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Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function

View Article: PubMed Central - PubMed

ABSTRACT

The chloride (Cl-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is defective in cystic fibrosis (CF), and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature. Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity. Few works used automated patch clamp (APC) for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. In this study, we evaluated the use of planar parallel APC technique for pharmacological search of CFTR-trafficking correctors and CFTR function modulators. Using optimized conditions, we recorded both wt- and corrected F508del-CFTR Cl- currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp. Also, the results showed good similarities of CFTR currents recording between the two methods. We showed that similar stimulation protocols could be used in both manual and automatic techniques allowing precise control of temperature, classic I/V relationship, and monitoring of current stability in time. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.

No MeSH data available.


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Cystic fibrosis transmembrane conductance regulator whole-cell recording by automated and MPC from corrected (VX809 24 h) BHK-F508del-CFTR. (A) Current densities (pA/pF) obtained by current–voltage relationships normalized by cell capacitance on BHK-F508del-CFTR corrected 24 h with 10 μM VX809. Whole-cell Cl- currents are recorded by APC (left traces; n = 16) or MPC (right traces; n = 7) by stepping from holding potential of -40 mV to a series of test potentials from -80 to +80 mV in 20 mV increments under basal (unstimulated) conditions or in the presence of 10 μM Fsk + 30 μM Gst or 10 μM CFTRInh 172 as indicated. (B) Corresponding distribution of current densities recorded at 0 mV. Error bars, SEM.
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Figure 3: Cystic fibrosis transmembrane conductance regulator whole-cell recording by automated and MPC from corrected (VX809 24 h) BHK-F508del-CFTR. (A) Current densities (pA/pF) obtained by current–voltage relationships normalized by cell capacitance on BHK-F508del-CFTR corrected 24 h with 10 μM VX809. Whole-cell Cl- currents are recorded by APC (left traces; n = 16) or MPC (right traces; n = 7) by stepping from holding potential of -40 mV to a series of test potentials from -80 to +80 mV in 20 mV increments under basal (unstimulated) conditions or in the presence of 10 μM Fsk + 30 μM Gst or 10 μM CFTRInh 172 as indicated. (B) Corresponding distribution of current densities recorded at 0 mV. Error bars, SEM.

Mentions: First, we used BHK-F508del-CFTR cells under control conditions (DMSO treatment for 24 h). As expected, whole-cell recording detected little for any Cl- current using either method (current densities at 0 mV: -0.67 ± 1.1 pA/pF, n = 10 with APC and 6.9 ± 2.9 pA/pF, n = 6 with MPC; data not shown). When the same experiments were performed after a 24-h pretreatment with the corrector VX809 (10 μM), CFTR current was detected. Figure 3A shows I/V relationships obtained using APC (left curves) and MPC (right curves) under basal conditions and during CFTR activation and inhibition. Figure 3B shows the corresponding distribution of each current density at 0 mV. As with wt-CFTR, Fsk + Gst elicited large F508del-CFTR currents that were nearly abolished by CFTRinh172 and, as previously observed, the APC current densities were somewhat larger and the reversal potential were shifted compared to MPC. Despite these differences, electrophysiological parameters were not significantly different (Table 2; p = 0.73 for Cm and p = 0.24 for Rs) nor were the current densities at 0 mV (Figure 3B; 27.70 ± 4.9 pA/pF, n = 16 for APC and 39.62 ± 9.3 pA/pF, n = 7 for MPC, p = 0.23). Taken together, these results indicate that, aside from a slight contamination by leak current with APC, similar results can be obtained when recording rescued F508del-CFTR using either method.


Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function
Cystic fibrosis transmembrane conductance regulator whole-cell recording by automated and MPC from corrected (VX809 24 h) BHK-F508del-CFTR. (A) Current densities (pA/pF) obtained by current–voltage relationships normalized by cell capacitance on BHK-F508del-CFTR corrected 24 h with 10 μM VX809. Whole-cell Cl- currents are recorded by APC (left traces; n = 16) or MPC (right traces; n = 7) by stepping from holding potential of -40 mV to a series of test potentials from -80 to +80 mV in 20 mV increments under basal (unstimulated) conditions or in the presence of 10 μM Fsk + 30 μM Gst or 10 μM CFTRInh 172 as indicated. (B) Corresponding distribution of current densities recorded at 0 mV. Error bars, SEM.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5383655&req=5

Figure 3: Cystic fibrosis transmembrane conductance regulator whole-cell recording by automated and MPC from corrected (VX809 24 h) BHK-F508del-CFTR. (A) Current densities (pA/pF) obtained by current–voltage relationships normalized by cell capacitance on BHK-F508del-CFTR corrected 24 h with 10 μM VX809. Whole-cell Cl- currents are recorded by APC (left traces; n = 16) or MPC (right traces; n = 7) by stepping from holding potential of -40 mV to a series of test potentials from -80 to +80 mV in 20 mV increments under basal (unstimulated) conditions or in the presence of 10 μM Fsk + 30 μM Gst or 10 μM CFTRInh 172 as indicated. (B) Corresponding distribution of current densities recorded at 0 mV. Error bars, SEM.
Mentions: First, we used BHK-F508del-CFTR cells under control conditions (DMSO treatment for 24 h). As expected, whole-cell recording detected little for any Cl- current using either method (current densities at 0 mV: -0.67 ± 1.1 pA/pF, n = 10 with APC and 6.9 ± 2.9 pA/pF, n = 6 with MPC; data not shown). When the same experiments were performed after a 24-h pretreatment with the corrector VX809 (10 μM), CFTR current was detected. Figure 3A shows I/V relationships obtained using APC (left curves) and MPC (right curves) under basal conditions and during CFTR activation and inhibition. Figure 3B shows the corresponding distribution of each current density at 0 mV. As with wt-CFTR, Fsk + Gst elicited large F508del-CFTR currents that were nearly abolished by CFTRinh172 and, as previously observed, the APC current densities were somewhat larger and the reversal potential were shifted compared to MPC. Despite these differences, electrophysiological parameters were not significantly different (Table 2; p = 0.73 for Cm and p = 0.24 for Rs) nor were the current densities at 0 mV (Figure 3B; 27.70 ± 4.9 pA/pF, n = 16 for APC and 39.62 ± 9.3 pA/pF, n = 7 for MPC, p = 0.23). Taken together, these results indicate that, aside from a slight contamination by leak current with APC, similar results can be obtained when recording rescued F508del-CFTR using either method.

View Article: PubMed Central - PubMed

ABSTRACT

The chloride (Cl-) channel cystic fibrosis transmembrane conductance regulator (CFTR) is defective in cystic fibrosis (CF), and mutation of its encoding gene leads to various defects such as retention of the misfolded protein in the endoplasmic reticulum, reduced stability at the plasma membrane, abnormal channel gating with low open probability, and thermal instability, which leads to inactivation of the channel at physiological temperature. Pharmacotherapy is one major therapeutic approach in the CF field and needs sensible and fast tools to identify promising compounds. The high throughput screening assays available are often fast and sensible techniques but with lack of specificity. Few works used automated patch clamp (APC) for CFTR recording, and none have compared conventional and planar techniques and demonstrated their capabilities for different types of experiments. In this study, we evaluated the use of planar parallel APC technique for pharmacological search of CFTR-trafficking correctors and CFTR function modulators. Using optimized conditions, we recorded both wt- and corrected F508del-CFTR Cl- currents with automated whole-cell patch clamp and compared the data to results obtained with conventional manual whole-cell patch clamp. We found no significant difference in patch clamp parameters such as cell capacitance and series resistance between automated and manual patch clamp. Also, the results showed good similarities of CFTR currents recording between the two methods. We showed that similar stimulation protocols could be used in both manual and automatic techniques allowing precise control of temperature, classic I/V relationship, and monitoring of current stability in time. In conclusion, parallel patch-clamp recording allows rapid and efficient investigation of CFTR currents with a variety of tests available and could be considered as new tool for medium throughput screening in CF pharmacotherapy.

No MeSH data available.


Related in: MedlinePlus