IonWorks® HT

Kv Potassium Channel Screening Assay

Potassium channels are perhaps the most ubiquitously expressed of all mammalian ion channels. Voltage-gated potassium channels are activated at depolarizing potentials and underlie the repolarization kinetics of action potentials. Potassium channels have been implicated in diverse disease states such as epilepsy, deafness and movement disorders.

• K_v1.5 potassium channels
• 4-AP dose response experiment completed in 45 minutes
• Consistent results from one PatchPlate to the next
• Consistent intra-plate results
• Conclusions
• References and recommended reading

K_v1.5 potassium channels

Voltage-gated potassium channels (K_v) are a subset of the ion channel superfamily containing six transmembrane spanning domains. A functional K_v channel requires four individual pore-forming α subunits, which includes an intrinsic voltage sensor, a K⁺ selectivity filter, as well as activation and inactivation gates. K_v1.5 is one member of the K_v family that is expressed in atrial myocytes, as well as pulmonary and vascular smooth muscle tissues. Here, a stably-transfected CHO cell line expressing the K_v1.5 channel was used in IonWorks HT experiments. Figure 1 shows a representative trace of K_v1.5 currents measured on the IonWorks HT system.

		Figure 1. Potassium channel currents measured in CHO cells expressing Kv1.5 channels. Pre-compound trace shown in black, 3.9 mM 4-aminopyridine (4-AP) shown in orange. Vertical bar is 500 pA, horizontal bar is 10 ms.

4-AP dose response experiment completed in 45 minutes

Experiments performed on the IonWorks HT system with an optimized CHO cell line expressing K_v1.5 had a success rate of 60-85%—thus generating over 230 data points in approximately 45 minutes. A 4-aminopyridine (4-AP) dose response curve, replicated 8-fold, was performed on the IonWorks HT system to compare variability among multiple PatchPlate™ experiments, as well as variability between compound plate rows used within a single PatchPlate experiment. The compound plate was made by serially diluting 4-AP in 10 columns of a 96-well plate (the last two columns were reserved for positive and negative controls). Using a multi-channel pipettor, stock solution of 4-AP was transferred to the first column (A1-H1) of the 96-well compound plate. One-to-three (1:3) serial dilutions were performed by transferring solution in column 1 to column 2, then mixing 10 times and changing tips. The process was repeated until 1:3 serial dilutions were made through column 10. Positive control was 3.5 x 10^-2 M 4-AP in all the wells of column 11 (A11-H11), and negative control was PBS buffer alone, in column 12 (A12-H12) (see Figure 2).

Solution from each well was transferred to four wells in the PatchPlate, thus resulting in quadruplicate sampling for every compound. This greatly increased the likelihood that all compounds are successfully tested and also increased the statistical validity of each data point.

		Figure 2. Compound plate layout for replicate dilution series of 4-AP. Highest concentration of 4-AP in all wells of Column 1 was 3.5 x 10-2 M. Each subsequent column (2-10) was serially diluted 1:3 and [4-AP] is indicated. Columns 11 and 12 contained 3.5 x 10-2 and 0 M 4-AP (positive and negative control), respectively.

consistent results from one PatchPlate to the next

Four PatchPlate experiments were performed using CHO cells expressing K_v1.5 channels and the compound plate described in Figure 2. Since each PatchPlate run is 45 minutes, it took approximately 4 hours to complete this experiment. It is possible to routinely conduct 10 PatchPlate runs per eight-hour shift. IC₅₀ curves were generated for each PatchPlate and shown in Figure 3. In this case, concentrations were pooled and each data point was assayed 32 times, since the dilution series is replicated eight-fold in the compound plate and four-fold each in the PatchPlate (8x4). Considering drop-outs, each data point consists of n<25.

		Figure 3. IC50 curves for four separate PatchPlate experiments demonstrating the ability of IonWorks HT to rapidly collect dose response data. Each curve consists of over 250 data points with individual data points obtained from separate cells (mean ± SEM). IC50 values are indicated.

consistent intra-plate results

IC₅₀ values were compared within a single PatchPlate by plotting each dilution series of the compound plate. Results are summarized in Figure 4. In this case, n=4 for each data point within a dilution series, and the eight dilution series are shown as individual lines (varying color). Statistical analysis of intra-plate comparisons is also shown.

		Figure 4. Sixteen IC50 curves obtained from two runs. Eight individual 10-point curves/run, from eight compound plate rows. All concentrations for each curve are from a maximum of four replicates or eight replicates for highest concentration (<45 minutes/run). IC50 values are mM.

conclusions

These experiments demonstrate the robustness of IonWorks HT for performing consistent, highly reliable and reproducible statistical analysis of K_v1.5 currents expressed in stably-transfected cell lines. Furthermore, IonWorks HT experiments are at least two orders of magnitude faster than comparable experiments using conventional techniques. A case in point, the experiments described on this page were collected in less than four hours and represent over 900 data points (230 x 4).

For more details on experiments described on this page, download our application note entitled K_v1.5 Potassium Channel Assay Using IonWorks™ HT (registration required).

references and recommended reading

Adda, S., B.K. Fleischmann, et al., Expression and function of voltage-dependent potassium channel genes in Human airway smooth muscle. J. Biol. Chem., 271:13239-13243, 1996.

Schram, G., M. Pourrier, et al., Differential distribution of cardiac ion channel expression as a basis for regional specialization in electrical function. Circ. Res., 90:939-950, 2002.

Foundations of Cellular Neurophysiology. Daniel Johnston and Samuel Miao-Sin Wu, The MIT Press, Cambridge, MA, 1995.

Ion Channels and Disease. Frances M. Ashcroft, Academic Press, San Diego, CA, 2000.

Ionic Channels of Excitable Membranes, third edition. Bertil Hille, Sinauer Associates Inc., Sunderland, MA, 2001.