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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.


Related in: MedlinePlus

Alternative application recorded with APC. Means of whole-cell Cl- current density time courses at 0 mV recorded on BHK-wt-CFTR (A,B) or BHK-F508del-CFTR (C) by APC. (A) Monitoring of wt channels rundown by long recording in presence of 10 μM Fsk + 30 μM Gst (n = 5). (B) Monitoring CFTR potentiation by sequential addition of 1 μM Fsk, and then 10 μM VX770 (n = 8). Black lines are representative linear regressions of the time course curves before and after potentiator addition. Means of slope values are indicated. (C) Effect of temperature on F508del channel function: recording of whole-cell current at room (RT; n = 6) or physiological temperature (35°C; n = 5) in presence of 10 μM Fsk + 30 μM Gst. For all conditions, 10 μM CFTRinh172 was added at the end of experiment. Colored symbols are the mean of current densities and gray dashed lines the corresponding error, SEM.
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Figure 4: Alternative application recorded with APC. Means of whole-cell Cl- current density time courses at 0 mV recorded on BHK-wt-CFTR (A,B) or BHK-F508del-CFTR (C) by APC. (A) Monitoring of wt channels rundown by long recording in presence of 10 μM Fsk + 30 μM Gst (n = 5). (B) Monitoring CFTR potentiation by sequential addition of 1 μM Fsk, and then 10 μM VX770 (n = 8). Black lines are representative linear regressions of the time course curves before and after potentiator addition. Means of slope values are indicated. (C) Effect of temperature on F508del channel function: recording of whole-cell current at room (RT; n = 6) or physiological temperature (35°C; n = 5) in presence of 10 μM Fsk + 30 μM Gst. For all conditions, 10 μM CFTRinh172 was added at the end of experiment. Colored symbols are the mean of current densities and gray dashed lines the corresponding error, SEM.

Mentions: The recording sequence above is useful when determining the efficiency of candidate corrector molecules or comparing the potencies of several correctors. However, with MPC, we are potentially able to collect information such as the stability of currents over long time periods and the temperature dependence of channel gating. We examined the capabilities of APC for such experiments with Patchliner®system (Figure 4).


Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function
Alternative application recorded with APC. Means of whole-cell Cl- current density time courses at 0 mV recorded on BHK-wt-CFTR (A,B) or BHK-F508del-CFTR (C) by APC. (A) Monitoring of wt channels rundown by long recording in presence of 10 μM Fsk + 30 μM Gst (n = 5). (B) Monitoring CFTR potentiation by sequential addition of 1 μM Fsk, and then 10 μM VX770 (n = 8). Black lines are representative linear regressions of the time course curves before and after potentiator addition. Means of slope values are indicated. (C) Effect of temperature on F508del channel function: recording of whole-cell current at room (RT; n = 6) or physiological temperature (35°C; n = 5) in presence of 10 μM Fsk + 30 μM Gst. For all conditions, 10 μM CFTRinh172 was added at the end of experiment. Colored symbols are the mean of current densities and gray dashed lines the corresponding error, SEM.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Alternative application recorded with APC. Means of whole-cell Cl- current density time courses at 0 mV recorded on BHK-wt-CFTR (A,B) or BHK-F508del-CFTR (C) by APC. (A) Monitoring of wt channels rundown by long recording in presence of 10 μM Fsk + 30 μM Gst (n = 5). (B) Monitoring CFTR potentiation by sequential addition of 1 μM Fsk, and then 10 μM VX770 (n = 8). Black lines are representative linear regressions of the time course curves before and after potentiator addition. Means of slope values are indicated. (C) Effect of temperature on F508del channel function: recording of whole-cell current at room (RT; n = 6) or physiological temperature (35°C; n = 5) in presence of 10 μM Fsk + 30 μM Gst. For all conditions, 10 μM CFTRinh172 was added at the end of experiment. Colored symbols are the mean of current densities and gray dashed lines the corresponding error, SEM.
Mentions: The recording sequence above is useful when determining the efficiency of candidate corrector molecules or comparing the potencies of several correctors. However, with MPC, we are potentially able to collect information such as the stability of currents over long time periods and the temperature dependence of channel gating. We examined the capabilities of APC for such experiments with Patchliner®system (Figure 4).

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