Characterization of Inhibitory Capability on Hyperpolarization-Activated Cation Current Caused by Lutein (β,ε-Carotene-3,3′-Diol), a Dietary Xanthophyll Carotenoid

Lutein (β,ε-carotene-3,3′-diol), a xanthophyll carotenoid, is found in high concentrations in the macula of the human retina. It has been recognized to exert potential effectiveness in antioxidative and anti-inflammatory properties. However, whether and how its modifications on varying types of plasmalemmal ionic currents occur in electrically excitable cells remain incompletely answered. The current hypothesis is that lutein produces any direct adjustments on ionic currents (e.g., hyperpolarization-activated cation current, Ih [or funny current, If]). In the present study, GH3-cell exposure to lutein resulted in a time-, state- and concentration-dependent reduction in Ih amplitude with an IC50 value of 4.1 μM. There was a hyperpolarizing shift along the voltage axis in the steady-state activation curve of Ih in the presence of this compound, despite being void of changes in the gating charge of the curve. Under continued exposure to lutein (3 μM), further addition of oxaliplatin (10 μM) or ivabradine (3 μM) could be effective at either reversing or further decreasing lutein-induced suppression of hyperpolarization-evoked Ih, respectively. The voltage-dependent anti-clockwise hysteresis of Ih responding to long-lasting inverted isosceles-triangular ramp concentration-dependently became diminished by adding this compound. However, the addition of 10 μM lutein caused a mild but significant suppression in the amplitude of erg-mediated or A-type K+ currents. Under current-clamp potential recordings, the sag potential evoked by long-lasting hyperpolarizing current stimulus was reduced under cell exposure to lutein. Altogether, findings from the current observations enabled us to reflect that during cell exposure to lutein used at pharmacologically achievable concentrations, lutein-perturbed inhibition of Ih would be an ionic mechanism underlying its changes in membrane excitability.


Introduction
Lutein (xanthophyll, β,ε-carotene-3,3 -diol), derived from a hydride of a (6 R)-β,εcarotene, is one of the few xanthophyll carotenoids that is believed to exist not only in vegetables and fruits, but also in high concentrations present in the macula of the human retina, where it is thought to act as a yellow filter [1][2][3][4][5]. Evidence from human studies suggests that dietary intake of lutein can lead to the accumulation of lutein in retinal neural tissue, thereby presumably promoting eye and brain health [6]. Moreover, the accumulation 2 of 18 of lutein in the retina has been reported to influence the early maturation of the retina [6]. Alternatively, the beneficial bioactive effects of lutein have been widely recognized to be attributable to its antioxidant and anti-inflammatory properties. Indeed, many basic and clinical studies have growingly reported the capability of lutein to exert anti-oxidative and anti-inflammatory properties in the eye, since this compound might act as an antioxidant, protecting cells against the damaging effects of free radicals [2,[4][5][6][7][8][9][10][11][12][13][14].
Moreover, lutein cotreatment with antiepileptic drugs was recently reported to be a promising strategy either to improve therapeutic efficacy in patients suffering from epilepsy [15] or to improve cognitive function [16]. This compound has been additionally revealed to ameliorate either ischemia-reperfusion-induced vasculitic neuropathic pain and neuropathy in rats, or photoreceptor degeneration in a mouse model of retinitis pigmentosa [17][18][19]. It was also recently reported to exert anti-neoplastic action [20,21]. Changes in oxidative stress or the activity of NADPH oxidase have been recently demonstrated to affect different types of plasmalemmal ionic currents [22]. However, to our surprise, the ionic mechanism of lutein-induced actions through which it affects the functional activities of excitable cells remains largely unanswered.
The hyperpolarization-activated cation current (I h or funny current [I f ]) is wellrecognized as being a key determinant of repetitive electrical activity in heart cells and in various types of neurons and neuroendocrine or endocrine cells [23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38]. This type of ionic current comprises a mixed inward Na + /K + current (i.e., unusual ion selectivity in that it can conduct both Na + and K + ions), which is vulnerable to inhibition by CsCl, ivabradine, or ZD7288 [23,26,[39][40][41][42][43]. It is believed that the activation of this current, which proceeds at resting membrane potential, is allowed to produce a net inwardly directed current carried largely by Na + ions, thereby bringing about membrane depolarization to the threshold critically involved in the generation of action potentials [23,25,32,44]. These currents are assumed to be carried by channels of the hyperpolarization-activated cyclic nucleotide-gated (HCN) gene family, which belongs to the superfamily of voltage-gated K + (K V ) channels and cyclic nucleotide-gated (CNG) channels [26,28,34,37,[44][45][46]. HCN2, HCN3, or mixed HCN2 + HCN3 channels were reported previously to be distributed in pituitary tumor (GH 3 ) cells [27,28,39]. The activities of CNG and HCN channels have also been thought to be closely linked to phototransduction intrinsically found in photosensitive retinal ganglion cells [47]. At present, the issue of whether and how lutein can interact with the HCN channel to modify the amplitude, gating properties, and hysteretic magnitude of I h remains unsolved.
In view of the foregoing considerations, the possible electrophysiological actions of lutein and other related compounds in pituitary GH 3 cells were explored in this study. We tried to study the effects of lutein on the amplitude and gating of I h residing in pituitary GH 3 somatolactotrophs. Findings from the current observations tempt us to reflect that the I h present in different cell types could be an unidentified but distinctive target through which the lutein molecule can act to affect the functional activities of the cells involved.

Suppressive Effect of Lutein on Hyperpolarization-Activated Cation Current (I h ) Measured from Pituitary Tumor (GH 3 ) Cells
In this study, we began to exploit the whole-cell configuration of the patch-clamp technique to evaluate any possible modifications of lutein on ionic currents identified in GH 3 cells. In order to record ionic current flowing through I h , we kept cells in Ca 2+ -free, Tyrode's solution containing 0.5 mM CdCl 2 and 1 µM tetrodotoxin (TTX), and the recording pipette that was used was filled up with a K + -containing (145 mM). TTX or CdCl 2 was used to block voltage-gated Na + or Ca 2+ currents, respectively. As the whole-cell mode was firmly achieved, we maintained the tested cell at a holding potential of −40 mV and a 2-sec hyperpolarizing step to −120 mV was thereafter imposed to evoke I h . Of interest, one minute after cell exposure to lutein, the amplitude of I h evoked in response to such 2-sec long step hyperpolarization from −40 to −120 mV became progressively lessened ( Figure 1A). For example, at the level of −120 mV, the application of the compound at a concentration of 3 µM resulted in a decrease of I h amplitude from 338 ± 27 to 201 ± 18 pA (n = 7, p < 0.05). After the lutein was removed, the current amplitude was returned to 332 ± 25 pA (n = 7). Concurrently, under GH 3 -cell exposure to 3 µM lutein, the value of activation time constant (τ act ) significantly arose from 1.4 ± 0.1 s to 2.8 ± 0.2 s (n = 7, p < 0.05).
ther collated, and the results are hence presented in Figure 1B. In this stage of measurements, we held the examined cell in a voltage clamp at −40 mV, and the hyperpolarizing pulse (2-sec duration) to −120 mV was thereafter imposed on it, while current amplitudes during cell exposure to varying lutein concentrations (0.1-100 μM) were measured at the end pulse of long-lasting hyperpolarizing command voltage. It became clear that cell exposure to lutein is able to suppress Ih in a concentration-dependent manner in these cells. By virtue of a sigmoidal least-square fit to the experimental data, the value of effective IC50 (i.e., the concentration required for a half-maximal inhibition) for the inhibitory effect of lutein on Ih was properly yielded at 4.1 μM. Additionally, the existence of lutein increased the τact value of Ih evoked by long-lasting membrane hyperpolarization ( Figure  1C). The results tempt us to reflect that, under our experimental conditions, lutein can bring about a depressant action of Ih in these cells in a time-and concentration-dependent manner. Figure 1. Inhibitory effect of lutein on hyperpolarization-activated cation current (Ih) identified from pituitary GH3 cells. In this stage of experiments, we placed cells to be immersed in Ca 2+ -free, Tyrode's solution, which contained 1 μM tetrodotoxin (TTX) and 0.5 mM CdCl2, and we then filled up the recording pipette by using a K + -containing solution (145 mM). (A) Superimposed current traces were obtained in the control period (a, absence of lutein, black color), during cell exposure to 3 μM lutein (b, red color), and after washout of lutein (c, blue color). The voltage protocol (i.e., 2-sec membrane hyperpolarization from −40 to −120 mV, blue color) used to evoke Ih is pointed out in the upper part. The lowest part in (A) demonstrates the best fit of digitized data points acquired in the Figure 1. Inhibitory effect of lutein on hyperpolarization-activated cation current (I h ) identified from pituitary GH 3 cells. In this stage of experiments, we placed cells to be immersed in Ca 2+ -free, Tyrode's solution, which contained 1 µM tetrodotoxin (TTX) and 0.5 mM CdCl 2 , and we then filled up the recording pipette by using a K + -containing solution (145 mM). (A) Superimposed current traces were obtained in the control period (a, absence of lutein, black color), during cell exposure to 3 µM lutein (b, red color), and after washout of lutein (c, blue color). The voltage protocol (i.e., 2-sec membrane hyperpolarization from −40 to −120 mV, blue color) used to evoke I h is pointed out in the upper part. The lowest part in (A) demonstrates the best fit of digitized data points acquired in the absence (a, open black circles) or presence (b, open red circles) of 3 µM lutein to single exponential (indicated in smooth continuous black or red line) with a time constant of 1395 or 2927 msec, respectively. (B) Concentration-dependent inhibitory effect of lutein on the amplitude of I h (mean ± SEM; n = 7 for each point). We measured current amplitude at the end of hyperpolarizing step, and the smooth gray line drawn was reliably fitted to a modified Hill function as described in Section 4. (C) Summary scatter graph showing the effect of lutein (1 or 3 µM) on the activation time constant (τ act ) of I h (mean ± SEM; n = 7 for each point). The slowly activating I h was evoked by 2 sec hyperpolarizing step from −40 to −120 mV and the I h 's τ act value was optimally estimated with single exponential. * Significantly different from control (p < 0.05) and ** significantly different from the lutein-alone (1 µM) group (p < 0.05).
The relationship between the concentration of lutein and the amplitude of I h was further collated, and the results are hence presented in Figure 1B. In this stage of measurements, we held the examined cell in a voltage clamp at −40 mV, and the hyperpolarizing pulse (2-sec duration) to −120 mV was thereafter imposed on it, while current amplitudes during cell exposure to varying lutein concentrations (0.1-100 µM) were measured at the end pulse of long-lasting hyperpolarizing command voltage. It became clear that cell exposure to lutein is able to suppress I h in a concentration-dependent manner in these cells. By virtue of a sigmoidal least-square fit to the experimental data, the value of effective IC 50 (i.e., the concentration required for a half-maximal inhibition) for the inhibitory effect of lutein on I h was properly yielded at 4.1 µM. Additionally, the existence of lutein increased the τ act value of I h evoked by long-lasting membrane hyperpolarization ( Figure 1C). The results tempt us to reflect that, under our experimental conditions, lutein can bring about a depressant action of I h in these cells in a time-and concentration-dependent manner.

Effect of Lutein on the Quasi-Steady-State Activation Curve of I h Identified in GH 3 Cells
To characterize the inhibitory effect of lutein on I h , an effort was made to ascertain if cell exposure to lutein might modify any changes in the steady-state activation curve of I h intrinsically in GH 3 cells. Figure 2C illustrates the steady-state activation curve of I h acquired without or with the presence of this compound at a concentration of 3 µM. The Boltzmann distribution (or Fermi-Dirac distribution) detailed in Section 4 was applied to fit the experimental data points appropriately. In the control (i.e., in the absence of lutein), V 1/2 = −82 ± 8 mV and q = 4.35 ± 0.07 e (n = 7), and during the exposure to 3 µM lutein, V 1/2 = −93 ± 9 mV and q = 4.29 ± 0.08 e (n = 7). Consequently, the emerging data led us to project that during GH 3 -cell exposure to lutein (3 µM), the steady-state activation curve of I h elicited under different preceding levels of conditioning step-pulse potential was shifted along the voltage axis toward more hyperpolarized potential (approximately 11 mV), despite no concurrent changes in the apparent gating charge (i.e., q) of the curve. of Ih (mean ± SEM; n = 7 for each point). The slowly activating Ih was evoked by 2 sec hyperpolarizing step from −40 to −120 mV and the Ih's τact value was optimally estimated with single exponential. * Significantly different from control (p < 0.05) and ** significantly different from the lutein-alone (1 μM) group (p < 0.05).

Effect of Lutein on the Quasi-Steady-State Activation Curve of Ih Identified in GH3 Cells
To characterize the inhibitory effect of lutein on Ih, an effort was made to ascertain if cell exposure to lutein might modify any changes in the steady-state activation curve of Ih intrinsically in GH3 cells. Figure 2C illustrates the steady-state activation curve of Ih acquired without or with the presence of this compound at a concentration of 3 μM. The Boltzmann distribution (or Fermi-Dirac distribution) detailed in Section 4 was applied to fit the experimental data points appropriately. In the control (i.e., in the absence of lutein), V1/2 = −82 ± 8 mV and q = 4.35 ± 0.07 e (n = 7), and during the exposure to 3 μM lutein, V1/2 = −93 ± 9 mV and q = 4.29 ± 0.08 e (n = 7). Consequently, the emerging data led us to project that during GH3-cell exposure to lutein (3 μM), the steady-state activation curve of Ih elicited under different preceding levels of conditioning step-pulse potential was shifted along the voltage axis toward more hyperpolarized potential (approximately 11 mV), despite no concurrent changes in the apparent gating charge (i.e., q) of the curve.

Comparisons among the Effect of Lutein, Lutein Plus Oxaliplatin and Lutein Plus Ivabradine on Ih Amplitude
We then investigated if the addition of oxaliplatin or ivabradine, but still in the continued exposure to 3 μM lutein, was able to adjust lutein-perturbed inhibition of Ih identified in GH3 cells. Oxaliplatin or ivabradine has been recently demonstrated to activate or suppress Ih, respectively [40,48,49]. In this set of current recordings, we placed cells in Ca 2+ -free, Tyrode's solution containing 1 μM TTX, and the Ih residing in each cell was activated by stepping to −120 mV for a duration of 2 sec from a holding potential of −40 mV. As can be seen in Figure 3A,B, the subsequent addition of 10 μM oxaliplatin or 3 μM ivabradine, but still in continued exposure to 3 μM lutein, was able to attenuate or further decrease Ih amplitude, respectively. It is, therefore, clear that the Ih magnitude susceptible to being inhibited by lutein presented in GH3 cells would be effectively reversed by further application of oxaliplatin; however, it was further reduced by adding ivabradine.

Comparisons among the Effect of Lutein, Lutein Plus Oxaliplatin and Lutein Plus Ivabradine on I h Amplitude
We then investigated if the addition of oxaliplatin or ivabradine, but still in the continued exposure to 3 µM lutein, was able to adjust lutein-perturbed inhibition of I h identified in GH 3 cells. Oxaliplatin or ivabradine has been recently demonstrated to activate or suppress I h , respectively [40,48,49]. In this set of current recordings, we placed cells in Ca 2+ -free, Tyrode's solution containing 1 µM TTX, and the I h residing in each cell was activated by stepping to −120 mV for a duration of 2 sec from a holding potential of −40 mV. As can be seen in Figure 3A,B, the subsequent addition of 10 µM oxaliplatin or 3 µM ivabradine, but still in continued exposure to 3 µM lutein, was able to attenuate or further decrease I h amplitude, respectively. It is, therefore, clear that the I h magnitude susceptible to being inhibited by lutein presented in GH 3 cells would be effectively reversed by further application of oxaliplatin; however, it was further reduced by adding ivabradine. , and lutein plus ivabradine on Ih amplitude (mean ± SEM; n = 7 for each point). We measured each current amplitude (acquired during cell exposure to different tested compounds) at the end-point of the 2-sec hyperpolarizing step from −40 to −120 mV. In the experiments on lutein plus OXAL or lutein or ivabradine, lutein was first added to the bath before OXAL or ivabradine was further applied. * Significantly different from control (p < 0.05) and **significantly different from the lutein-alone (3 μM) group (p < 0.05).

Effect of Lutein on Voltage-Dependent Hysteresis (Hys(V)) of Ih Triggered by Inverted Isosceles-Triangular Ramp Voltage (Vramp)
Previous observations disclosed the capability of Ih strength evoked by Vramp to modify the varying patterns of bursting firing in different types of excitable cells, including central neurons [29,35,[50][51][52]. Cyclic nucleotide-gated (CNG) channels (e.g., CNGA2 ion channels) were previously demonstrated to exhibit Hys(V) behavior [53]. We hence continued to evaluate how lutein could have any propensity to adjust Ih's Hys(V) responding to long-lasting triangular Vramp, the protocol of which was designed and then imposed on the tested cells through digital-to-analog conversion. In the current measurements, the tested cell was held at −40 mV, and we then applied a long-lasting inverted Vramp to it. The Vramp protocol comprises the downsloping (forward) ramp from −40 to −150 mV followed by the upsloping (backward) limb back to −40 mV with a total duration of 3.2 sec (i.e., a ramp speed of ±69 mV/sec) as indicated in the upper part of Figure 4A. As demonstrated in Figure 4, the voltage-dependent hysteresis (Hys(V)) of Ih (i.e., the relationship of forward or backward Ih versus membrane potential) was robustly observed upon activation by triangular double Vramp. That is, the Ih amplitude triggered by the downsloping limb of the inverted triangular Vramp was overly lower than that by the upsloping end of Vramp. For example, in the control period (i.e., lutein was not present), the Ih amplitude at −120 mV taken during the downsloping or upsloping end of Vramp was markedly different (p < 0.05) (i.e., 48 ± 8 pA [downsloping] versus 249 ± pA [upsloping]; n = 7, p < 0.05). Under cell exposure to lutein (3 μM), Ih evoked in the downsloping limb of the long-lasting inverted , and lutein plus ivabradine on I h amplitude (mean ± SEM; n = 7 for each point). We measured each current amplitude (acquired during cell exposure to different tested compounds) at the end-point of the 2-sec hyperpolarizing step from −40 to −120 mV. In the experiments on lutein plus OXAL or lutein or ivabradine, lutein was first added to the bath before OXAL or ivabradine was further applied. * Significantly different from control (p < 0.05) and **significantly different from the lutein-alone (3 µM) group (p < 0.05).

Effect of Lutein on Voltage-Dependent Hysteresis (Hys (V) ) of I h Triggered by Inverted Isosceles-Triangular Ramp Voltage (V ramp )
Previous observations disclosed the capability of I h strength evoked by V ramp to modify the varying patterns of bursting firing in different types of excitable cells, including central neurons [29,35,[50][51][52]. Cyclic nucleotide-gated (CNG) channels (e.g., CNGA2 ion channels) were previously demonstrated to exhibit Hys (V) behavior [53]. We hence continued to evaluate how lutein could have any propensity to adjust I h 's Hys (V) responding to long-lasting triangular V ramp , the protocol of which was designed and then imposed on the tested cells through digital-to-analog conversion. In the current measurements, the tested cell was held at −40 mV, and we then applied a long-lasting inverted V ramp to it. The V ramp protocol comprises the downsloping (forward) ramp from −40 to −150 mV followed by the upsloping (backward) limb back to −40 mV with a total duration of 3.2 sec (i.e., a ramp speed of ±69 mV/sec) as indicated in the upper part of Figure 4A. As demonstrated in Figure 4, the voltage-dependent hysteresis (Hys (V) ) of I h (i.e., the relationship of forward or backward I h versus membrane potential) was robustly observed upon activation by triangular double V ramp . That is, the I h amplitude triggered by the downsloping limb of the inverted triangular V ramp was overly lower than that by the upsloping end of V ramp . For example, in the control period (i.e., lutein was not present), the I h amplitude at −120 mV taken during the downsloping or upsloping end of V ramp was markedly different (p < 0.05) (i.e., 48 ± 8 pA [downsloping] versus 249 ± pA [upsloping]; n = 7, p < 0.05). Under cell exposure to lutein (3 µM), I h evoked in the downsloping limb of the long-lasting inverted V ramp was noticed to decline to a less extent than that measured from the upsloping end of the triangular V ramp . Moreover, the strength of lutein-induced current inhibition at the downsloping (forward) and upsloping (reverse) limbs of triangular V ramp differ significantly. As demonstrated by the dashed arrows in Figure 4A, upon the difference (i.e., ∆area) in the shaded area under the curve in the downsloping and upsloping direction, we further quantified the degree of I h 's Hys (V) residing in GH 3 cells. The experimental observations disclosed that the amount of Hys (V) responding to the long-lasting inverted V ramp became conceivably decreased during cell exposure to lutein. Figure 4B summarized the data demonstrating the effects of lutein and lutein plus ivabradine on the ∆area (i.e., the shaded region in (A)) under such curve. For example, in addition to its depressive action on I h magnitude, the addition of 3 µM lutein resulted in a reduction in the area responding to inverted triangular V ramp , as demonstrated by a reduction of ∆area from 17.7 ± 2.4 to 8.8 ± 1.6 mV·nA (n = 7, p < 0.05). After the lutein was removed, the Hys (V) 's area returned to 17.2 ± 2.3 mV·nA. As cells were continually exposed to 3 µM lutein, subsequent application of 3 µM ivabradine measurably decreased the ∆area of Hys (V) further. Vramp was noticed to decline to a less extent than that measured from the upsloping end of the triangular Vramp. Moreover, the strength of lutein-induced current inhibition at the downsloping (forward) and upsloping (reverse) limbs of triangular Vramp differ significantly. As demonstrated by the dashed arrows in Figure 4A, upon the difference (i.e., ∆area) in the shaded area under the curve in the downsloping and upsloping direction, we further quantified the degree of Ih's Hys(V) residing in GH3 cells. The experimental observations disclosed that the amount of Hys(V) responding to the long-lasting inverted Vramp became conceivably decreased during cell exposure to lutein. Figure 4B summarized the data demonstrating the effects of lutein and lutein plus ivabradine on the ∆area (i.e., the shaded region in (A)) under such curve. For example, in addition to its depressive action on Ih magnitude, the addition of 3 μM lutein resulted in a reduction in the area responding to inverted triangular Vramp, as demonstrated by a reduction of ∆area from 17.7 ± 2.4 to 8.8 ± 1.6 mV·nA (n = 7, p < 0.05). After the lutein was removed, the Hys(V)'s area returned to 17.2 ± 2.3 mV·nA. As cells were continually exposed to 3 μM lutein, subsequent application of 3 μM ivabradine measurably decreased the ∆area of Hys(V) further.

Mild Modification by Lutein of A-Type K + Current (I K(A) ) in GH 3 Cells
We also continued to study if the presence of lutein could influence macroscopic K + currents (e.g., I K(A) ) identified in these cells. These experiments were conducted in cells which were bathed in Ca 2+ -free, Tyrode's solution containing 1 µM TTX, and we filled up the recording pipette by adding K + -enriched (145 mM) solution. As depicted in Figure 5A, with cell exposure to lutein at a concentration of 10 µM, the amplitudes of I K(A) were robustly reduced, which were activated upon different levels of voltage steps with a rapidly activating and slowing inactivating time course of the current [54,55]. The average I-V relationship of peak and sustained I K(A) in the control period (i.e., lutein was not present) or during cell exposure to 10 µM lutein was collated and is hence illustrated in Figure 5B or Figure 5C, respectively. For example, cell exposure to 10 µM lutein markedly decreased the peak I K(A) at 0 mV from 1076 ± 122 to 992 ± 112 pA (n = 7, p < 0.05). Therefore, the presence of lutein (10 µM) brought about a mild suppression in the peak and sustained amplitudes of I K(A) with no marked change in the inactivation time course of the current.

Mild Modification by Lutein of A-Type K + Current (IK(A)) in GH3 Cells
We also continued to study if the presence of lutein could influence macroscopic K + currents (e.g., IK(A)) identified in these cells. These experiments were conducted in cells which were bathed in Ca 2+ -free, Tyrode's solution containing 1 μM TTX, and we filled up the recording pipette by adding K + -enriched (145 mM) solution. As depicted in Figure 5A, with cell exposure to lutein at a concentration of 10 μM, the amplitudes of IK(A) were robustly reduced, which were activated upon different levels of voltage steps with a rapidly activating and slowing inactivating time course of the current [54,55]. The average I-V relationship of peak and sustained IK(A) in the control period (i.e., lutein was not present) or during cell exposure to 10 μM lutein was collated and is hence illustrated in Figure 5B or 5C, respectively. For example, cell exposure to 10 μM lutein markedly decreased the peak IK(A) at 0 mV from 1076 ± 122 to 992 ± 112 pA (n = 7, p < 0.05). Therefore, the presence of lutein (10 μM) brought about a mild suppression in the peak and sustained amplitudes of IK(A) with no marked change in the inactivation time course of the current.

Effect of Lutein on erg-Mediated K + Current (IK(erg)) in GH3 Cells
We also explored whether cell exposure to lutein could modify another type of K + currents (e.g., IK(erg)). To amplify IK(erg), we placed cells in a high-K + , Ca 2+ -free solution, and the recording pipette was filled with K + -containing solution. As shown in Figure 6A, when we held the tested cells at −10 mV, and a 1 sec hyperpolarizing step to −90 mV was imposed to activate the deactivating IK(erg) with a slowly decaying time course [56][57][58]. One

Effect of Lutein on erg-Mediated K + Current (I K(erg) ) in GH 3 Cells
We also explored whether cell exposure to lutein could modify another type of K + currents (e.g., I K(erg) ). To amplify I K(erg) , we placed cells in a high-K + , Ca 2+ -free solution, and the recording pipette was filled with K + -containing solution. As shown in Figure 6A, when we held the tested cells at −10 mV, and a 1 sec hyperpolarizing step to −90 mV was imposed to activate the deactivating I K(erg) with a slowly decaying time course [56][57][58].
One minute after cells were exposed to 10 µM lutein, upon membrane hyperpolarization from −10 to −90 mV, the peak amplitude of I K(erg) decreased from 891 ± 83 to 771 ± 79 pA (n = 7, p < 0.05). After washout of the compound, I K(erg) amplitude returned to 885 ± 22 pA (n = 7). The average I-V relationship of peak I K(erg) with or without cell exposure to 10 µM lutein was constructed and is hence illustrated in Figure 6B. Therefore, similar to the results described above in I K(A) , the data showed that lutein led to a mild inhibitory effect on I K(erg) activated in response to long-lasting membrane hyperpolarization. minute after cells were exposed to 10 μM lutein, upon membrane hyperpolarization from −10 to −90 mV, the peak amplitude of IK(erg) decreased from 891 ± 83 to 771 ± 79 pA (n = 7, p < 0.05). After washout of the compound, IK(erg) amplitude returned to 885 ± 22 pA (n = 7). The average I-V relationship of peak IK(erg) with or without cell exposure to 10 μM lutein was constructed and is hence illustrated in Figure 6B. Therefore, similar to the results described above in IK(A), the data showed that lutein led to a mild inhibitory effect on IK(erg) activated in response to long-lasting membrane hyperpolarization. Figure 6. Effect of lutein on erg-mediated K + current (IK(erg)) recorded from GH3 cells. In these experiments of amplifying IK(erg), we placed cells in high-K + (145 mM), Ca 2+ -free solution, and the recording pipette was filled with K + -containing solution. (A) Representative current traces activated by the hyperpolarizing step from −10 to −90 mV (indicated in the upper part, blue color). Current trace labeled a is the control (i.e., lutein was not present, black color), and that labeled b was recorded during the exposure of 10 μM lutein (brown color). (B) Average I-V relationship of IK(erg) taken in the absence (filled black squares) and presence (open brown squares) of 10 μM lutein (mean ± SEM; n = 7 for each point). Current amplitude (i.e., deactivating IK(erg)) was acquired at the beginning of each voltage step from a holding potential of −10 mV.

Effect of Lutein on Sag Potential Measured from GH3 Cells
We further switched to current-clamp potential recordings in an attempt to examine if lutein could result in any changes on sag potential in these cells. Sag potential evoked in response to hyperpolarizing current stimulus has been demonstrated previously to be linked to the occurrence of Ih in different types of excitable cells [59][60][61]. In this set of experiments, GH3 cells were placed in normal Tyrode's solution containing 1.8 mM CaCl2, and the pipette was filled with K + -enrich solution. Current-clamp configuration with a holding current of 0 pA was then established. As shown in Figure 7, under our experimental conditions, when the whole-cell potential recordings were achieved, a long-step 2sec hyperpolarizing current injection with the amplitude of around 25 pA was strikingly noticed to induce the occurrence of sag potential (i.e., an abrupt drop-down to a lower level in the membrane potential upon hyperpolarizing current injection) [62]. Cell exposure to ivabradine (3 μM) was effective at suppressing the amplitude of sag potential. Furthermore, the application of 1 or 3 μM lutein resulted in a conceivable depression of Figure 6. Effect of lutein on erg-mediated K + current (I K(erg) ) recorded from GH 3 cells. In these experiments of amplifying I K(erg) , we placed cells in high-K + (145 mM), Ca 2+ -free solution, and the recording pipette was filled with K + -containing solution. (A) Representative current traces activated by the hyperpolarizing step from −10 to −90 mV (indicated in the upper part, blue color). Current trace labeled a is the control (i.e., lutein was not present, black color), and that labeled b was recorded during the exposure of 10 µM lutein (brown color). (B) Average I-V relationship of I K(erg) taken in the absence (filled black squares) and presence (open brown squares) of 10 µM lutein (mean ± SEM; n = 7 for each point). Current amplitude (i.e., deactivating I K(erg) ) was acquired at the beginning of each voltage step from a holding potential of −10 mV.

Effect of Lutein on Sag Potential Measured from GH 3 Cells
We further switched to current-clamp potential recordings in an attempt to examine if lutein could result in any changes on sag potential in these cells. Sag potential evoked in response to hyperpolarizing current stimulus has been demonstrated previously to be linked to the occurrence of I h in different types of excitable cells [59][60][61]. In this set of experiments, GH 3 cells were placed in normal Tyrode's solution containing 1.8 mM CaCl 2 , and the pipette was filled with K + -enrich solution. Current-clamp configuration with a holding current of 0 pA was then established. As shown in Figure 7, under our experimental conditions, when the whole-cell potential recordings were achieved, a longstep 2-sec hyperpolarizing current injection with the amplitude of around 25 pA was strikingly noticed to induce the occurrence of sag potential (i.e., an abrupt drop-down to a lower level in the membrane potential upon hyperpolarizing current injection) [62]. Cell exposure to ivabradine (3 µM) was effective at suppressing the amplitude of sag potential. Furthermore, the application of 1 or 3 µM lutein resulted in a conceivable depression of sag potential evoked in response to hyperpolarizing current stimulus. For example, lutein at a concentration of 3 µM measurably decreased the amplitude of sag potential from 43.5 ± 7.0 to 20.5 ± 34.9 mV (n = 7, p < 0.05). It is conceivable, therefore, that the sag potential presented in GH 3 cells is associated with the magnitude of I h and that the depression of such potential produced by the presence of lutein could be largely ascribed from its inhibitory effectiveness in the I h amplitude of these cells. sag potential evoked in response to hyperpolarizing current stimulus. For example, lutein at a concentration of 3 μM measurably decreased the amplitude of sag potential from 43.5 ± 7.0 to 20.5 ± 34.9 mV (n = 7, p < 0.05). It is conceivable, therefore, that the sag potential presented in GH3 cells is associated with the magnitude of Ih and that the depression of such potential produced by the presence of lutein could be largely ascribed from its inhibitory effectiveness in the Ih amplitude of these cells.

Figure 7.
Effect of lutein on sag potential recorded from GH3 cells. In this set of current-clamp potential recordings, we bathed cells in normal Tyrode's solution, which contained 1.8 mM CaCl2 and 1 μM TTX, and a long-last hyperpolarizing current stimulus with a duration of 2 sec was then applied to the tested cell. (A) Representative potential traces acquired in the control period (a, black color) and during cell exposure to 1 μM lutein (b, blue color) or 3μM lutein (c, red color). The top part indicates the long-step current injection imposed over the tested cell. (B) Summary scatter graph demonstrating the capability of lutein to alter the amplitude of sag potential (mean ± SEM; n = 7 for each point). Under the current-clamp configuration, the magnitude of sag potential (i.e., the difference between the start and end of hyperpolarizing current stimulus) was acquired with or without cell exposure to lutein (1 or 3 μM). * Significantly different from control (p < 0.05) and ** significantly different from the lutein-alone (1 μM) group (p < 0.05).

Discussion
The principal findings in this work are that GH3-cell exposure to lutein is capable of suppressing Ih in a concentration-, state-, voltage-, and Hys(V)-dependent manner. A hyperpolarizing shift of the steady-state activation curve of Ih was observed in the presence of this compound. The strength of Vramp-evoked Hys(V) was also decreased by adding it. Under current-clamp configuration, the amplitude of sag potential observed in GH3 cells was also decreased during exposure to lutein. HCN2, HCN3, or mixed HCN2+HCN3 channels that can be encoded for generation of macroscopic Ih were reported to be distributed in pituitary GH3 cells [27,28,39]. Mild inhibition of IK(erg) or IK(A) by adding lutein was observed in this study. Under the current-clamp configuration, the exposure to lutein can result in a decrease in the magnitude of sag potential. Together, findings from the present study can thus be interpreted to mean that, besides its antioxidative or anti-inflammatory properties, the existence of lutein can inhibit the amplitude as well as alter gating and Figure 7. Effect of lutein on sag potential recorded from GH 3 cells. In this set of current-clamp potential recordings, we bathed cells in normal Tyrode's solution, which contained 1.8 mM CaCl 2 and 1 µM TTX, and a long-last hyperpolarizing current stimulus with a duration of 2 sec was then applied to the tested cell. (A) Representative potential traces acquired in the control period (a, black color) and during cell exposure to 1 µM lutein (b, blue color) or 3µM lutein (c, red color). The top part indicates the long-step current injection imposed over the tested cell. (B) Summary scatter graph demonstrating the capability of lutein to alter the amplitude of sag potential (mean ± SEM; n = 7 for each point). Under the current-clamp configuration, the magnitude of sag potential (i.e., the difference between the start and end of hyperpolarizing current stimulus) was acquired with or without cell exposure to lutein (1 or 3 µM). * Significantly different from control (p < 0.05) and ** significantly different from the lutein-alone (1 µM) group (p < 0.05).

Discussion
The principal findings in this work are that GH 3 -cell exposure to lutein is capable of suppressing I h in a concentration-, state-, voltage-, and Hys (V) -dependent manner. A hyperpolarizing shift of the steady-state activation curve of I h was observed in the presence of this compound. The strength of V ramp -evoked Hys (V) was also decreased by adding it. Under current-clamp configuration, the amplitude of sag potential observed in GH 3 cells was also decreased during exposure to lutein. HCN2, HCN3, or mixed HCN2+HCN3 channels that can be encoded for generation of macroscopic I h were reported to be distributed in pituitary GH 3 cells [27,28,39]. Mild inhibition of I K(erg) or I K(A) by adding lutein was observed in this study. Under the current-clamp configuration, the exposure to lutein can result in a decrease in the magnitude of sag potential. Together, findings from the present study can thus be interpreted to mean that, besides its antioxidative or anti-inflammatory properties, the existence of lutein can inhibit the amplitude as well as alter gating and Hys (V) behavior of I h , and that lutein's actions presented herein would engage in the modi-fications on spontaneous actions potentials present in electrically excitable cells (e.g., GH 3 cells), presuming that similar in vivo observations occur.
In this study, a left shift in the steady-state activation curve of I h with no concurrent modifications in the number of apparent gating charge in the curve was demonstrated with the existence of lutein. The τ act value estimated from the slowly activating I h during cell exposure to lutein also turned out to be larger because of a concurrent slowing in the activation time course of the current. Cell exposure to lutein was thus capable of altering hyperpolarization-activated I h in a time-, concentration-and voltage-dependent manner. Whatever the ionic mechanism of the active lutein, these results can be interpreted to mean that the responsiveness in different cell types to this compound would heavily rely either on the lutein concentration achieved, pre-existing levels of resting membrane potential, different patterns of action potential firing, or a combination of the three.
The Hys (V) properties of I h activated in response to triangular V ramp have been viewed to play a role in perturbing the electrical behaviors of various excitable cells [29,38,50,51,53,63]. Voltage-sensing domain relaxation has also been noticed to have a unique impact on such Hys (V) behavior observed in the proteins (i.e., HCNx channels) [29,50,64,65]. In other words, the observed "inertia" in the responsiveness of HCNx channels can be driven by changes in their electrical sensitivity, which was allowed to resemble that of ferromagnetic materials displaying Hys (V) behaviors [52,66]. Of considerable interest, in accordance with previous observations [29,49,63], the I h intrinsically residing in GH 3 cells was noticed to undergo a non-equilibrium property of instantaneous I h -that is, an anti-clockwise Hys (V) loop responding to the isosceles-triangular V ramp . Such change has been viewed to be dynamically linked to a mode shift in situations where the voltage sensitivity of gating charge movements (i.e., voltage-sensing domain relaxation) relies on the previous state (or conformation) of the channel [50,52,63,64]. Notably, GH 3 -cell exposure to lutein presented herein resulted in a significant reduction in Hys (V) strength (i.e., shaded region in Figure 4) of I h evoked by long-lasting triangular V ramp . However, whether the lutein molecule can interact with the voltage-sensing domains of HCNx channels remains to be further delineated.
In previous studies, different HCN isoforms have been demonstrated to combine to constitute macroscopic I h existing in different types of excitable cells [26,28,67]. Because HCN2, HCN3, or mixed HCN2+HCN3 channels were reported to be functionally expressed in GH 3 cells [28,39], it appears unlikely that lutein-induced block of I h inherently in native cells is isoform-specific. Of additional note, it has been reported that the blocker of HCN channels could be linked to changes in the phosphene perception of the retina [68], which might potentially account for lutein-mediated amelioration in the retina (e.g., macular degeneration) [4,7,8,10,37].
Earlier investigations have shown that the cyclic nucleotide-gated (CNG) channels, the behavior of which was noticed to show Hys (V) behavior in their gating mechanism, are regarded to mediate receptor potentials involved in olfaction and vision [53]. The HCN channels, another family of ionic channels gated by cyclic nucleotides, have been previously demonstrated to be linked to phototransduction in photosensitive retinal ganglion cells [47]. Its activity was found to alter the electroretinographic ON and OFF responses or to delay photoreceptor degeneration [19,[68][69][70]. To what extent do lutein-mediated changes in magnitude, gating kinetics and Hys (V) behavior of I h presented herein engage in lutein-mediated action on age-related diseases (e.g., macular degeneration or retinitis pigmentosa) [8,19,[71][72][73][74] still needs to be further delineated.
In this study, the lutein concentration required for the half-maximal inhibition (i.e., IC 50 ) of I h detected in GH 3 cells was calculated to be 4.1 µM. From the previous pharmacokinetic studies, it has been previously reported that plasma lutein concentration could reach around 2.5 µM after daily doses of lutein (20.5 mg) for 42 days, and that it could increase 3.5-and 10-fold on average, respectively, after the long-term intake of 4.1 and 20.5 mg lutein [75]. In comparison, it is, therefore, reasonable to assume that the observed effects by lutein on the amplitude, gating and Hys (V) of I h (or HCNx-encoded currents) may achieve the concentration of therapeutic or pharmacological requirements [75,76].
In this study, we additionally investigated how the protein of HCN channel could be docked with the lutein molecule through PyRx software. The protein structure of HCN channel was acquired from PDB (PDB ID: 5V4S) [77]. The predicted binding sites of the lutein with which the amino-acid residues can interact are presented in Figure 8.  [43]. It is thus likely that the interaction of the lutein molecules with HCN channels could be located at the cytosolic side of the membrane, although the detailed ionic mechanisms of lutein's actions are currently unknown. effects by lutein on the amplitude, gating and Hys(V) of Ih (or HCNx-encoded currents) may achieve the concentration of therapeutic or pharmacological requirements [75,76].
In this study, we additionally investigated how the protein of HCN channel could be docked with the lutein molecule through PyRx software. The protein structure of HCN channel was acquired from PDB (PDB ID: 5V4S) [77]. The predicted binding sites of the lutein with which the amino-acid residues can interact are presented in Figure 8.  [43]. It is thus likely that the interaction of the lutein molecules with HCN channels could be located at the cytosolic side of the membrane, although the detailed ionic mechanisms of lutein's actions are currently unknown.
The HEPES-buffered normal Tyrode's solution contained (in mM): NaCl 136.5, KCl 5.4, MgCl 2 0.53, CaCl 2 1.8, glucose 5.5, HEPES 5.5, and the pH was adjusted to 7.4 by adding NaOH. For measurements of I h or I K(A) across the cell membrane, GH 3 cells were placed in Ca 2+ -free, Tyrode's solution in order to avoid the contamination of voltage-gated Ca 2+ currents and Ca 2+ -activated K + currents. To record I K(erg) , we bathed cells in high-K + , Ca 2+ -free solution containing: KCl 145, MgCl 2 0.53 and HEPES-KOH 5 (in mM and pH 7.4).
In whole-cell current or potential recordings, we filled up the recording pipette with an internal solution, which contained: K-aspartate 140, KH 2 PO 4 1, Na 2 ATP 3, Na 2 GTP 0.1, EGTA 0.1, and HEPES 4 (in mM), and the pH was adjusted to 7.4 with KOH. The chemicals or reagents used to make these solutions were acquired from Sigma-Aldrich. The twicedistilled water was deionized through a Millipore-Q system and used in all experiments.

Electrophysiological Measurements
Shortly before the recordings, we carefully dispersed the GH 3 with 1% trypsin-EDTA solution, and thereafter placed a few aliquots of cell suspension containing clumps of cells into a home-made chamber tightly mounted on the working stage of a DM-II inverted microscope (Leica; Major Instruments, Tainan, Taiwan). Cells were immersed at room temperature (20-25 • C) in HEPES-buffered normal Tyrode's solution, ionic composition of which was elaborated above. The electrode that we used to record were prepared from Kimax ® -51 capillaries with 1.5-1.8 mm outer diameter (#34500; Kimble; Dogger, New Taipei City, Taiwan) by using a vertical puller (PP-83; Narishige, Major Instruments). Being filled with different internal solutions stated above, the recording electrodes had tip resistances of 3-5 MΩ. We measured ionic currents or membrane potential in the whole-cell current or potential recording of a modified patch-clamp technique, respectively, by using either a MultiClamp 700B (Molecular Devices, Sunnyvale, CA) or an RK-400 amplifier (Bio-Logic, Claix, France), as described elsewhere [24,49,55]. GΩ-seals were established in an all-or-nothing fashion and hence resulted in a dramatic improvement in the signal-tonoise ratio. The liquid junction potential, which occurred when the ionic compositions in the pipette solution and those of the external solution were different, was zeroed shortly before GΩ-seal formation was made, and the whole-cell data were then corrected. During measurements, we exchanged the solution through a homemade gravity-driven type of bath perfusion. In order to measure the I h , we voltage-clamped the examined cell at a holding potential of −40 mV before a long-lasting hyperpolarizing step was delivered.
The signals, comprising both potential and current traces, were monitored at a given interval and digitally stored online at 10 kHz in an ASUS ExpertBook laptop computer (P2451F; Yuan-Dai, Tainan, Taiwan). For efficient analog-to-digital (A/D) and digital-toanalog (D/A) conversion to proceed during the measurements, a low-noise Digidata ® 1440A equipped with an ASUS computer was controlled by pClamp TM 10.6 software run under Microsoft Windows 7 (Redmond, WA, USA). We low-pass filtered current signals at 2 kHz with a FL-4 four-pole Bessel filter (Dagan, Minneapolis, MN, USA). A variety of pClamp-generated voltage-clamp protocols with various rectangular or ramp waveforms were designed, and they were then applied to the tested cells through D/A conversion in attempts to assess the current-voltage (I-V) relation or steady-state activation curve of the currents.

Data Analyses
To assess concentration-dependent inhibition of lutein on I h amplitude, we put GH 3 cells in Ca 2+ -free Tyrode's solution. During the measurements, we voltage-clamped each cell at a holding potential of −40 mV and a long-lasting hyperpolarizing pulse to −120 mV for a duration of 2 sec was delivered to evoke I h . The I h amplitudes were thereafter measured at the end pulse of 2-sec hyperpolarizing command voltage during cell exposure to different lutein concentrations (0.1-100 µM). The concentration-dependent inhibition by lutein of I h in GH 3  To assess lutein-induced modifications on the quasi-steady-state activation of I h , the relationship between the normalized amplitude of I h and the conditioning potential acquired with or without cell exposure to 3 µM lutein was constructed, and the data were thereafter fitted by chi-squared goodness of fit with a Boltzmann function of the following form:

RT/qF
where I max = the maximal I h magnitude acquired with or without cell exposure to the lutein (3 µM) at the conditioning potential of −30 mV; V 1/2 = the voltage at which half-maximal activation (i.e., midpoint potential) occurs; q = the apparent gating charge; F = the Faraday constant, R the idea gas constant; T = the absolute temperature.

Curve-Fitting Approximations and Statistical Analyses
Curve-fitting (linear or non-linear [e.g., Hill or Boltzmann equation] to experimental data sets was dealt with the goodness of fit by using different analytical procedures, which include the Microsoft "Solver" built into Excel ® 2019 (Microsoft) and OriginPro ® 2021 program (Microcal; Scientific Formosa, Kaohsiung, Taiwan). The electrophysiological data (whole-cell current or potential data) are presented as the mean ± standard error of the mean (SEM), with the size of experimental observations (n) indicative of cell numbers from which the data were obtained. The data distribution was found to satisfy the tests for normality. We performed Student's t-tests (for paired or unpaired samples) between the two different groups, while for more than two different groups, analysis of variance (for one-way ANOVA or two-way ANOVA) with or without repeated measured followed by a post-hoc Fisher's least-significance difference test was utilized to determine the significance. A p-value of less than 0.05 was considered to indicate statistical difference (marked with * or ** in the figures).

Conclusions
These results reveal that lutein profoundly leads to a reduction in hyperpolarizationevoked I h (or HCNx-encoded currents) in a concentration-, time-, voltage-, and in a Hys (V)dependent manner. Our findings on GH 3 cells thus shed light on the evidence revealing the effectiveness of lutein in modifying specific ionic currents (e.g., I h ), and these data may be linked to its additional effects, which are potentially useful pharmacological applications in different excitable cells occurring in vivo.

Data Availability Statement:
The original data is available upon reasonable request to the corresponding author.