Introduction
CLC-Ka and CLC-Kb are members of the CLC family of chloride channels and transporters, crucial for maintaining chloride ion homeostasis across cell membranes. CLC-Ka is predominantly found in the thin ascending limb of Henle’s loop, while CLC-Kb is expressed in both the thick ascending limb of Henle’s loop and the distal convoluted tubule. These channels facilitate the reabsorption of chloride ions from the filtrate back into the bloodstream, essential for maintaining the body’s fluid and electrolyte balance. Additionally, these channels are expressed in the inner ear, where they are vital for normal hearing and balance.
Figure 1. Schematic representation of CLC-Ka and CLC-Kb structure together with their auxillary subunit β-barrtin.
Given their vital role in renal function, CLC-Ka and CLC-Kb present promising targets for therapeutic intervention in various kidney-related disorders. Modulation of these channels can potentially treat conditions characterized by improper chloride handling and disrupted electrolyte balance, such as hypertension, edema, congestive heart failure, Bartter syndrome, and chronic kidney disease.
Supporting Your Research
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Methods
HEK cells stably expressing CLC-Ka and CLC-Kb were produced by SB Drug Discovery. Whole cell patch-clamp experiments were carried out at room temperature using multi-hole chips on the SynchroPatch 384i automated electrophysiology platform. Currents were elicited by using repeated ramps, steadily increasing from -140 mV to + 60 mV over 500 ms, from a holding potential of – 30 mV.
Data analysis was performed using Data Control 384 V2.3 (Nanion) and GraphPad Prism V10.1.
Figure 2. Voltage Protocol. CLC-Ka and CLC-Kb currents were elicited by using repeated ramps, steadily increasing from -140 mV to + 60 mV over 500ms, from a holding potential of -30 mV. The red cursors indicate the area of analysis at – 140 mV, which is used to measure inward chloride currents
CLC-Ka Electrophysiology
Figure 3. Representative current trace, time course and concentration response curve showing the effect of activator against CLC-Ka and reproducibility across 3 plates. An example of superimposed CLC-Ka current traces under control conditions (grey line), in the presence of 100 μM Flufenamic acid (blue line) and a saturating concentration of 300 μM Flufenamic acid (black line).
CLC-Kb Electrophysiology
Figure 4. Representative current trace, time course and concentration response curve showing the effect of activator against CLC-Kb and reproducibility across 3 plates. An example of superimosed CLC-Kb current traces under control conditions (grey line), in the presence of 30 μM Flufenamic acid (blue line) and a saturating concentration of 300 μM Flufenamic acid (black line).
