ABCMdb

PMID: 17849169

Liu X
A possible role for intracellular GSH in spontaneous reaction of a cysteine (T338C) engineered into the Cystic Fibrosis Transmembrane Conductance Regulator.
Biometals. 2008 Jun;21(3):277-87. Epub 2007 Sep 12., [PubMed]

Sentences

No. Mutations Sentence Comment

0 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys A possible role for intracellular GSH in spontaneous reaction of a cysteine (T338C) engineered into the Cystic Fibrosis Transmembrane Conductance Regulator Xuehong Liu Received: 26 December 2006 / Accepted: 27 August 2007 / Published online: 12 September 2007 Ó Springer Science+Business Media B.V. 2007 Abstract The conductance of oocytes expressing T338C CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) exhibits variable responses to dithiothreitol (DTT) and 2-mercaptoethanol (2-ME) that we proposed might be due to the extraction of copper from an adventitious binding site (Liu et al. Login to comment 2 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys In order to study the origins of variability in chemical reactivity of T338C CFTR channels, oocytes expressing T338C CFTR were exposed to BCNU (bischloroethylnitrosourea), an inhibitor of glutathione reductase. Login to comment 4 ABCC7 p.Thr338Ala ABCC7 p.Thr338Cys Single-channel recordings indicated that T338C CFTR channels not exposed to 2-ME or DTT exhibited multiple conductance levels not seen in T338A CFTR channels. Login to comment 6 ABCC7 p.Thr338Cys These results suggest that the altered chemical state of T338C channels is associated with a decreased single-channel conductance and that intracellular factors (most likely GSH) may modulate the propensity of the channel to form these altered states. Login to comment 9 ABCC7 p.Thr338Cys However, the origin of the variability in the chemical reactivity among oocytes expressing T338C CFTR was not fully understood, nor was the basis for the changes in macroscopic conductance discerned, i.e., change in single-channel conductance or gating (open probability). Login to comment 10 ABCC7 p.Thr338Cys Because intracellular GSH has been demonstrated to be an important determinant of the disposition of intracellular copper (Freedman et al. 1989; Ciriolo et al. 1990; Ascone et al. 1993; Ferreira et al. 1993), I used two-electrode-voltage-clamp (TEVC) and single-channel recording to examine the effects of changes in intracellular GSH on the spontaneous reactions of T338C CFTR. Login to comment 12 ABCC7 p.Thr338Cys Exposure to BCNU decreased the initial conductance of oocytes expressing T338C CFTR and increased the magnitude of the response to 2-ME or DTT, as if a lower level of cellular GSH promoted the modified state of the cysteine. Login to comment 13 ABCC7 p.Thr338Cys These results are consistent with the idea that intracellular GSH might be responsible, at least in part, for the variability in the chemical state of T338C CFTR. Login to comment 48 ABCC7 p.Thr338Cys Results The conductance of oocytes expressing T338C CFTR and the response to 2-ME or DTT were altered by BCNU, an inhibitor of glutathione reductase GSH is the most abundant free thiol in cells and it has high affinity for metals (Rabenstein 1989). Login to comment 49 ABCC7 p.Thr338Cys I thus considered the possibility that variable intracellular GSH concentrations might contribute to the variability in initial conductance and responses to 2-ME or DTT seen in oocytes expressing T338C CFTR (Liu et al. 2006) by altering the fractional distribution of channels containing copper. Login to comment 51 ABCC7 p.Thr338Ala ABCC7 p.Thr338Cys Summarized in Fig. 1 are results obtained from oocytes expressing T338C or T338A CFTRs that were either untreated, or exposed to 100 lM BCNU for 72 h prior to electrophysiological recording. Login to comment 52 ABCC7 p.Thr338Cys Oocytes expressing T338C CFTR and exposed to 100 lM BCNU exhibited a significantly lower initial steady state conductance (22 ± 4 lS) than untreated oocytes (92 ± 14, lS, P-value \ 0.05). Login to comment 54 ABCC7 p.Thr338Cys The conductances after exposure to 2-ME were not significantly different between treated (119 ± (( lS) and untreated oocytes expressing T338C CFTR (98 ± 13 lS). Login to comment 56 ABCC7 p.Thr338Cys In this population, oocyte pre-exposed to BCNU did not differ Fig. 1 BCNU altered T338C CFTR conductance and its response to 2-ME. Login to comment 57 ABCC7 p.Thr338Cys (A) The initial steady state conductance of oocytes expressing T338C CFTR (black bars) and the conductance after exposure to 1 mM 2-ME (white bars) were summarized for the control oocytes and oocytes maintained in the incubation solution (MBSH) containing 100 lM BCNU since injection of cRNA. Login to comment 59 ABCC7 p.Thr338Ala (B) The initial steady state conductance of oocytes expressing T338A CFTR (black bars) and the conductance after exposure to 1 mM 2-ME (white bars) were summarized for the control oocytes and oocytes maintained in the storage solution (MBSH) containing 100 lM BCNU since injection of cRNA significantly from untreated controls, suggesting that the treatment may mimic the ''naturally modified`` state. Login to comment 60 ABCC7 p.Thr338Ala Oocytes expressing T338A CFTR (Fig. 1B) exhibited no response to 2-ME with or without exposure to BCNU. Login to comment 61 ABCC7 p.Thr338Ala The mean initial conductance of T338A CFTR was slightly lower in BCNU treated oocytes (67 ± 6 lS) than untreated ones (52 ± 9 lS), but the difference was not statistically significant. Login to comment 62 ABCC7 p.Thr338Cys These results are consistent with the hypothesis that BCNU- induced responses in T338C CFTR are specific to the cysteine at position 338. Login to comment 64 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys BCNU altered the fractional distribution of single-channel current amplitudes in oocytes expressing T338C CFTR To determine if BCNU treatment altered open probability or single-channel conductance, I recorded single-channel currents from inside-out patches detached from oocytes expressing T338C CFTR that were either untreated or exposed to BCNU. Login to comment 68 ABCC7 p.Thr338Cys To mitigate this potential contamination by non-CFTR channels, a patch was operationally defined as containing T338C CFTR channels if the events were activated by PKA and ATP. Login to comment 70 ABCC7 p.Thr338Cys Summarized in Fig. 2 are fractional distributions of current amplitudes extracted from patches obtained from oocytes expressing T338C CFTR at pH 7.4 (extracellular, Vm = -100 mV). Login to comment 72 ABCC7 p.Thr338Cys We have shown previously that events with 0.6 pA amplitude represent the full conductance of T338C channels at pH 7.4 in the presence of 2-ME or DTT (Liu et al. 2004). Login to comment 79 ABCC7 p.Thr338Cys If events with different current amplitudes represent T338C CFTR channels in different chemical states, be it oxidation or metal complexes, some of these channels might be sensitive to DTT, a strong reducing agent and a potent metal ligand (Krezel et al. 2001). Login to comment 86 ABCC7 p.Thr338Cys We reported previously that the single-channel conductance of T338C CFTR is larger at pH 6.0 (*9 pS) than at pH 7.4 (Liu et al. 2004). Login to comment 90 ABCC7 p.Thr338Ala ABCC7 p.Thr338Cys To verify that a cysteine was required for the multiple current amplitudes observed in T338C CFTR, I recorded single-channel currents of T338A CFTR. Login to comment 91 ABCC7 p.Thr338Cys The single-channel conductance of this construct is greater than that of T338C CFTR (Linsdell et al. 1998; Liu et al. 2004). Login to comment 93 ABCC7 p.Thr338Ala Thus exposure to BCNU did not result in any significant change in the fractional distribution of 0.9 pA events in T338A CFTR channels. Login to comment 94 ABCC7 p.Thr338Ala No difference was detected among the apparent open probabilities (NPo /N) of T338A CFTR channels (pH 7.4) under control or BCNU treated conditions using records obtained at 500 lM intracellular ATP. Login to comment 98 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys Low concentration of GSH reverses sponstanous and copper-modified states at T338C locus The impact of BCNU on the chemical state of a cysteine at 338 suggests that in the event of reduction Fig. 2 BCNU altered the fractional distribution of current amplitudes of single T338C CFTR channels at pH 7.4. Login to comment 99 ABCC7 p.Thr338Cys Fractional distribution of single-channel current amplitudes at pHextra = 7.4 from patches obtained from T338C CFTR expressing oocytes that were: (A) incubated in MBSH, (B) incubated in MBSH containing 100 lM BCNU since injection of cRNA, (C) incubated in MBSH and exposed to 10 mM DTT for about 1 to 24 hours before patching or MBSH, (D) incubated in MBSH containing 100 lM BCNU since injection of cRNA and exposed to 10 mM DTT for about 1 to 24 hours before patching. The sample current traces obtained at Vm = -100 mV for each group are shown above the bars of cytoplasmic GSH the cysteine at 338 is more likely to be chemically altered, perhaps by coordinating copper. Login to comment 102 ABCC7 p.Thr338Cys Alternatively, efflux of GSH and GSH-conjugates via an endogenous pathway in Xenopus oocytes (Ballatori et al. 1996) may lead to a higher local concentration of GSH in the extracellular space between the plasma membrane and the follicular membrane bringing GSH into proximity with T338C. Login to comment 103 ABCC7 p.Thr338Cys Because it is impossible at present to assay the intracellular concentration of GSH in intact cells in real time, I chose to use a functional assay to characterize the impact of externally applied GSH on the spontaneously-altered state and copper-modified state of T338C CFTR. Login to comment 104 ABCC7 p.Thr338Cys A naive oocyte expressing T338C CFTR (Fig. 5A), was first exposed to 1 lM and then to 1 mM GSH, resulting in rapid, dose-dependent increases in conductance, similar to those seen after treatment with DTT or 2-ME, indicating a reversal of the modified state of this engineered cysteine. Login to comment 108 ABCC7 p.Thr338Cys Under copper modified condition, exposure to 1 lM GSH and Fig. 3 BCNU altered the fractional distribution of current amplitudes of single T338C CFTR channels at pH 6.0. Login to comment 109 ABCC7 p.Thr338Cys Fractional distribution of single-channel current amplitudes at pHextra = 6.0 from patches obtained from T338C CFTR expressing oocytes that were: (A) incubated in MBSH, (B) incubated in MBSH containing 100 lM BCNU since injection of cRNA, (C) incubated in MBSH and exposed to 10 mM DTT for about 1 to 24 hours before patching, (D) incubated in MBSH containing 100 lM BCNU since injection of cRNA and exposed to 10 mM DTT for about 1 to 24 hours before patching. The sample current traces obtained at Vm = -100 mV for each group are shown above the bars 1 mM GSH also resulted in dose-dependent increases in conductance. Login to comment 111 ABCC7 p.Thr338Cys ABCC7 p.Thr338Cys The similar efficacies of GSH on T338C CFTR conductance under naive and external copper-bound state strongly suggest a similar, if not identical chemical modification of T338C under the two conditions. Login to comment 112 ABCC7 p.Thr338Cys The above result suggests that extracellular GSH can perturb copper binding at T338C locus with an affinity in the micromolar range. Login to comment 116 ABCC7 p.Thr338Ala Regardless, a cysteine at position 338 was essential for the GSH effect because at concentrations as high as 10 mM, GSH had no effect on conductance of oocytes expressing Cys-less CFTR (Fig. 5B, n = 2) or T338A CFTR (Fig. 5C, n = 2). Login to comment 118 ABCC7 p.Thr338Cys Results shown in Fig. 6A indicated that GSH could only partially reverse the mixed disulfide bond between T338C and MTSET+ and could do so only at a concentration nearly 1,000 fold higher than that needed to perturb the copper binding site. Login to comment 119 ABCC7 p.Arg334Cys Similar behavior was observed in R334C CFTR (Fig. 6B). Login to comment 120 ABCC7 p.Thr338Ala These results indicate that although GSH is capable of breaking a mixed disulfide bond at 338, the reaction precedes at a much lower rate and required a much higher Fig. 4 BCNU had no effect on single T338A CFTR conductance. Login to comment 121 ABCC7 p.Thr338Ala Fractional distribution of single-channel current amplitudes at pH 7.4 from patches obtained from T338A CFTR expressing oocytes that were: (A) incubated in MBSH, (B) incubated in MBSH containing 100 lM BCNU since injection of cRNA. Login to comment 122 ABCC7 p.Thr338Ala Fractional distribution of single-channel current amplitudes at pH 6.0 from patches obtained from T338A CFTR expressing oocytes that were: (C) incubated in MBSH, (D) incubated in MBSH containing 100 lM BCNU since injection of cRNA. Login to comment 124 ABCC7 p.Thr338Cys These experiments support the notion that GSH is an importamt determinate of chemical reactivity of T338C in naı¨ve oocytes. Login to comment 133 ABCC7 p.Thr338Cys In addition, GSH is known to coordinate copper (Rabenstein 1989) and to be an important determinant of the disposition of intracellular copper (Freedman et al. 1989; Ciriolo et al. 1990; Ascone Fig. 5 Extracellular GSH could remove copper from T338C locus. Login to comment 134 ABCC7 p.Thr338Cys (A) Following activation by stimulatory cocktail (10 lM Isop and 1 mM IBMX, hatched bar and crosshair), a naive oocyte expressing T338C CFTR was exposed to: 1 lM and then 1 mM GSH (open triangles), 1 mM DTT (open circles), 1 mM CuCl2 (grey circles), 1 lM GSH and 1 mM GSH, 1 mM 2-ME (open circles), (n = 4). Login to comment 136 ABCC7 p.Thr338Ala (C) Following activation (hatched bar and crosshair), an oocyte expressing T338A CFTR was exposed to 10 mM GSH (open triangles) et al. 1993; Ferreira et al. 1993). Login to comment 137 ABCC7 p.Thr338Cys The influence of BCNU on the 2-ME/DTT-sensitive conductance of oocyte expressing T338C CFTR suggests that decreasing cytosolic GSH increases the likelihood that copper will be bound by the adventitious metal center that is inadvertently created in the cysteine-substituted channel. Login to comment 151 ABCC7 p.Thr338Cys (A) Following activation by stimulatory cocktail (10 lM Isop and 1 mM IBMX, hatched bar and crosshair), a naive oocyte expressing T338C CFTR was exposed to: 1 mM DTT (open circles), 1 mM MTSET+ (black circles), 100 lM GSH and 1 mM GSH (open triagles), 1 mM 2-ME (open squares). Login to comment 152 ABCC7 p.Arg334Cys (B) Following activation by stimulatory cocktail (10 lM Isop and 1 mM IBMX, hatched bar and crosshair), an oocyte expressing R334C CFTR was exposed to: 1 mM MTSET+ (black circles), 100 lM GSH and 10 mM GSH (open triagles) could, in principle, remove bound copper from the adventitious site. Login to comment 157 ABCC7 p.Thr338Cys The dose-dependent response to extracellular GSH is consistent with an equilibrium mechanism in which increasing GSH shifted the equilibrium towards a state where the copper at the T338C locus was either freed or the coordination geometry was perturbed. Login to comment