed pronounced inward rectification. Currents evoked by voltage steps to potentials below ؊40 mV partially inactivated with a biexponential time course. This inactivation was less pronounced if Ba 2؉ or Sr 2؉ replaced Ca 2؉ and was absent in Ca 2؉ -free solutions. ECaC showed an anomalous mole fraction behavior. The permeability ratio P Ca :P Na calculated from the reversal potential at 30 mM [Ca 2؉ ] o was larger than 100. The divalent cation selectivity profile is Ca 2؉ > Mn 2؉ > Ba 2؉ ϳ Sr 2؉ . Repetitive stimulation of ECaCexpressing cells induced a decay of the current response, which was greatly reduced if Ca 2؉ was replaced by Ba 2؉ and was virtually abolished if [Ca 2؉ ] o was lowered to 1 nM. In conclusion, ECaC is a Ca 2؉ selective channel, exhibiting Ca 2؉ -dependent autoregulatory mechanisms, including fast inactivation and slow down-regulation.
Changes in ultrastructure and endogenous ionic channels activity during culture of HEK 293 cell line
European Journal of Pharmacology, 2007
Human embryonic kidney (HEK) 293 cells were characterised as an expression system for voltage-activated cationic channels. Current density for cationic channels intrinsically expressed in HEK 293 cells as well as cell ultrastructure was described after 7-11, 29-30 and 49-63 days of cell culture. Slowly activating outward potassium current with the current density varying between +10 and + 26 pA/pF was observed in 72% to 95% of investigated cells. Rapidly inactivating outward potassium current with the current density varying between + 7 and +10 pA/pF was present in 38% to 48% of all cells. 30% of cells exhibited voltage-activated calcium channel with the current density less than -1 pA/pF. Tetrodotoxinsensitive sodium current with amplitudes between −1.4 and −2.2 pA/pF was initially present in 5% of cells, nevertheless, after 49-63 days of cell culture this proportion increased to 35%. Ultrastructure of HEK 293 cell surface, but not of cell's interior changed during cell culture. The longer the time after thawing the more microvilli and protrusions appear on the cell surface. Irregular cell contours hinder the cells to appose and only small patches of membranes form attachments. Staining of cells with a polycationic dye ruthenium red initially increased and decreased again following prolonged period of time in culture indicating regression of negatively charged layers of the cell surface coat. We suggest that the optimal time window for patch clamp experiment is between days 7 and 63 of cell culture due to alterations of cell surface.
The Journal of Physiology, 1999
Voltage-dependent calcium channels (VDCCs) are heteromeric complexes consisting of a channel-forming á1 subunit and accessory á2-ä and â subunits. There are at least eight cloned and expressed á1 subunits (Perez-Reyes & Schneider, 1994; Perez-Reyes et al. 1998), at least six of which (á1A-E and G) are found in the nervous system. The á1 subunit determines the characteristics of the channel and some of the cloned channels have been attributed to functionally identified channels. The N-type channel is believed to be encoded by the á1B clone (De Waard et al. 1994), PÏQ-type channels by á1A (Gillard et al. 1997) and L-type channels by á1C and á1D (Birnbaumer et al. 1994). The assignment of the á1E clone to a native channel has been controversial. It has been suggested that it encodes either an R-type (residual) (Randall & Tsien, 1995) or a subset of low voltageactivated T-type channels (Bourinet et al. 1996). The á1G subunit encodes a T-type channel (Perez-Reyes et al. 1998). The accessory subunits, particularly the intracellular â subunit, have been shown to have marked effects on the properties of á1 subunits (apart from á1G), including modification of kinetics, amplitude and targeting of the complex to the plasma membrane (Singer et al. 1991; Brice et al. 1997). There are four â subunits, all of which are expressed in the nervous system (Perez-Reyes & Schneider, 1994). We have shown previously that the â subunit has a chaperone-like effect, promoting functional expression of the VDCC á1A subunit at the plasma membrane of COS_7 cells (Brice et al. 1997), and similar results have been obtained for cardiac á1C with â2a (Chien et al. 1995). In neurons, and other polarized cells, VDCCs must be sorted to the correct membrane domain for the proper functioning of the cell, as the different subtypes of VDCC have very different biophysical properties and potential for modulation (Dolphin, 1998). For example, in neurons certain subtypes of VDCC are essential to provide Ca¥ for neurotransmitter release. Studies have shown that the N-and PÏQ-type channels are particularly involved, either individually or in combination with each other (Turner et al. 1993; Wheeler et al. 1994), indicating a presynaptic localization for these channels. Nevertheless, these VDCCs have also been
The Journal of …, 2006
TRPC6 is thought to be a Ca 2+ -permeable cation channel activated following stimulation of G-protein-coupled membrane receptors linked to phospholipase C (PLC). TRPC6 current is also activated by exogenous application of 1-oleoyl-acetyl-sn-glycerol (OAG) or by inhibiting 1,2-diacylglycerol (DAG) lipase activity using RHC80267. In the present study, both OAG and RHC80267 increased whole-cell TRPC6 current in cells from a human embryonic kidney cell line (HEK 293) stably expressing TRPC6, but neither compound increased cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) when the cells were bathed in high-K + buffer to hold the membrane potential near 0 mV. These results suggested that TRPC6 channels have limited Ca 2+ permeability relative to monovalent cation permeability and/or that Ca 2+ influx via TRPC6 is greatly attenuated by depolarization. To evaluate Ca 2+ permeability, TRPC6 currents were examined in extracellular buffer in which Ca 2+ was varied from 0.02 to 20 mM. The results were consistent with a pore-permeation model in which Ca 2+ acts primarily as a blocking ion and contributes only a small percentage (∼4%) to whole-cell currents in the presence of extracellular Na + . Measurement of single-cell fura-2 fluorescence during perforated-patch recording of TRPC6 currents showed that OAG increased [Ca 2+ ] i 50-100 nM when the membrane potential was clamped at between −50 and −80 mV, but had little or no effect if the membrane potential was left uncontrolled. These results suggest that in cells exhibiting a high input resistance, the primary effect of activating TRPC6 will be membrane depolarization. However, in cells able to maintain a hyperpolarized potential (e.g. cells with a large inwardly rectifying or Ca 2+ -activated K + current), activation of TRPC6 will lead to a sustained increase in [Ca 2+ ] i . Thus, the contribution of TRPC6 current to both the kinetics and magnitude of the Ca 2+ response will be cell specific and dependent upon the complement of other channel types.
Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26
Molecular biology of the cell, 2000
This study was undertaken to obtain direct evidence for the involvement of gap junctions in the propagation of intercellular Ca(2+) waves. Gap junction-deficient HeLa cells were transfected with plasmids encoding for green fluorescent protein (GFP) fused to the cytoplasmic carboxyl termini of connexin 43 (Cx43), 32 (Cx32), or 26 (Cx26). The subsequently expressed GFP-labeled gap junctions rendered the cells dye- and electrically coupled and were detected at the plasma membranes at points of contact between adjacent cells. To correlate the distribution of gap junctions with the changes in [Ca(2+)](i) associated with Ca(2+) waves and the distribution of the endoplasmic reticulum (ER), cells were loaded with fluorescent Ca(2+)-sensitive (fluo-3 and fura-2) and ER membrane (ER-Tracker) dyes. Digital high-speed microscopy was used to collect a series of image slices from which the three-dimensional distribution of the gap junctions and ER were reconstructed. Subsequently, intercellular C...
Pflügers Archiv : European journal of physiology, 2003
The family of epithelial Ca(2+) channels (ECaC) is a unique group of highly Ca(2+)-selective channels consisting of two members, ECaC1 and ECaC2. We used carboxyl terminal truncations and mutants to delineate the molecular determinants of the Ca(2+)-dependent inhibition of ECaC. To this end, rabbit ECaC1 was expressed heterologously with green fluorescent protein (GFP) in human embryonic kidney 293 (HEK293) cells using a bicistronic vector. Deletion of the last 30 amino acids of the carboxyl terminus of ECaC1 (G701X) decreased the Ca(2+) sensitivity significantly. Another critical sequence for Ca(2+)-dependent inactivation of ECaC1 was found upstream in the carboxyl terminus. Analysis of truncations at amino acid 635, 639, 646, 649 and 653 disclosed a critical sequence involved in Ca(2+)-dependent inactivation at positions 650-653. C653X showed decreased Ca(2+) sensitivity, comparable to G701X, while E649X lacked Ca(2+)-dependent inactivation. Interestingly, the number of green fluo...