Molly Rowlett, Thomas Hill, Scott Maidment, Luke Marrington, Ayesha Jinat, Alexandra Pinggera, Katie Puddefoot, Gary Clark, Zeki Ilkan, Robert Kirby, Graham Smith
Metrion Biosciences Ltd, Granta Centre, Granta Park, Cambridge, CB21 6AL, United Kingdom
Introduction
TRPML1-4A and Metrion’s expertise in ion channel screening
TRPML1 (transient receptor potential mucolipin subfamily 1) is a non-selective cation channel primarily localised in late endosomes and lysosomes. Channel activity is regulated by both calcium (Ca2+) and pH. TRPML1 facilitates Ca2+ efflux from the lysosomal lumen and is, therefore, vital for vesicular trafficking processes such as exocytosis and autophagy1.
Mutations in the gene encoding the channel protein (MCOLN1) results in lysosomal storage diseases, such as Mucolipidosis type IV (ML4) which presents as motor deficits, neurodegeneration and visual impairments2.
The development and validation of ion channel screening assays to study TRPML1 is important to understand the function and pharmacology of the channel. Here, we used a TRPML1 variant that lacks the endo-lysosomal retention sequences (TRPML1-4A), enabling the channel to express at the plasma membrane3. As such channel behaviour can be characterised by means of whole-cell patch-clamp and fluorescence-based techniques.
Aim
Developing advanced TRPML1-4A screening assays with automated patch clamp and FLIPR technologies
To develop a suite of screening assays using manual patch-clamp, automated patch-clamp and fluorescence-based platforms capable of identifying modulators of the TRPML1-4A channel.
Materials and Methods
Metrion’s innovative electrophysiological assay development
Cell culture: Establishing the HEK-TRPML1-4A stable cell line
TRPML1 channel sequence contains two di-leucine sorting motifs that are responsible for endo-lysosomal trafficking. Mutations at these sites gives rise to the -4A mutant, which allows accumulation of TRPML1 at the plasma membrane3.
An inducible monoclonal human embryonic kidney 293 (HEK293) cell line expressing the TRPML1-4A variant (HEK-TRPML1-4A) was generated by Metrion’s cell biology team.
TRPML1 channel expression was induced by the addition of doxycycline into culture media (0.25 µg/ml to 1 µg/ml), 24 to 48 hours prior to experiments.
Compounds: Reagents for TRPML1-4A patch clamp and fluorescence assays
TRPML1-4A currents were elicited using the agonist, ML-SA5 (Alomone Labs), stocks of which were prepared in 100% DMSO to 30 mM. Test concentrations were prepared in the appropriate solution each experimental day to a final DMSO concentration of either 0.1% or 0.3% v/v.
Automated and manual patch clamp protocols: TRPML1-4A electrophysiology
Manual patch-clamp recordings were performed on a Multiclamp 700B and 1400A digitiser device (Molecular Devices) and automated patch-clamp recordings were performed on Qube 384 (Sophion).
The following voltage protocol and assay paradigms were utilised in patch-clamp recordings:
Figure 1. Currents were elicited using the voltage protocol shown in (A), applied at 0.1 Hz, with peak inward current amplitudes measured at -100 mV (indicated by green cursor). Assay paradigms used in the manual patch-clamp assay are depicted in (B). Assay paradigms used for the automated patch-clamp assay are shown in (C). For both MPC and APC, whole-cell conformation is obtained in pH 7.4. Inward currents were stabilised in either pH 7.4 or 5.0.
Patch-clamp solutions: Optimised buffers for TRPML1-4A assays
The intracellular (ICS) and extracellular solutions (ECS) used in patch-clamp assays are summarised below:
1. Validation of the TPRML1-4A stable cell line using manual patch-clamp technique
Figure 2. Comparison of ML-SA5 modulation of TRPML1-4A in different pH conditions (MPC). (A) Representative current traces showing TRPML1-4A activation by increasing concentrations of ML-SA5 (0.01 – 1 µM). (B) Corresponding current vs. time plots from representative traces shown in (A). (C) Concentration-response curves of TRPML1-4A activation with ML-SA5 showing an increase in potency at pH 5.0 compared to pH 7.4. Data is normalised to the maximal current elicited by 1 µM ML-SA5. (D) Plot displaying current amplitudes for individual cells following application of 1 µM ML-SA5 in both pH conditions. TRPML1-4A currents were significantly larger in pH 5.0 compared to pH 7.4 (p=0.0055). Statistical significance was determined using an unpaired Student’s t-test (** p<0.01). Data represent mean ± S.D.
2. Development of a TPRML1-4A assay for use on the automated patch-clamp platform, Qube 384
Figure 3. Comparison of ML-SA5 modulation of TRPML1-4A in different pH conditions. (A) Representative current traces compiled from five recordings where each cell received one concentration of ML-SA5. (B) Representative current vs. time plot of one concentration of ML-SA5 (1 µM) followed by maximal current response elicited using 10 µM ML-SA5. (C) Concentration-response curves for TRPML1-4A activation by ML-SA5 showed an increase in potency at pH 5.0 compared to pH 7.4. Data was normalised to maximum current activation with 10 µM. (D) Plot displaying maximum current amplitudes elicited with 10 µM ML-SA5 from individual cells showed a significant increase in current size at pH 5.0 compared to pH 7.4. Statistical significance was determined using an unpaired Student’s t-test (**** p<0.0001). Data represent mean ± S.D.
3. Development of a fluorescence-based TPRML1-4A assay using the FLIPR platform
The reference agonist, ML-SA5 was tested at 10 concentrations (0.0003 to 10 µM) at pH 7.4.
Two assay runs were performed to ensure reproducibility between experiments.
Area under curve was calculated for each well and concentration-response curves were constructed.
Figure 4. TRPML1-4A FLIPR assay using the agonist ML-SA5. (A) Example screening plate consisting of assay standard (highlighted) and test compounds of unknown properties. (B) A summary of statistics for day 1 and day 2 plates including signal to background ratio and Z’ value. (C) Example kinetics traces for ML-SA5 (0.0003 -10 µM) extracted from day 1 plate data. Data corresponds to the ML-SA5-containing wells indicated by the labelled boxes in (A). (D) Concentration-response curves of TRPML1-4A activation by the agonist ML-SA5 in pH 7.4. Data show excellent reproducibility across two experimental days. Data represent mean ± S.D.
Conclusions
Metrion’s breakthrough in TRPML1-4A automated patch clamp and fluorescence-based electrophysiology
We successfully generated a HEK-TRPML1-4A stable cell line which can be functionally characterised using electrophysiological techniques.
Leveraging both patch-clamp electrophysiology and fluorescence-based assays, we were able to optimise screening services capable of studying TRPML1 modulators with excellent cross-platform correlation.
The EC50 values we obtained for ML-SA5 were in line with literature for FLIPR5 andQube6 platforms.
The pH sensitivity of TRPML1 was confirmed in our patch clamp assays showing greater response to ML-SA5 in acidic pH conditions.
The cardiac late Na+ current (late INa) generates persistent inward currents throughout the plateau phase of the ventricular action potential and is an important determinant of repolarisation rate, EADs and arrythmia risk¹. As inhibition of late INa can offset drug effects on hERG and other repolarising K⁺conductances, it is one of the key cardiac channels in the Comprehensive in vitro Pro-arrythmia Assay (CiPA) panel being developed by the FDA to improve human clinical arrythmia risk assessment²̛ ³.
Cardiac toxicity remains the leading cause of new drug safety side-effects. Current preclinical cardiac safety assays rely on in vitro cell-based ion channel assays and ex vivo and in vivo animal models⁽¹⁾. These assays provide an indication of acute risk but they do not always predict the effect of chronic compound exposure, as recently seen with oncology drugs. Therefore, new assays are required to characterise chronic structural and functional effects in human cells earlier in drug discovery. Impedance-based technology can provide more accurate chronic cardiotoxicity measurements in an efficient manner using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs).
