Automated patch clamp analysis of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B K_ATP currents

Abstract

ATP-sensitive potassium (K_ATP) channels are therapeutic targets for numerous metabolic, cardiovascular, and neurological disorders. Drug development for K_ATP channels requires electrophysiology assays for detailed compound characterization. Parallel automated patch clamp (APC) techniques offer considerable advantages over low-throughput manual patch clamp electrophysiology. Here, we characterized the functional properties and pharmacological sensitivity of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B using a SyncroPatch 384PE APC instrument. Ruptured-membrane and perforated-patch whole-cell recordings in potassium fluoride and fluoride-free assay buffers and electrophysiology chips were evaluated for both subtypes. Effects of internal ATP and ADP and magnesium (Mg²⁺) addition were also assessed. Kir6.2/SUR1 currents were constitutively active in all potassium fluoride-based recordings, insensitive to activation by the SUR1 agonist, VU0071063, and variably inhibited by glibenclamide. Success rates, current run-down, and glibenclamide sensitivity were associated with internal buffer composition. Recordings in fluoride-free buffers revealed a minor population of constitutively active Kir6.2/SUR1 currents and a larger population of currents exhibiting low basal activity and activation by VU0071063. Success rate and stability were associated with internal buffer composition. Kir6.1/SUR2B currents, which were most readily assayed in ruptured-membrane and potassium fluoride-based conditions, were stable, activatable with pinacidil, and inhibited by glibenclamide. Our study sheds new light on the behavior of Kir6.2/SUR1 and Kir6.1/SUR2B currents under available APC conditions and represents an important step toward developing truly high-throughput APC techniques for K_ATP.

New & Noteworthy

Highly parallel automated patch clamp (APC) methods have revolutionized the way electrophysiology is performed in the pharmaceutical and biotech industries and increasingly in academic labs. Here, we characterized the functional and pharmacological properties of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B using a SyncroPatch 384PE APC instrument. The results of our studies highlight heretofore unappreciated effects of fluoride-base internal solutions on Kir6.2/SUR1 and provide foundational support for developing truly high-throughput electrophysiology methods for both drug targets.

Graphical Abstract

Introduction

ATP-sensitive potassium channels (K_ATP), composed of inward rectifier potassium (Kir) pore-forming Kir6.1 or Kir6.2 subunits and regulatory sulfonylurea receptor (SUR) SUR1, SUR2A, or SUR2B subunits, are critical elements of organismal integrative physiology as they link cellular metabolic status to electrical excitability.^1–5 Kir6.2/SUR1 channels are primarily found in pancreatic beta cells and neurons channels, whereas Kir6.1/SUR2B are most prominently expressed in vascular smooth muscle cells.^{2, 6–11} The cell- and tissue-specific expression of distinct channel subtypes creates important opportunities for developing targeted therapeutics to treat conditions such as neonatal diabetes, congenital hyperinsulinism, and hypertension.^{2, 12, 13}

K_ATP channels have been the targets of extensive drug discovery efforts since the 1940s^{2, 14}. However, there remain unmet and newly emerging needs for developing novel antagonists and agonists for the channel family. For example, Kir6.1/SUR2B inhibition is a promising new strategy for treating Cantú syndrome, a multi-organ disorder caused by gain-of-function Kir6.1/SUR2B mutations.^{2, 15, 16} Activation of Kir6.1/SUR1 channels holds promise for controlling appetite in patients with hyperphagic obesity.^{17, 18}

High-throughput quantitative methods enable the seamless integration of the various stages of drug development, from early-stage library screening to later stage compound characterization and optimization. We previously employed a quantitative fluorescence-based, high-throughput thallium flux assay for library screening against Kir6.2/SUR1 and Kir6.1/SUR2B and discovered novel modulators of both channel subtypes^{17, 19, 20}. Thallium assays are ideally suited for driving compound optimization with medicinal chemistry. Pairing K_ATP channel thallium assays with parental cell-type-matched assays of other channels allow for the rapid assessment of compound ancillary pharmacology. These assays benefit from their high throughput, robustness and scalability, but suffer from a lack of detailed kinetic information, voltage control, and reversibility.

More detailed characterization of K_ATP channel pharmacology requires voltage-clamp electrophysiology techniques, as they enable direct measurement of K⁺ ion conduction through the channel of interest. Voltage-dependency and kinetics and reversibility of drug action are easily assessed, as are potential effects of intracellular composition on channel pharmacology. Single-cell MPC techniques are considered the gold standard in the field, but suffer from low-throughput, reliance on highly specialized scientists, and consequently, high cost per data point. Highly parallel automated patch clamp (APC) methods have revolutionized the way electrophysiology is performed in the pharmaceutical and biotech industries and increasingly in academic laboratories^21–25. Planar electrodes fabricated from borosilicate glass separate independently perfused upper and lower chambers corresponding to the extracellular and intracellular compartments, respectively. Each well contains one or more laser-etched apertures of defined geometry and resistance that vacuum-trap cells dispensed into the upper chamber. Whole-cell access is gained by either rupturing the membrane with negative pressure or with the addition of perforating reagent to the internal buffer.

The SyncroPatch 384PE is a high-throughput automated patch clamp (APC) platform that combines scalability with high-fidelity electrophysiological measurements, enabling simultaneous recordings from up to 384 wells with giga-ohm (GΩ) seal quality. The platform replaces conventional glass pipettes with planar substrates containing small apertures, where cells in suspension are captured and sealed using suction. An automated liquid handling system ensures precise compound delivery, reducing variability and enhancing experimental reproducibility. This approach ensures precise and reproducible recordings while significantly increasing throughput.

Here, we characterized the functional and pharmacological properties of heterologously expressed Kir6.2/SUR1 and Kir6.1/SUR2B using a SyncroPatch 384PE APC instrument. The results of our studies highlight heretofore unappreciated effects of fluoride-base internal solutions on Kir6.2/SUR1 and provide foundational support for developing truly high-throughput electrophysiology methods for both drug targets.

Methods

Cell culture

T-REx^™-HEK-293 cell lines stably expressing Kir6.1/SUR2B and Kir6.2/SUR1 channels were generated and cultured as described previously^{19, 20}. Briefly, cells were cultured in 175 cm² flasks (Thermo Scientific, 159910) using DMEM media (Gibco, 11965092) with 10% FBS (bio-techne, MN S11150H), 1% PenStrep (Gibco, 16140), 250 μg/mL Hygromycin B (CORNING, 3-240-CR), and 5 μg/mL Blasticidine S HCl (Gibco A11139-03). The day of recordings, cells were dissociated with TrypLE reagent (Gibco, 12604-013) for 2-4 min at 37°C and then quenched with CHO-S-SFM II media (Gibco, 12052114) to stop dissociation. The cells were centrifuged and resuspended in external buffer (Table 1) at a density of 1-1.5 x10⁶ cells/mL, transferred to a cooled (10°C) reservoir on the SyncroPatch deck, and stirred at 200 rpm to prevent cell settling. Cells were transferred to NPC-384 electrophysiological recording chips with a robotic liquid handler (Biomek; Beckman Coulter).

Table 1. APC buffer composition.

Internal and external buffer composition for KF and FF APC recordings.

	External				Internal
Concentration (mM)	Divalent-free	Physiological	NMDG 60	NMDG 140	KF internal	FF internal
NaCl	140	140	80		10	10
KCl	4	4	4	4	10	10
CaCl2		2	2	4
MgCl2		1	1	1
HEPES	10	10	10	10	10	10
Glucose	5	5	5	5
NMDG-Cl			60	140
KF					110
K-gluconate						120
EGTA					10	5
pH	7.4	7.4	7.4	7.4	7.2	7.2
Osmolarity (mOsm)	290±3	298±3	298±3	285±3	285±3	285±3

Solutions and drugs

Buffer compositions are provided in Table 1. Glibenclamide (SUR1/SUR2 inhibitor) (Cat # 10238-21-8) and pinacidil (SUR2 activator) (Cat # 85371-64-8) were purchased from Millipore Sigma. The SUR1 agonist, VU0071063¹⁹, was purchased from Tocris (Cat # 7057). Glibenclamide, pinacidil, and VU0071063 stock solutions were prepared in DMSO and diluted immediately before experiments. Drug stocks were acoustically transferred from a source plate to a destination plate (Greiner 781280) using an Echo 555 Omics Liquid Handler (Labcyte) before dilution to a 2X concentration with NMDG 60 using a Multidrop Combi reagent dispenser (Fisher). Final 1X doses were achieved with a 1:1 volume dilution in the NPC384 chip well during experiments.

APC recordings

K_ATP currents were recorded at room temperature in both ruptured membrane (RM) and perforated patch (PP) modes using Nanion Synchropatch384PE. Figure 1A shows a representative screen shot of the user interface used to monitor the progress of recordings from all 384 wells simultaneously. Four-hole, medium-resistance NPC-384 chips (Nanion, 08 3007) were used for KF-based recordings, and single-hole NPC-384 chips (Nanion, 223121/51) were used for FF recordings. KF chips were filled with internal and external buffers (Table 1) before 20 μL of cell suspension (~1-1.5 ×10⁶ cells/mL) was transferred to each well from the cell reservoir. Negative pressure and seal enhancer solution (Table 1) were used to promote cell capture and sealing, respectively. FF chips were first primed with NaOH according to the manufacturer’s instructions but were otherwise used identically to KF chips. The RM configuration was achieved with three successive pulses of −200 mBar. PP mode was achieved by adding 5 μM escin (Sigma Millipore, E1378) to the internal buffer. Whole-cell access in RM and PP modes was assessed by monitoring membrane capacitance. K_ATP currents were evoked by voltage clamping cells at a holding potential of −75 mV, stepping the membrane potential (V_m) to −120 mV for 200 msec, ramping V_m to 120 mV over 2 sec, holding V_m at 120 mV for 100 msec, and then stepping V_m back to −75 mV. This step-ramp protocol was repeated every 20 sec. Baseline currents were measured for 2 min before adding 10 μM (1X) VU0071063 or pinacidil for 10 min before a final addition of 10 μM (1X) glibenclamide to terminate the experiment. Currents measured at 120mV were used for data analysis.

Fig. 1: Data acquisition and analysis.

(A) Screenshot of the automated patch clamp recording software. Raw current traces from each of the 384 wells of the patch clamp chip are displayed on the top left and individual highlighted wells are on the right. Inclusion criteria of ≥100MΩ seal resistance was used (blue wells 100MΩ-1G Ω and green wells ≥ 1GΩ). Cells constitutively expressing Kir6.1/SUR2B K_ATP channels were used. Each well shows the first 30 current amplitudes versus time traces, with the baseline trace in black and blue traces after application of Kir6.1/SUR2B specific activator 10 μM Pinacidil, followed by the blocker 10 μM Glibenclamide (not shown). (B) Analyzed data shows the average percent current activation with 10 μM Pinacidil from baseline (top left), average current response at +120mV over time (10 μM Pinacidil application highlighted in blue; middle left), and average current vs time traces before (black) and after 10uM Pinacidil (blue) application (bottom left) of all included wells. Responses from a few individual wells are shown on the right.

APC data analysis and statistics

SyncroPatch data analysis was performed using DataControl384 software (Nanion Technologies GmbH) (Fig. 1B). Current recordings were included in the analysis only if they were of at least −1 nA at −95 mV and exhibited clear inhibition by 10 μM glibenclamide. Each channel was manually inspected to ensure that it met inclusion criteria. Data are presented as means ± SEM. N represents the number of wells and success rate is the percentage of cells out of 192 (half-plate) or 384 (full plate) that met inclusion criteria. Concentration-response relationships were evaluated in single-dose experiments in which each well received only one dose of activator (i.e., VU0071063 or pinacidil) or inhibitor (i.e., glibenclamide). The 50% activation concentration (EC₅₀) or 50% inhibitor concentration (IC₅₀) were derived from single-site, four-parameter logistical fits to dose-response data in GraphPad Prism 9 (GraphPad Software). Statistical comparisons of current amplitudes were performed using an unpaired t-test or ANOVA, with p-value of less than 0.05 was considered as statistically significant.

Measurement of magnesium concentration

The soluble magnesium concentration in KF and FF internal buffers was measured using a Magnesium Reagent kit (Pointe Scientific, HM929-1000).

Results

APC analysis of Kir6.2/SUR1 currents in KF buffers

Kir6.2/SUR1 currents were first measured using the KF-based buffers and four-hole NPC384 chips. A monoclonal T-Rex-HEK-293-Kir6.2/SUR cell line previously validated for HTS was used ^{19, 26}. The internal KF buffer was supplemented with 1 mM ADP and 3 mM Mg²⁺ (KF-ADP/Mg²⁺). In ruptured-membrane (RM) mode, voltage clamp experiments revealed large-amplitude, glibenclamide-inhibitable currents that were insensitive to the SUR1 agonist, VU0071063 (Fig. 2A–B). Of the approximately 70% of wells meeting inclusion criteria (see Methods), 100% exhibited spontaneously active currents. The current amplitude at 120 mV declined by approximately 20% over 10 mins in the presence of VU0071063 (Fig. 2A–B and Table 2). Similarly, about 75% of wells met inclusion criteria using the perforated-patch (PP) mode, and 100% of those contained constitutively active currents (Fig. 2C–D and Table 2).

Fig. 2. Large spontaneous Kir6.2/SUR1 currents were observed using KF system in RM and PP configurations.

(A) Average current trace recorded at 120 mV in KF RM mode (n=223 wells, 2 independent experiments). (C) Average current trace recorded at 120 mV in KF PP mode (n=242 wells, 2 independent experiments). (B, D) Quantification of average current in KF at baseline, following stimulation with 10 μM VU0071063, and subsequent inhibition with 10 μM glibenclamide in RM and PP mode, respectively. Both experimental conditions had 1 mM ADP and 3 mM MgCl₂ in the internal KF solution.

Table 2.

Kir6.2/SUR1 currents with 1mM ADP and 3mM Mg²⁺ in internal buffer.

	KF chips		FF chip
	RM	PP	RM	PP
% Constitutively active (n)	70 (223)	75 (242)	2 (4)	7 (42)
% VU007163 activated	0	0	7 (13)	23 (179)
Baseline (nA)	23 ± 0.707	32.3 ± 0.69	1.9* ± 0.322, 9.9** ± 0.781	2.3* ± 0.190, 11.8** ± 1.324
VU0071063 activation (nA)	19.8 ± 0.651	30.6 ± 0.61	3.5* ± 0.632, 8.6** ± 0.867	7.0* ± 0.383, 9.8** ± 1.211
Glibenclamide inhibition (nA)	6.4 ± 0.427	10.5 ± 0.39	1.4* ± 0.215, 1.0** ± 0.2	2.2* ± 0.169, 2.3** ± 0.363
% Run-down	19.4	19.3	0*, 14.1**	0*, 20.5**
% Success Rate (n)	70 (112)	75 (242)	9 (17)	30 (221)

^*VU007163 activated current.

^**Constitutively active current.

() indicated samples size.

Data presented as mean ± SEM.

Because Kir6.2/SUR1 channels are opened by ADP binding to SUR1 in the presence of Mg²⁺, we explored whether their inclusion in KF-ADP/Mg²⁺ buffer led to constitutive channel activation.^{27, 28} Toward this end, Kir6.2/SUR1 currents were recorded in four additional buffer conditions: KF buffer (KF), KF with 3 mM Mg²⁺ (KF-Mg²⁺), KF with 1 mM ADP (KF-ADP), or KF with 1 mM ATP and 3 mM Mg²⁺ (KF-ATP/Mg²⁺). Success rate differed among conditions and had a rank-order of KF-Mg²⁺ (94.7%) > KF-ATP/Mg²⁺ (75%) > KF (67.7%) > KF-ADP (48%) (Table 3). Spontaneously active Kir6.2/SUR1 currents were observed in 100% of wells meeting inclusion criteria under all four experimental conditions (Fig. 3A to 3C). Rundown ranged from 14% to 23% with a rank-order of KF (22.65%) > KF-Mg²⁺ (21.74%) > KF-ADP (16%) > KF-ATP/Mg²⁺ (14%). (Table 3).

Table 3.

Success rate and % rundown of Kir6.2/SUR1 currents.

Chip type	Internal condition	% Success rate (n)	% Rundown
KF (RM)	No supplement	67.7 (260)	22.65
	+1mM ADP	48 (89)	16.1
	+1mM ATP + 3mM Mg2+	75 (144)	13.5
	+3mM Mg2+	94.7 (364)	21.74
FF (PP)	No supplement (VU0071063 activated)	16.5 (57)	0
	+ADP (VU0071063 activated)	22.5 (173)	0
	+Mg2+ (VU0071063 activated)	26 (202)	0
	No supplement (Spontaneous active)	9 (31)	48.9
	+ADP (Spontaneous active)	13 (101)	30.4
	+Mg2+ (Spontaneous active)	17 (135)	17.2

Fig. 3. Effects of Mg²⁺ and nucleotides in the internal solution on Kir6.2/SUR1 currents with KF system.

(A) Average current trace with KF internal solution supplemented with either 1 mM ATP + 3 mM MgCl₂ (black circles, n=89 wells, 2 independent experiments) or 1 mM ADP alone (grey squares, n=144 wells, 2 independent experiments). (B) Average current trace with KF internal solution supplemented with either 3 mM MgCl₂ alone (black circles, n=364 wells, 3 independent experiments) or no supplement (grey squares, n=260 wells, 3 independent experiments). (C) Quantification of average current (nA) at baseline, after stimulation with 10 μM VU063, and following inhibition with 10 μM glibenclamide for each internal buffer condition. (D) Dose-response curve of glibenclamide, recorded in KF RM configuration with 1 mM ADP supplemented in the internal solution.

Interestingly, the apparent sensitivity to 10 μM glibenclamide differed between conditions, ranging from approximately 75% inhibition in KF-ATP/Mg²⁺ and KF-ADP buffers to less than 50% in KF and KF-Mg²⁺ solutions (Fig. 3A–C). Mean ± SEM reversal potentials under KF and KF-Mg²⁺ conditions were −82.4 ± 0.3 (n=546) and −82.4 ± 0.3 (n=322), respectively, which is close to the calculated K⁺ Nernst potential of −84 mV (22 ºC). In addition, 1 mM Ba²⁺ addition following glibenclamide treatment blocked 90.9 ± 0.9% (n=128 wells) and 76.1 ± 2.9% (n=173 wells) of the baseline current at −120mV in KF and KF-Mg²⁺ conditions, respectively. Taken together, these data support the conclusion that while a small component of the glibenclamide-insensitive current may be attributable to leak, the larger residual current reflects an authentic reduction in Kir6.2/SUR1 sensitivity to glibenclamide. Glibenclamide concentration-response experiments in KF-ADP buffer yielded an IC₅₀ value of 29 nM (Fig. 3D), which is close to that reported from MPC experiments.²⁹

APC analysis of Kir6.2/SUR1 currents in FF buffers

Because Kir6.1/SUR1 channels were constitutively active in KF buffers, we next evaluated a FF buffer system recently developed by Nanion Technologies. Single-hole NPC384 chips and FF internal buffer containing 1 mM ADP and 3 mM MgCl₂ (FF-ADP/Mg²⁺) were initially used. In RM mode, the success rate in FF-ADP/Mg²⁺ buffer (~8%) was much lower than that observed with any of the KF buffers. However, unlike KF-based recordings, in which only spontaneously active currents were observed, we found both constitutively active (2%) and VU0071063-sensitive (7%) currents in RM FF-ADP/Mg²⁺ conditions (Fig. 4A). VU0071063 increased Kir6.2/SUR1 currents at 120 mV by approximately 2-fold (Fig. 4B and Table 2).

Fig. 4. Spontaneous and VU0071063-induced Kir6.2/SUR1 currents using FF system.

Average current traces showing spontaneous (grey squares) and VU0071063-induced (black circles) Kir6.2/SUR1 currents in FF RM mode (A, n=13 wells for VU007163-induced, n=4 wells for spontaneous current, 2 independent experiments) and FF PP mode (C, n=179 wells for VU007163-induced, n=42 wells for spontaneous current, 3 independent experiments). Quantification of average current (nA) in FF RM mode at baseline, after stimulation with 10 μM VU0071063, and following inhibition with 10 μM glibenclamide in FF RM mode (B) and FF PP mode (D). Internal solution with both experimental conditions contained 1 mM ADP and 3 mM MgCl₂.

We next measured Kir6.2/SUR1 currents in PP conditions in the hope of improving the assay’s success rate. Indeed, success rate increased modestly from 8% in RM FF-ADP/Mg²⁺ conditions to 30% in PP FF-ADP/Mg²⁺ conditions. Once again, two current populations were observed. 7% of wells contained constitutively active currents, whereas 23% of wells exhibited VU0071063-sensitive currents (Fig. 4C). VU0071063 activated Kir6.2/SUR1 by approximately 3-fold in PP FF-ADP/Mg²⁺ conditions. Both current populations were strongly inhibited by glibenclamide (Fig. 4D and Table 2).

We next evaluated the internal nucleotide and Mg²⁺ dependency in the PP FF-based system. Kir6.2/SUR1 currents were measured in FF buffer (FF), FF with 1 mM ADP (FF-ADP), or FF with 3 mM Mg²⁺ (FF-Mg²⁺). Overall, there was no substantial improvement in the percentage of wells containing spontaneously active vs VU0071063-activated currents or the amplitude of VU0071063-activated currents across the three conditions (Fig. 5A–C). However, one notable difference was the degree of channel rundown. The spontaneously active current ran down with a rank-order of FF (48.9%) > FF-ADP (30.4%) > FF-Mg2+ (17.2%), whereas the VU0071063-inducible current did not exhibited rundown (Table 3). Dose-response experiments with FF-ADP/Mg²⁺ internal buffer yielded a VU0071063 EC₅₀ of 5.2 μM (Fig. 4D), which is close to that reported from MPC recordings¹⁹.

Fig. 5. Effect of different internal solution conditions on Kir6.2/SUR1 currents using FF system in PP configuration.

Average traces of spontaneous (grey squares) and VU0071063-induced (black circles) currents with FF internal solution supplemented with 1 mM ADP alone (A, n=173 wells for VU007163-induced, n=101 wells for spontaneous current, 2 independent experiments), supplemented with 3 mM MgCl₂ alone (B, n=202 wells for VU007163-induced, n=135 wells for spontaneous current, 2 independent experiments), and without supplements (C, n=57 wells for VU007163-induced, n=31 wells for spontaneous current, 2 independent experiments). (D) Dose-response curve of VU0071063, recorded in FF PP configuration with 1 mM ADP and 3 mM MgCl₂ supplemented in the internal solution.

APC analysis of Kir6.1/SUR2B currents in FF and KF buffers

We next evaluated FF and KF assay systems for measuring Kir6.1/SUR2B channel activity. A monoclonal T-Rex-HEK-293-Kir6.1/SUR2B cell line previously validated for HTS was used for experiments²⁰. RM FF recordings failed to meet quality control criteria (data not shown). Despite a low success rate (9%) in PP FF recordings, Kir6.1/SUR2B currents exhibited modest activation with pinacidil and strong inhibition by glibenclamide (Fig. 6A, B). Success rate was also low in PP KF (~5%, Fig. 6C, D and Table 4) conditions but increased to 57% in the RM KF system (Fig. 6E, F and Table 4). Kir6.1/SUR2B currents exhibited 2-fold activation with pinacidil and robust inhibition with glibenclamide (Fig. 6E, F). Changing internal nucleotide and Mg²⁺ concentrations did not improve the success rate (Fig. 7A–E and Table 5). Concentration-response experiments in RM KF-ADP/Mg2+ conditions yielded an EC₅₀ of 7.62 μM (Fig. 7F), which is close to published values. ²⁶

Fig. 6. Kir6.1/SUR2B currents in FF and KF systems.

(A) Average current traces recorded in FF PP mode (A, n=44 wells, 2 independent experiments), KF PP mode (C, n=10 wells, 2 independent experiments), and KF RM mode (E, n=222 wells, 2 independent experiments). Quantification of average current (nA) at baseline, following stimulation with 10 μM Pinacidil, and subsequent inhibition with 10 μM glibenclamide in FF PP mode (B), KF PP mode (D), and KF RM mode (F). All 3 experimental conditions contained 1 mM ADP and 3 mM MgCl₂ in the internal solution.

Table 4.

Kir6.1/SUR2B currents with 1mM ADP and 3mM Mg²⁺ in internal buffer.

	KF chips		FF chip
	RM	PP	RM	PP
% Constitutively active	0	0	\	0
% Pinacidil activated (n)	57.7 (222)	5.2 (10)	\	9 (44)
Baseline (nA)	2.2 ± 0.126	3.1 ± 0.300	\	1.1 ± 0.242
Pinacidil activation (nA)	3.2 ± 0.142	4.2 ± 0.330	\	1.9 ± 0.323
Glibenclamide inhibition (nA)	2.1 ± 0.130	2.7 ± 0.250	\	1.3 ± 0.224
% Run-down	0	0	\	0
% Success Rate (n)	57.7 (222)	5.2 (10)	0	9 (44)

Parentheses indicated samples size. Data presented as mean ± SEM.

Fig. 7. Effect of different internal solution conditions on Kir6.2/SUR1 current recorded using KF system.

Average current traces following stimulation with 10 μM Pinacidil, and subsequent inhibition with 10 μM glibenclamide using KF internal solution supplemented with 1 mM ATP and 3 mM MgCl₂ (A, n=33 wells, 2 independent experiments), supplemented with 3 mM MgCl₂ alone (B, n=20 wells, 2 independent experiments), and without supplementation (C, n=39 wells, 2 independent experiments). (D) Percent Pinacidil activation in with internal supplements as described in A-C. (E) Dose-response curve of Pinacidil in KF RM configuration with 1 mM ADP and 3 mM MgCl₂ supplemented in the internal solution.

Table 5. Success rate and % rundown of Kir6.1/SUR2B currents.

Success rate and % rundown of Kir6.1/SUR2B recordings with KF PP mode in 3mM Mg²⁺ only, 1mM ADP only, and 3mM Mg²⁺ only with 1mM ADP conditions.

Chip type	Internal condition	% Success rate (n)	% Rundown
KF (PP)	No supplement	12.5 (33)	0
	+ADP	5 (20)	0
	+Mg2+	20.3 (39)	0
FF (RM)	No supplement	0	\
	+ADP	0	\
	+Mg2+	0	\

Free Mg²⁺ concentrations in KF and FF buffers

Given the apparent association of some assay metrics with internal Mg²⁺ supplementation, we measured the concentration of free Mg²⁺ in KF and FF buffers. For FF internal solution samples, supplementing the internal solutions with 1 mM, 2 mM, and 3 mM concentrations resulted in 0.9 mM, 2.1 mM, and 2.9 mM free Mg²⁺ concentrations, respectively. However, the concentration of Mg²⁺ in all KF-based buffers was below the 10 μM limit of detection (Table 6).

Table 6.

Free Mg²⁺ concentrations in KF and FF buffers

Solution	Free Mg2+ concentration
KF	Below sensitivity range (<10 μM)
KF + 1 mM MgCl2	Below sensitivity range (<10 μM)
KF + 2 mM MgCl2	Below sensitivity range (<10 μM)
KF + 3 mM MgCl2	Below sensitivity range (<10 μM)
K Gluconate	Below sensitivity range (<10 μM)
K Gluconate + 1 mM MgCl2	900 μM
K Gluconate + 2 mM MgCl2	2.1 mM
K Gluconate + 3 mM MgCl2	2.9 mM
QC sample 500 μM	548 μM
QC sample 1 mM	1.1 mM

Discussion

Here we evaluated the functional and pharmacological properties of the two major K_ATP channel subtypes, Kir6.2/SUR1 and Kir6.1/SUR2B, in APC electrophysiology assays, as critical first steps toward developing high-throughput methods for these two important drug targets. Robust K_ATP currents could be measured from both channel subtypes, however, their properties were strongly dependent on the buffer system used.

In all KF-based recordings, Kir6.2/SUR1 currents were constitutively active and insensitive to further activation with VU00710630. Kir6.2/SUR1 channels are regulated by intracellular nucleotides and Mg²⁺ in complex ways. In the absence of nucleotides, K_ATP channels are constitutively open due in part to relief of Kir6.2 pore block by free ATP. The IC₅₀ for ATP-dependent inhibition of Kir6.2/SUR1 is approximately 10 μM³⁰, which is 100-fold lower than the concentration of ATP used in KF-based experiments. However, we saw no evidence of ATP-dependent inhibition of Kir6.2/SUR1 in our experiments. Mg²⁺ complexes of either ATP or ADP stimulate Kir6.2/SUR1 activity through interactions with separate binding sites on SUR1. Importantly, nucleotide-dependent channel activation does not occur in the absence of Mg²⁺³¹ . The free Mg²⁺ concentration in KF buffers supplemented with 3 mM Mg²⁺ is below 10 μM due to MgF₂ complex formation. It is plausible that under KF-based recording conditions, Kir6.2/SUR1 can be activated by Mg²⁺-nucleotide complexes at doses lower than those studied using more physiological buffers. Dose-response experiments in which intracellular Mg²⁺-nucleotide concentrations are varied using the internal perfusion capabilities of the SyncroPatch 384PE may help answer this question.

Internal buffer supplementation experiments revealed a modest association of success rate and run-down with internal nucleotides and Mg²⁺. One observation of potentially significant importance for assay development is the apparent dependency of glibenclamide sensitivity on the internal buffer composition. Each experiment was terminated with the addition of 10 μM glibenclamide to block K_ATP. The residual, glibenclamide-insensitive current at 120 mV varied widely between ~2 nA to ~13 nA with a rank order of KF ~ KF/Mg²⁺ > KF-ADP > KF-ADP/Mg²⁺ > KF-ATP/Mg²⁺. The simplest explanation, and one we favor, is that Kir6.2/SUR1 sensitivity to glibenclamide is enhanced in the presence of intracellular nucleotides. Indeed, there is good evidence that Kir6.2/SUR1 sensitivity to tolbutamide, whose binding site overlaps with that of glibenclamide, is enhanced by Mg²⁺/nucleotides^{32, 33,34}. Glibenclamide dose-response experiments across different internal buffers will clarify if inhibitor sensitive to internal nucleotides and Mg²⁺.

In FF-based experiments, we identified a small spontaneously active population of Kir6.2/SUR1 channels and a second larger population characterized by low basal current and sensitivity to VU00710630. The FF system thus offers the advantage over the KF system of enabling studies of Kir6.2/SUR1 activators. However, this advantage comes at the expense of lower success rates, which ranged from 2-23% in FF- vs 46-99% in KF-based conditions. Gaining whole-cell access in FF buffers with membrane rupture was not very successful (data not shown) and limited assay throughput. Success rate was threefold higher in perforated vs ruptured patch. Whether inadvertent membrane rupture and cytosolic dialysis under “perforated patch” conditions accounts for the observed spontaneous channel activation awaits further study. At present, the FF system is not suitable for high-throughput APC screening for Kir6.2/SUR1.

Internal Mg²⁺ and nucleotide supplementation experiments in PP FF conditions yielded at least two notable observations. First, the spontaneously active Kir6.2/SUR1 current was mostly stable in FF buffer containing both ADP and Mg²⁺, and highly unstable in the absence of both supplements. The rank-order of run down was FF(48.9%)> FF-ADP (30.4%)> FF-ADP/Mg²⁺(20.5%) FF-Mg²⁺ (17.2%)(Tables 2 and 3). We postulate that the current stability observed in FF-ADP/Mg²⁺ conditions is due to continuous channel stimulation by ADP/Mg²⁺, whereas current rundown reflects the depletion of these activating complexes in the other conditions. Second, unlike the KF-based system, the glibenclamide-insensitive residual current was not Mg²⁺ or nucleotide dependent in spontaneously active or VU00710630-activated channel populations. Therefore, any of the internal buffers should be suitable for inhibitor studies in the FF system.

We did not observe constitutively active Kir6.1/SUR2B currents under any recording condition tested. Pinacidil-activated currents were observed under both KF and FF conditions. Success rate was only 5-9% in perforated patch mode with either buffer system, but up to 65% in ruptured-patch KF-based recordings. The pinacidil-activated current was larger in KF-ADP/Mg²⁺ vs. KF-Mg²⁺ buffer, suggesting a role of ADP in pinacidil sensitivity.

In conclusion, we have characterized the functional and pharmacological properties of Kir6.2/SUR1 and Kir6.2/SUR2B currents under different buffer conditions using APC electrophysiology. Kir6.2/SUR1 currents were constitutively active in all KF-based recordings and inhibitable by glibenclamide but exhibited low basal activity and sensitivity to VU063 in a majority of wells in the FF system. The KF- and FF-based systems are therefore best suited for studying Kir6.2/SUR1 inhibitors and activators, respectively. Without further optimization, the use of the FF-based system comes at the expense of lower success rates. Kir6.1/SUR2B currents exhibited low basal activity and sensitivity to pinacidil activation glibenclamide inhibition in all assay conditions tested. The substantially higher success rate using ruptured-patch KF-based conditions makes this the preferred system for Kir6.1/SUR2B. As sophisticated APC instrumentation is used routinely in the pharmaceutical and biotechnology industries, and increasingly in academic laboratories, this study provides an important framework for developing truly high-throughput electrophysiology methods for these two important channels.