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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

Experimental procedure for automated patch clamp (APC) cystic fibrosis transmembrane conductance regulator (CFTR) recording. (A) Tree of events for whole-cell configuration establishing (gray rectangle 1) and personalized CFTR recording (gray rectangle 2). (B) The two different protocols applied for CFTR recording: I/V protocol (blue dots) for current/voltage relationship visualization and drug time course (green dots) protocol to monitor the effect of compound in time.
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Figure 1: Experimental procedure for automated patch clamp (APC) cystic fibrosis transmembrane conductance regulator (CFTR) recording. (A) Tree of events for whole-cell configuration establishing (gray rectangle 1) and personalized CFTR recording (gray rectangle 2). (B) The two different protocols applied for CFTR recording: I/V protocol (blue dots) for current/voltage relationship visualization and drug time course (green dots) protocol to monitor the effect of compound in time.

Mentions: Similar experiments were performed using APC and MPC. The holding potential was maintained at -40 mV throughout the experiment, and two voltage-clamp protocols were used to measure whole-cell CFTR currents. Figure 1 shows the sequence of events (called tree) used on the Nanion software (Figure 1A) and protocols applied for CFTR recording (Figure 1B). The tree of events shows all steps leading to the whole-cell configuration (gray rectangle 1) followed by the CFTR recording parameters (gray rectangle 2). The current–voltage (I–V) relationship (I/V protocol, blue dots) was determined by pulsing from the holding potential of -40 mV to test potentials between -80 and +80 mV increasing in 20 mV increments. To monitor the current evolution under drugs application and confirm the absence of significant leak current; a single depolarization from -40 to 0 mV was applied every 5 s for 6–7 min (drug time course protocol, green dots).


Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function
Experimental procedure for automated patch clamp (APC) cystic fibrosis transmembrane conductance regulator (CFTR) recording. (A) Tree of events for whole-cell configuration establishing (gray rectangle 1) and personalized CFTR recording (gray rectangle 2). (B) The two different protocols applied for CFTR recording: I/V protocol (blue dots) for current/voltage relationship visualization and drug time course (green dots) protocol to monitor the effect of compound in time.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Experimental procedure for automated patch clamp (APC) cystic fibrosis transmembrane conductance regulator (CFTR) recording. (A) Tree of events for whole-cell configuration establishing (gray rectangle 1) and personalized CFTR recording (gray rectangle 2). (B) The two different protocols applied for CFTR recording: I/V protocol (blue dots) for current/voltage relationship visualization and drug time course (green dots) protocol to monitor the effect of compound in time.
Mentions: Similar experiments were performed using APC and MPC. The holding potential was maintained at -40 mV throughout the experiment, and two voltage-clamp protocols were used to measure whole-cell CFTR currents. Figure 1 shows the sequence of events (called tree) used on the Nanion software (Figure 1A) and protocols applied for CFTR recording (Figure 1B). The tree of events shows all steps leading to the whole-cell configuration (gray rectangle 1) followed by the CFTR recording parameters (gray rectangle 2). The current–voltage (I–V) relationship (I/V protocol, blue dots) was determined by pulsing from the holding potential of -40 mV to test potentials between -80 and +80 mV increasing in 20 mV increments. To monitor the current evolution under drugs application and confirm the absence of significant leak current; a single depolarization from -40 to 0 mV was applied every 5 s for 6–7 min (drug time course protocol, green dots).

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