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Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions a
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Haijun Chen , Dongchuan Zuo , Jianing Zhang , Min Zhou & Liqun Ma
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Department of Biological Sciences; University at Albany; State University of New York; Albany, NY USA b
Department of Neuroscience; The Ohio State University Wexner Medical Center; Columbus, OH USA Published online: 23 Jan 2015.
Click for updates To cite this article: Haijun Chen, Dongchuan Zuo, Jianing Zhang, Min Zhou & Liqun Ma (2014) Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions, Channels, 8:6, 503-508, DOI: 10.4161/19336950.2014.973779 To link to this article: http://dx.doi.org/10.4161/19336950.2014.973779
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Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions Haijun Chen1,*, Dongchuan Zuo1, Jianing Zhang1, Min Zhou2, and Liqun Ma1 1
Department of Biological Sciences; University at Albany; State University of New York; Albany, NY USA; 2Department of Neuroscience; The Ohio State University
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Wexner Medical Center; Columbus, OH USA
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Keywords: *Correspondence to: [email protected]
Haijun
Chen;
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Submitted: 04/23/2014 Revised: 07/30/2014 Accepted: 10/02/2014 http://dx.doi.org/10.4161/19336950.2014.973779 www.landesbioscience.com
t is generally expected that 2-pore domain KC (K2P) channels are open or outward rectifiers in asymmetric physiological KC gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K2P subfamilies in transfected Chinese hamster ovary cells with patchclamp technique, and examine whether their I-V relationships are described by the GHK current equation. K2P channels display 2 distinct types of I-V curves in asymmetric physiological KC gradients. Two K2P isoforms in the TWIK subfamily conduct large inward KC currents and have a nearly linear I-V curve. Ten isoforms from 5 other K2P subfamilies conduct small inward KC currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K2P channels. The Rectification Index, a ratio of limiting IV slopes for outward and inward currents, is used to quantitatively describe open rectification of each K2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K2P channels and suggest that TWIK KC channels have a unique feature in regulating cellular function. Channels
Introduction Background or leak KC conductance has been observed in excitable cells for several decades. They play a key role in setting the rest membrane potential. Twopore domain KC (K2P) channels mediate leak KC conductance.1-4 Mammalian K2P channels are divided into 6 subfamilies: TWIK, TREK, TALK, TASK, THIK, and TRESK, based on the sequence homology and functional similarity2 (Fig. 1A). They share the same topology in the plasma membrane and each K2P subunit contains 4 transmembrane segments and 2 asymmetric pore-forming loops (P1 and P2)5,6 (Fig. 1B). K2P channels operate with simple electrochemical diffusion and participate in the maintenance of the resting potential. They regulate cellular function in response to various physiological stimuli such as pH, neurotransmitters, and volatile anesthetics.3 As leak KC conductance previously recorded in neurons is outward rectifying and follows the Goldman-Hodgkin-Katz (GHK) current equation,7-9 it is generally expected that K2P channels are open or outward rectifiers in asymmetric physiological KC gradients.1,10 Since the first mammalian K2P channel was cloned in 1996, K2P channels have been studied extensively. Among 15 mammalian K2P channels, TWIK-3, TASK-5, and THIK2 channels do not produce detectable currents in heterologous expression systems; 503
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they have the same topology in the plasma membrane. Two isoforms in the TWIK subfamily have a linear or nearly linear IV curve, which does not exhibit the GHK-like open rectification, whereas 10 isoforms from other 5 K2P subfamilies are open rectifiers and their I-V curves follow the GHK current equation either completely or very closely. The Rectification Index, a ratio of the I-V slopes of K2P outward and inward currents, is employed to quantitatively describe the extent of open rectification of these K2P isoforms. These results precisely classify a basic property of K2P channels and imply a unique functional feature of TWIK KC channels.
Results and Discussion
Figure 1. Current-voltage relationships of TWIK KC channels under asymmetric physiological KC gradients. (A) 15 mammalian K2P isoforms in 6 subfamilies. TRESK-1 and TRESK-2 represent human and mouse TRESK, respectively. (B) Topology of a K2P subunit. (C and D) Whole-cell ramp currents (black lines) of rat TWIK-1 (C) and TWIK-2 (D) KC channels heterologously expressed in CHO cells are shown (n D 5). TWIK currents were blocked by quinine, a KC channel blocker (purple lines). Red lines are fits with the GHK current equation for K2P currents in test voltages from -140 mV to C80 mV with 10 mV increment (except 0 mV), when PNa/PK values were held at measured values in experiments. Blue lines are fits for the same data points with straight lines. Gray boxes indicate relatively large inward KC currents.
other 12 functional K2P channels are defined as open or outward rectifying KC channels in asymmetric physiological KC gradients.2 TASK-1 KC channels are perfect leak KC channels, as their functional properties match very well with leak KC conductance previously discovered in neurons.7 Whole-cell currents of TASK-1 KC channels heterologously expressed in mammalian cell lines are perfectly fitted with the GHK current equation.10 TWIK-1, the first cloned mammalian K2P channel, was first defined in the Xenopus oocyte expression system as a weakly inward rectifying KC channel because of its weak rectification at test voltages above C20 mV,11 but how TWIK-1 KC channels behave at the voltages more negative than the reversal potential was not addressed. It becomes more complicated to define TWIK-1 KC channels due to contradictory observations whether
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TWIK-1 KC channels show weakly inward rectification.12,13 Single channel recordings in another study suggested that TWIK-1 KC channels are open rectifiers between -100 and C80 mV.12 However, recent studies indicate that whole-cell currents of TWIK-1 and TWIK-2 KC channels heterologously expressed in mammalian cell lines seem not exhibiting the GHK-like open rectification,14-17 as their inward KC currents are relatively large. In this study, we systematically investigate the I-V relationships of all isoforms from 6 K2P subfamilies, which are functionally expressed in Chinese hamster ovary (CHO) cells, and then examine whether their I-V curves are described by the GHK current equation. We demonstrated that K2P channels display 2 distinct types of I-V relationships in asymmetric physiological KC gradients, even though
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TWIK-1 and TWIK-2 KC channels have a linear current-voltage relationship under physiological KC gradients We first examined whether whole-cell currents of TWIK KC channels, which were heterologously expressed in CHO cells, exhibit open rectification under asymmetric physiological KC gradients, since previously reports imply that TWIK KC channels seem not to show such a behavior.14-17 We use whole-cell currents of K2P channels in transfected CHO cells to assess their open rectification with the GHK current equation, based on 2 basic assumptions. First, recorded whole-cell currents are KC-selective currents via K2P channels. Endogenous currents in CHO cells are very small and neglected.18 Measured reversal potentials and calculated NaC to KC relative permeability of K2P channels support the assumption. Second, gating and ionic block, or fundamental asymmetry structures of the K2P openpore do not contribute to open rectification, since I-V curves of most K2P channels are very consistent in different voltage-clamp recording configurations such as whole-cell and inside-out recordings; all K2P channels share similar structures of the pore; it is generally accepted that the gating process do not affect I-V shapes of K2P channels. In asymmetric physiological KC gradients, TWIK-1 and TWIK-2 KC channels conduct relatively large inward KC currents in the voltages
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more negative than the reversal potential (gray boxes in Fig. 1C and D), so their IV curves are not open rectifying at all. After establishment of stable whole-cell configurations, outward KC currents of TWIK-1 KC channels are fitted closely with the GHK current equation and it may be the reason why a previous study defined TWIK-1 KC channels as open rectifying channels in test voltages between -100 and C80 mV.12 However, in a full range of test voltages, the GHK open rectification does not occur in I-V curves of TWIK-1 KC channels. Instead, TWIK-1 I-V relationships are nearly linear (blue and black lines, Fig. 1C). The behavior of TWIK-2 KC channels is more obvious. TWIK-2 I-V curves do not have any rectification, consistent with previous observations.16,17 They can be fitted very well with a straight line in all tested voltages (blue and black lines, Fig. 1D). Thus TWIK currents in the voltages more negative than the reversal potentials do not exhibit the GHK open rectification. To quantitatively describe the rectification of whole-cell currents of K2P channels, we employ a parameter, the Rectification Index, which is a relative ratio of the slopes of outward currents at test voltages between C20 and C80 mV and inward currents at test voltages
between ¡140 and ¡80 mV (column 1 and 2, Table 1)(Fig. 2). These 2 slopes actually reflect outward and inward conductance of K2P channels, respectively. TWIK-1 and TWIK-2 KC channels have a Rectification Index close to 1 (column 3, Table 1), supporting that their I-V curves are nearly linear. It is also worth to note that both TWIK-1 and TWIK-2 KC channels do not show weakly inward rectification at test voltages above C20 mV after establishment of stable whole-cell configurations, in disagreement with previous observations on weakly inward rectification of TWIK-1 KC channels heterologously expressed in Xenopus oocytes.11 K2P channels in TREK, TALK, TASK, THIK, and TRESK subfamilies show the GHK-like open rectification under physiological KC gradients We further similarly studied other 10 K2P isoforms in transfected CHO cells. Consistent with previous studies,10,19 TASK-1 and TASK-3 KC channels conduct small inward KC currents in the membrane potentials more negative than the reversal potential (Fig. 2D and E) and exhibit weakly open rectification in 5 mM extracellular KC. Their I-V relationships are perfectly fitted by the GHK current
equation and have a Rectification Index of »10 and »20, respectively. TRAAK and TASK-2 (a member of the TALK subfamily) KC channels also have I-V curves with Reflection Index of »19 and »15, respectively, which are nearly described by the GHK current equation, so their currents are also weakly open rectifying (Fig. 2C and G). Alkaline pH-activated TALK-1 and TALK-2 KC channels did not produce detectable whole-cell currents in transfected CHO cells at bath solutions with pH 9. Previous reports showed that their whole-cell currents, which were recorded in COS cells, are open rectifying,20 and TALK-1 I-V curve is fitted well with the GHK current equation (Fig. 2F). Other 4 tested K2P channels are basically open rectifiers, although their currents are not exactly overlapped with the lines predicted by the GHK current equation. Compared to TASK and TALK KC channels, TREK-1, TREK-2, and TRESK2 KC channels exhibit stronger open rectification and conduct much less inward KC currents in the membrane potentials more negative than the reversal potential (Fig. 2A, B and I). Their Rectification Indexes are »70, »68, and »44, respectively. Previously studies defined TREK-1 and TREK-2 channels as outward rectifiers,19 because under recording solutions
Table 1. Quantitative classification of K2P channels based on rectification. Slopes for recorded outward and inward currents were listed for all tested K2P isoforms in 6 subfamilies, while Rectification Index and PNa/PK values were compared side-by-side from the GHK fits and recorded K2P currents. Outward and inward current slopes were obtained as described in details in Figure 2. The Rectification Index of recorded K2P currents represents a ratio of outward and inward slopes of each K2P isoform. The Rectification Index of the GHK fits was similarly obtained from those fitted lines and its value is »9 for all K2P channels. The PNa/PK values predicted by the GHK fits were calculated by 2 parameters, PK(F2/RT) and PNa(F2/RT), which were generated by the GHK fits. Measured PNa/PK values were calculated from reversal potentials of recorded K2P currents. *: The Rectification Index for K2P isoforms is significantly different from that for TASK-1 or TASK-3 (P < 0.005). Hold: the GHK fits were completed when PNa/PK values were held at those measured in experiments. 1. Outward and inward current slopes, Rectification Index, and PNa/PK of K2P channels Current slope (10¡9 A/V) K2P subfamily
TWIK TREK
TALK TASK THIK TRESK
K2P Isoform
TWIK-1/K2P1 TWIK-2/K2P6 TREK-1/K2P2 TREK-2/K2P10 TRAAK/K2P4 TASK-2/K2P5 TASK-1/K2P3 TASK-3/K2P9 THIK-1/K2P13 TRESK-2/K2P18
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Outward
Inward
6.6 § 1.1 27.2 § 9.6 107.2 § 18.4 230.2 § 29.4 253.9 § 31.3 74.4 § 17.0 26.5 § 4.2 80.6 § 15.5 50.5 § 9.4 59.1 § 11.1
5.7 § 0.8 23.4 § 11.5 2.1 § 0.6 3.4 § 0.7 12.7 § 0.2 5.8 § 2.3 2.6 § 0.4 3.9 § 0.4 8.6 § 1.1 1.5 § 0.4
Recorded current Rectification Index
GHK fit Rectification Index
Measured P Na/P K
GHK fit predicted P Na/PK
Measured cell number
1.2 § 0.1* 1.4 § 0.2* 69.8 § 11.8* 67.6 § 12.3* 19.1 § 3.7 15.4 § 1.9 10.3 § 0.8 20.5 § 2.3 5.7 § 0.5* 44.3 § 5.2*
8.9 § 0.1 8.9 § 0.1 9.1 § 0.1 9.0 § 0.1 8.9 § 0.2 8.6 § 0.2 8.9 § 0.2 9.2 § 0.1 8.8 § 0.1 9.3 § 0.1
0.006 § 0.001 0.007 § 0.001 0.004 § 0.001 0.002 § 0.001 0.009 § 0.002 0.008 § 0.001 0.005 § 0.002 0.002 § 0.001 0.003 § 0.001 0.004 § 0.001
Hold Hold 0.005 § 0.002 0.003 § 0.001 0.006 § 0.001 0.005 § 0.001 0.004 § 0.001 0.002 § 0.001 0.005 § 0.001 0.002 § 0.001
5 5 9 5 5 5 7 6 5 7
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Figure 2. Current-voltage curves of other 10 K2P channels under asymmetric physiological KC gradients. Whole-cell ramp currents (black lines) of rat TREK-1 (A), human TREK-2 (B), human TRAKK (C), human TASK-1 (D), human TASK-3 (E), human TASK-2 (G), mouse THIK-1 (H), and mouse TRESK-2 (I) KC channels heterologously expressed in CHO cells are shown (n D 5–9). Red curves are fits with the GHK current equation for K2P currents in test voltages from -140 mV to C80 mV with 10 mV increment (except 0 mV), yielding PNa/PK values for each K2P isoform (column 6, Table 1). Blue and orange lines are fits with straight lines for K2P currents in test voltages from C20 mV to C80 mV and from -140 mV to -80 mV with 20 mV increments, respectively, yielding slopes of outward and inward currents (column 1 and 2, Table 1). Data points (open circles) in F are adapted from the whole-cell (COS cell) ramp currents in test voltages from -100 mV to C80 mV with 20 mV increment (except 0 mV) in Ref. 20.
with symmetrical 140 mM KC these channels may still show outward rectification. In contrast, THIK-1 KC channels show the weakest open rectification and conduct relatively larger inward KC currents in the membrane potentials more negative than the reversal potential (Fig. 2H), so THIK-1 KC channels have an I-V curve with a Rectification Index of »6. These 10 K2P isoforms from 5 subfamilies are open rectifiers under quasiphysiological ionic conditions. Several GHK fits have clear deviations from recorded K2P currents such as
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sharper curvature (Fig. 2H and I). These deviations are not measuring errors, as they can be repeated. Under our recordings ionic conditions the GHK fits give an ideal reversal potential of -81 to -83 mV for all K2P channels, the deviations at test voltages between the reversal potential and C80 mV reflect the differences of K2P outward conductance from the GHK prediction, which are partially implied by K2P outward current slopes (Table 1); the deviations at test voltages between ¡140 mV and the reversal potential reflect the differences of K2P inward
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conductance from the GHK prediction, which actually indicates the extent of open rectification in different K2P isoforms. We have indicated that all 12 functional K2P channels show 2 different types of I-V curves in asymmetric physiological KC gradients, although they have the same topology. First, 10 K2P isoforms from TREK, TALK, TASK, and THIK, and TRESK subfamilies are open rectifiers that completely or nearly follow the GHK current equation. As K2P channels operate with simple electrochemical diffusion, in asymmetric physiological KC gradients
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(5 mM extracellular and 140 mM intracellular KC in our recording bath solutions) driving forces of KC against the KC electrochemical concentration differences should result in the GHK open rectification. However, some isoforms in these 5 K2P subfamilies still show clear differences in the extent of open rectification, as their Rectification Indexes suggest. We hypothesize that such differences are determined by slight diversities in molecular and structural bases of the ion-conductance pathway among K2P isoforms because the homology among K2P isoforms is low. The unique extracellular Cap structure of K2P channels may be a major contributor as it creates an extracellular ion pathway that is markedly different from that observed in other KC channel structures.5,6 For example, TRESK-2 KC channels have an extracellular domain in P1-looping significantly different from those in other K2P isoforms,21 and show strong open rectification with the smallest slope of inward currents (Table 1). In a contrast, TWIK-1 and TWIK-2 KC channels have a linear IV curve that does not show the GHK-like open rectification. They not only conduct large outward KC currents, but also conduct large inward KC currents and exhibit anomalous behaviors, reminiscent of conventional inward rectifying KC channels. Therefore, TWIK KC channels could play a unique role in regulation of cellular function.
Materials and Methods All tested K2P cDNAs were cloned in pMAX or pRAT vectors described previously,14,15 except that TWIK-2 cDNA was cloned into pEYFP-C1 vectors. CHO cells at least 80% confluence were transfected by Lipofectamine 2000 (Invitrogen) with 3 mg of K2P channel plasmids and 1 mg of pEGFP plasmids and studied 24 hours later. GFP expression was used to identify effectively transfected CHO cells. Whole-cell patch-clamp recordings were performed and data were analyzed, as described previously.14 Briefly, wholecell ramp currents of K2P channels in transfected CHO cells were recorded with a standard 2.2 s voltage ramp from
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-140 mV to C80 mV each 15 s. The pipette solution contained (in mM): 140 KCl, 1 MgCl2, 10 EGTA, and 10 HEPES (pH7.4). The bath solution contained (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, and 10 HEPES (pH 7.4). Whole-cell currents of K2P channels were fitted with the GHK current equation with the IgorPro program (WaveMetrics), yielding the NaC to KC relative permeability PNa/PNa:
Acknowledgments
We thank A. Patel for providing rat TWIK-2 plasmids.
Funding
This work was supported by the National Institute of General Medical Sciences, NIH, under Award Number R01GM102943 (to HC) and the American Heart Association under Award 11GRNT7270014 (to HC).
I D PK z2 .EF2 =RT/.[K C ]i ¡ [K C ]o exp. ¡ zFE=RT//= .1 ¡ exp. ¡ zFE=RT// C PNa z2 .EF2 =RT/.[Na C ]i ¡ [Na C ]o exp. ¡ zFE=RT//= .1 ¡ exp. ¡ zFE=RT// Where PK and PNa are the permeability of KC and NaC, respectively; z, F, R, and T have their usual meanings; I is the measured current and E is the voltage that the current is measured. The GHK fits were completed with the measured K2P currents at test voltages from ¡140 to C80 mV with 10 increments (except 0 mV) by employing the GHK current equation as the algorithm. Since I-V curves of TWIK KC channels do not follow the GHK current equation, their GHK fits had to be performed by holding the PNa/PNa values at experimentally measured values. Otherwise their GHK fits would give unreasonable PNa/PNa values. The GHK fits of other K2P channels produce 2 parameters, PK(F2/RT) and PNa(F2/RT), which are used to calculate the GHK predicted PNa/ PNa values, comparable to the PNa/PNa values obtained from measured reversal potentials in experiments. Whole-cell currents of K2P channels at test voltages between C20 and C80 mV and between ¡140 and ¡80 mV with 20 mV increments were fitted with straight lines, respectively, yielding slopes of outward and inward K2P currents (Table 1). All data are presented as means § SEM. Two-tailed Student’s t-tests were used to check for significant differences between 2 groups of data.
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Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions a
a
a
b
Haijun Chen , Dongchuan Zuo , Jianing Zhang , Min Zhou & Liqun Ma
a
a
Department of Biological Sciences; University at Albany; State University of New York; Albany, NY USA b
Department of Neuroscience; The Ohio State University Wexner Medical Center; Columbus, OH USA Published online: 23 Jan 2015.
Click for updates To cite this article: Haijun Chen, Dongchuan Zuo, Jianing Zhang, Min Zhou & Liqun Ma (2014) Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions, Channels, 8:6, 503-508, DOI: 10.4161/19336950.2014.973779 To link to this article: http://dx.doi.org/10.4161/19336950.2014.973779
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SHORT COMMUNICATION Channels 8:6, 503--508; November/December 2014; © 2014 Taylor & Francis Group, LLC
Classification of 2-pore domain potassium channels based on rectification under quasi-physiological ionic conditions Haijun Chen1,*, Dongchuan Zuo1, Jianing Zhang1, Min Zhou2, and Liqun Ma1 1
Department of Biological Sciences; University at Albany; State University of New York; Albany, NY USA; 2Department of Neuroscience; The Ohio State University
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Wexner Medical Center; Columbus, OH USA
I
Keywords: *Correspondence to: [email protected]
Haijun
Chen;
Email:
Submitted: 04/23/2014 Revised: 07/30/2014 Accepted: 10/02/2014 http://dx.doi.org/10.4161/19336950.2014.973779 www.landesbioscience.com
t is generally expected that 2-pore domain KC (K2P) channels are open or outward rectifiers in asymmetric physiological KC gradients, following the Goldman-Hodgkin-Katz (GHK) current equation. Although cloned K2P channels have been extensively studied, their current-voltage (I-V) relationships are not precisely characterized and previous definitions are contradictory. Here we study all the functional channels from 6 mammalian K2P subfamilies in transfected Chinese hamster ovary cells with patchclamp technique, and examine whether their I-V relationships are described by the GHK current equation. K2P channels display 2 distinct types of I-V curves in asymmetric physiological KC gradients. Two K2P isoforms in the TWIK subfamily conduct large inward KC currents and have a nearly linear I-V curve. Ten isoforms from 5 other K2P subfamilies conduct small inward KC currents and exhibit open rectification, but fits with the GHK current equation cannot precisely reveal the differences in rectification among K2P channels. The Rectification Index, a ratio of limiting IV slopes for outward and inward currents, is used to quantitatively describe open rectification of each K2P isoform, which is previously qualitatively defined as strong or weak open rectification. These results systematically and precisely classify K2P channels and suggest that TWIK KC channels have a unique feature in regulating cellular function. Channels
Introduction Background or leak KC conductance has been observed in excitable cells for several decades. They play a key role in setting the rest membrane potential. Twopore domain KC (K2P) channels mediate leak KC conductance.1-4 Mammalian K2P channels are divided into 6 subfamilies: TWIK, TREK, TALK, TASK, THIK, and TRESK, based on the sequence homology and functional similarity2 (Fig. 1A). They share the same topology in the plasma membrane and each K2P subunit contains 4 transmembrane segments and 2 asymmetric pore-forming loops (P1 and P2)5,6 (Fig. 1B). K2P channels operate with simple electrochemical diffusion and participate in the maintenance of the resting potential. They regulate cellular function in response to various physiological stimuli such as pH, neurotransmitters, and volatile anesthetics.3 As leak KC conductance previously recorded in neurons is outward rectifying and follows the Goldman-Hodgkin-Katz (GHK) current equation,7-9 it is generally expected that K2P channels are open or outward rectifiers in asymmetric physiological KC gradients.1,10 Since the first mammalian K2P channel was cloned in 1996, K2P channels have been studied extensively. Among 15 mammalian K2P channels, TWIK-3, TASK-5, and THIK2 channels do not produce detectable currents in heterologous expression systems; 503
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they have the same topology in the plasma membrane. Two isoforms in the TWIK subfamily have a linear or nearly linear IV curve, which does not exhibit the GHK-like open rectification, whereas 10 isoforms from other 5 K2P subfamilies are open rectifiers and their I-V curves follow the GHK current equation either completely or very closely. The Rectification Index, a ratio of the I-V slopes of K2P outward and inward currents, is employed to quantitatively describe the extent of open rectification of these K2P isoforms. These results precisely classify a basic property of K2P channels and imply a unique functional feature of TWIK KC channels.
Results and Discussion
Figure 1. Current-voltage relationships of TWIK KC channels under asymmetric physiological KC gradients. (A) 15 mammalian K2P isoforms in 6 subfamilies. TRESK-1 and TRESK-2 represent human and mouse TRESK, respectively. (B) Topology of a K2P subunit. (C and D) Whole-cell ramp currents (black lines) of rat TWIK-1 (C) and TWIK-2 (D) KC channels heterologously expressed in CHO cells are shown (n D 5). TWIK currents were blocked by quinine, a KC channel blocker (purple lines). Red lines are fits with the GHK current equation for K2P currents in test voltages from -140 mV to C80 mV with 10 mV increment (except 0 mV), when PNa/PK values were held at measured values in experiments. Blue lines are fits for the same data points with straight lines. Gray boxes indicate relatively large inward KC currents.
other 12 functional K2P channels are defined as open or outward rectifying KC channels in asymmetric physiological KC gradients.2 TASK-1 KC channels are perfect leak KC channels, as their functional properties match very well with leak KC conductance previously discovered in neurons.7 Whole-cell currents of TASK-1 KC channels heterologously expressed in mammalian cell lines are perfectly fitted with the GHK current equation.10 TWIK-1, the first cloned mammalian K2P channel, was first defined in the Xenopus oocyte expression system as a weakly inward rectifying KC channel because of its weak rectification at test voltages above C20 mV,11 but how TWIK-1 KC channels behave at the voltages more negative than the reversal potential was not addressed. It becomes more complicated to define TWIK-1 KC channels due to contradictory observations whether
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TWIK-1 KC channels show weakly inward rectification.12,13 Single channel recordings in another study suggested that TWIK-1 KC channels are open rectifiers between -100 and C80 mV.12 However, recent studies indicate that whole-cell currents of TWIK-1 and TWIK-2 KC channels heterologously expressed in mammalian cell lines seem not exhibiting the GHK-like open rectification,14-17 as their inward KC currents are relatively large. In this study, we systematically investigate the I-V relationships of all isoforms from 6 K2P subfamilies, which are functionally expressed in Chinese hamster ovary (CHO) cells, and then examine whether their I-V curves are described by the GHK current equation. We demonstrated that K2P channels display 2 distinct types of I-V relationships in asymmetric physiological KC gradients, even though
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TWIK-1 and TWIK-2 KC channels have a linear current-voltage relationship under physiological KC gradients We first examined whether whole-cell currents of TWIK KC channels, which were heterologously expressed in CHO cells, exhibit open rectification under asymmetric physiological KC gradients, since previously reports imply that TWIK KC channels seem not to show such a behavior.14-17 We use whole-cell currents of K2P channels in transfected CHO cells to assess their open rectification with the GHK current equation, based on 2 basic assumptions. First, recorded whole-cell currents are KC-selective currents via K2P channels. Endogenous currents in CHO cells are very small and neglected.18 Measured reversal potentials and calculated NaC to KC relative permeability of K2P channels support the assumption. Second, gating and ionic block, or fundamental asymmetry structures of the K2P openpore do not contribute to open rectification, since I-V curves of most K2P channels are very consistent in different voltage-clamp recording configurations such as whole-cell and inside-out recordings; all K2P channels share similar structures of the pore; it is generally accepted that the gating process do not affect I-V shapes of K2P channels. In asymmetric physiological KC gradients, TWIK-1 and TWIK-2 KC channels conduct relatively large inward KC currents in the voltages
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more negative than the reversal potential (gray boxes in Fig. 1C and D), so their IV curves are not open rectifying at all. After establishment of stable whole-cell configurations, outward KC currents of TWIK-1 KC channels are fitted closely with the GHK current equation and it may be the reason why a previous study defined TWIK-1 KC channels as open rectifying channels in test voltages between -100 and C80 mV.12 However, in a full range of test voltages, the GHK open rectification does not occur in I-V curves of TWIK-1 KC channels. Instead, TWIK-1 I-V relationships are nearly linear (blue and black lines, Fig. 1C). The behavior of TWIK-2 KC channels is more obvious. TWIK-2 I-V curves do not have any rectification, consistent with previous observations.16,17 They can be fitted very well with a straight line in all tested voltages (blue and black lines, Fig. 1D). Thus TWIK currents in the voltages more negative than the reversal potentials do not exhibit the GHK open rectification. To quantitatively describe the rectification of whole-cell currents of K2P channels, we employ a parameter, the Rectification Index, which is a relative ratio of the slopes of outward currents at test voltages between C20 and C80 mV and inward currents at test voltages
between ¡140 and ¡80 mV (column 1 and 2, Table 1)(Fig. 2). These 2 slopes actually reflect outward and inward conductance of K2P channels, respectively. TWIK-1 and TWIK-2 KC channels have a Rectification Index close to 1 (column 3, Table 1), supporting that their I-V curves are nearly linear. It is also worth to note that both TWIK-1 and TWIK-2 KC channels do not show weakly inward rectification at test voltages above C20 mV after establishment of stable whole-cell configurations, in disagreement with previous observations on weakly inward rectification of TWIK-1 KC channels heterologously expressed in Xenopus oocytes.11 K2P channels in TREK, TALK, TASK, THIK, and TRESK subfamilies show the GHK-like open rectification under physiological KC gradients We further similarly studied other 10 K2P isoforms in transfected CHO cells. Consistent with previous studies,10,19 TASK-1 and TASK-3 KC channels conduct small inward KC currents in the membrane potentials more negative than the reversal potential (Fig. 2D and E) and exhibit weakly open rectification in 5 mM extracellular KC. Their I-V relationships are perfectly fitted by the GHK current
equation and have a Rectification Index of »10 and »20, respectively. TRAAK and TASK-2 (a member of the TALK subfamily) KC channels also have I-V curves with Reflection Index of »19 and »15, respectively, which are nearly described by the GHK current equation, so their currents are also weakly open rectifying (Fig. 2C and G). Alkaline pH-activated TALK-1 and TALK-2 KC channels did not produce detectable whole-cell currents in transfected CHO cells at bath solutions with pH 9. Previous reports showed that their whole-cell currents, which were recorded in COS cells, are open rectifying,20 and TALK-1 I-V curve is fitted well with the GHK current equation (Fig. 2F). Other 4 tested K2P channels are basically open rectifiers, although their currents are not exactly overlapped with the lines predicted by the GHK current equation. Compared to TASK and TALK KC channels, TREK-1, TREK-2, and TRESK2 KC channels exhibit stronger open rectification and conduct much less inward KC currents in the membrane potentials more negative than the reversal potential (Fig. 2A, B and I). Their Rectification Indexes are »70, »68, and »44, respectively. Previously studies defined TREK-1 and TREK-2 channels as outward rectifiers,19 because under recording solutions
Table 1. Quantitative classification of K2P channels based on rectification. Slopes for recorded outward and inward currents were listed for all tested K2P isoforms in 6 subfamilies, while Rectification Index and PNa/PK values were compared side-by-side from the GHK fits and recorded K2P currents. Outward and inward current slopes were obtained as described in details in Figure 2. The Rectification Index of recorded K2P currents represents a ratio of outward and inward slopes of each K2P isoform. The Rectification Index of the GHK fits was similarly obtained from those fitted lines and its value is »9 for all K2P channels. The PNa/PK values predicted by the GHK fits were calculated by 2 parameters, PK(F2/RT) and PNa(F2/RT), which were generated by the GHK fits. Measured PNa/PK values were calculated from reversal potentials of recorded K2P currents. *: The Rectification Index for K2P isoforms is significantly different from that for TASK-1 or TASK-3 (P < 0.005). Hold: the GHK fits were completed when PNa/PK values were held at those measured in experiments. 1. Outward and inward current slopes, Rectification Index, and PNa/PK of K2P channels Current slope (10¡9 A/V) K2P subfamily
TWIK TREK
TALK TASK THIK TRESK
K2P Isoform
TWIK-1/K2P1 TWIK-2/K2P6 TREK-1/K2P2 TREK-2/K2P10 TRAAK/K2P4 TASK-2/K2P5 TASK-1/K2P3 TASK-3/K2P9 THIK-1/K2P13 TRESK-2/K2P18
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Outward
Inward
6.6 § 1.1 27.2 § 9.6 107.2 § 18.4 230.2 § 29.4 253.9 § 31.3 74.4 § 17.0 26.5 § 4.2 80.6 § 15.5 50.5 § 9.4 59.1 § 11.1
5.7 § 0.8 23.4 § 11.5 2.1 § 0.6 3.4 § 0.7 12.7 § 0.2 5.8 § 2.3 2.6 § 0.4 3.9 § 0.4 8.6 § 1.1 1.5 § 0.4
Recorded current Rectification Index
GHK fit Rectification Index
Measured P Na/P K
GHK fit predicted P Na/PK
Measured cell number
1.2 § 0.1* 1.4 § 0.2* 69.8 § 11.8* 67.6 § 12.3* 19.1 § 3.7 15.4 § 1.9 10.3 § 0.8 20.5 § 2.3 5.7 § 0.5* 44.3 § 5.2*
8.9 § 0.1 8.9 § 0.1 9.1 § 0.1 9.0 § 0.1 8.9 § 0.2 8.6 § 0.2 8.9 § 0.2 9.2 § 0.1 8.8 § 0.1 9.3 § 0.1
0.006 § 0.001 0.007 § 0.001 0.004 § 0.001 0.002 § 0.001 0.009 § 0.002 0.008 § 0.001 0.005 § 0.002 0.002 § 0.001 0.003 § 0.001 0.004 § 0.001
Hold Hold 0.005 § 0.002 0.003 § 0.001 0.006 § 0.001 0.005 § 0.001 0.004 § 0.001 0.002 § 0.001 0.005 § 0.001 0.002 § 0.001
5 5 9 5 5 5 7 6 5 7
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Figure 2. Current-voltage curves of other 10 K2P channels under asymmetric physiological KC gradients. Whole-cell ramp currents (black lines) of rat TREK-1 (A), human TREK-2 (B), human TRAKK (C), human TASK-1 (D), human TASK-3 (E), human TASK-2 (G), mouse THIK-1 (H), and mouse TRESK-2 (I) KC channels heterologously expressed in CHO cells are shown (n D 5–9). Red curves are fits with the GHK current equation for K2P currents in test voltages from -140 mV to C80 mV with 10 mV increment (except 0 mV), yielding PNa/PK values for each K2P isoform (column 6, Table 1). Blue and orange lines are fits with straight lines for K2P currents in test voltages from C20 mV to C80 mV and from -140 mV to -80 mV with 20 mV increments, respectively, yielding slopes of outward and inward currents (column 1 and 2, Table 1). Data points (open circles) in F are adapted from the whole-cell (COS cell) ramp currents in test voltages from -100 mV to C80 mV with 20 mV increment (except 0 mV) in Ref. 20.
with symmetrical 140 mM KC these channels may still show outward rectification. In contrast, THIK-1 KC channels show the weakest open rectification and conduct relatively larger inward KC currents in the membrane potentials more negative than the reversal potential (Fig. 2H), so THIK-1 KC channels have an I-V curve with a Rectification Index of »6. These 10 K2P isoforms from 5 subfamilies are open rectifiers under quasiphysiological ionic conditions. Several GHK fits have clear deviations from recorded K2P currents such as
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sharper curvature (Fig. 2H and I). These deviations are not measuring errors, as they can be repeated. Under our recordings ionic conditions the GHK fits give an ideal reversal potential of -81 to -83 mV for all K2P channels, the deviations at test voltages between the reversal potential and C80 mV reflect the differences of K2P outward conductance from the GHK prediction, which are partially implied by K2P outward current slopes (Table 1); the deviations at test voltages between ¡140 mV and the reversal potential reflect the differences of K2P inward
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conductance from the GHK prediction, which actually indicates the extent of open rectification in different K2P isoforms. We have indicated that all 12 functional K2P channels show 2 different types of I-V curves in asymmetric physiological KC gradients, although they have the same topology. First, 10 K2P isoforms from TREK, TALK, TASK, and THIK, and TRESK subfamilies are open rectifiers that completely or nearly follow the GHK current equation. As K2P channels operate with simple electrochemical diffusion, in asymmetric physiological KC gradients
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(5 mM extracellular and 140 mM intracellular KC in our recording bath solutions) driving forces of KC against the KC electrochemical concentration differences should result in the GHK open rectification. However, some isoforms in these 5 K2P subfamilies still show clear differences in the extent of open rectification, as their Rectification Indexes suggest. We hypothesize that such differences are determined by slight diversities in molecular and structural bases of the ion-conductance pathway among K2P isoforms because the homology among K2P isoforms is low. The unique extracellular Cap structure of K2P channels may be a major contributor as it creates an extracellular ion pathway that is markedly different from that observed in other KC channel structures.5,6 For example, TRESK-2 KC channels have an extracellular domain in P1-looping significantly different from those in other K2P isoforms,21 and show strong open rectification with the smallest slope of inward currents (Table 1). In a contrast, TWIK-1 and TWIK-2 KC channels have a linear IV curve that does not show the GHK-like open rectification. They not only conduct large outward KC currents, but also conduct large inward KC currents and exhibit anomalous behaviors, reminiscent of conventional inward rectifying KC channels. Therefore, TWIK KC channels could play a unique role in regulation of cellular function.
Materials and Methods All tested K2P cDNAs were cloned in pMAX or pRAT vectors described previously,14,15 except that TWIK-2 cDNA was cloned into pEYFP-C1 vectors. CHO cells at least 80% confluence were transfected by Lipofectamine 2000 (Invitrogen) with 3 mg of K2P channel plasmids and 1 mg of pEGFP plasmids and studied 24 hours later. GFP expression was used to identify effectively transfected CHO cells. Whole-cell patch-clamp recordings were performed and data were analyzed, as described previously.14 Briefly, wholecell ramp currents of K2P channels in transfected CHO cells were recorded with a standard 2.2 s voltage ramp from
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-140 mV to C80 mV each 15 s. The pipette solution contained (in mM): 140 KCl, 1 MgCl2, 10 EGTA, and 10 HEPES (pH7.4). The bath solution contained (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 15 glucose, and 10 HEPES (pH 7.4). Whole-cell currents of K2P channels were fitted with the GHK current equation with the IgorPro program (WaveMetrics), yielding the NaC to KC relative permeability PNa/PNa:
Acknowledgments
We thank A. Patel for providing rat TWIK-2 plasmids.
Funding
This work was supported by the National Institute of General Medical Sciences, NIH, under Award Number R01GM102943 (to HC) and the American Heart Association under Award 11GRNT7270014 (to HC).
I D PK z2 .EF2 =RT/.[K C ]i ¡ [K C ]o exp. ¡ zFE=RT//= .1 ¡ exp. ¡ zFE=RT// C PNa z2 .EF2 =RT/.[Na C ]i ¡ [Na C ]o exp. ¡ zFE=RT//= .1 ¡ exp. ¡ zFE=RT// Where PK and PNa are the permeability of KC and NaC, respectively; z, F, R, and T have their usual meanings; I is the measured current and E is the voltage that the current is measured. The GHK fits were completed with the measured K2P currents at test voltages from ¡140 to C80 mV with 10 increments (except 0 mV) by employing the GHK current equation as the algorithm. Since I-V curves of TWIK KC channels do not follow the GHK current equation, their GHK fits had to be performed by holding the PNa/PNa values at experimentally measured values. Otherwise their GHK fits would give unreasonable PNa/PNa values. The GHK fits of other K2P channels produce 2 parameters, PK(F2/RT) and PNa(F2/RT), which are used to calculate the GHK predicted PNa/ PNa values, comparable to the PNa/PNa values obtained from measured reversal potentials in experiments. Whole-cell currents of K2P channels at test voltages between C20 and C80 mV and between ¡140 and ¡80 mV with 20 mV increments were fitted with straight lines, respectively, yielding slopes of outward and inward K2P currents (Table 1). All data are presented as means § SEM. Two-tailed Student’s t-tests were used to check for significant differences between 2 groups of data.
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