IK Channel Confers Fine-tuning of Rod Bipolar Cell Excitation and Synaptic Transmission in the Retina

Abstract

During retinal visual processing, rod bipolar cells (RBC) transfer scotopic signals from rods to AII amacrine cells as second-order neurons. Elucidation of the RBC’s excitation/inhibition is essential for understanding the visual signal transmission. Excitation mechanisms via mGluR6 and voltage-gated Ca²⁺ channels in the RBCs and GABAergic inhibitory synaptic inputs have been studied in previous studies. However, its intrinsic inhibitory mechanisms like K⁺ and Cl⁻ channels remain unclear. We focused on RBC’s prominent K⁺ current, which exhibits voltage and Ca²⁺ dependence. We isolated and confirmed the expression of intermediate-conductance Ca²⁺-activated K⁺ channels (IK) in RBCs using the patch-clamp method with IK inhibitors (clotrimazole and TRAM34) and immunohistochemistry. The regulation of the IK channel primarily relies on Ca²⁺ influx via low-threshold Ca²⁺ channels during RBC’s excitation. Additionally, IK mediates late repolarization and suppresses excessive oscillation of the membrane potential in the RBCs, enabling fast and transient synaptic transmission to AII amacrine cells. Our findings highlight the unique role of the IK channel in RBCs, suggesting that it plays a critical role in the scotopic pathway by fine-tuning RBC activity.

Graphical Abstract

Open in new tabDownload slide

Introduction

Rod bipolar cells (RBCs) are the central mediator of the scotopic pathway in the retina. RBCs receive a scotopic visual signal from the rod photoreceptors and transmit it to the AII amacrine cells, which contacts the cone bipolar cell terminal to convey the scotopic signal to the ganglion cells.^1–6 RBCs have distinct excitatory components compared with other conventional neurons in the central nervous system (CNS). That is, it expresses metabotropic glutamate receptor 6 (mGluR6) and transient receptor potential cation channel subfamily M member 1 (TRPM1), instead of ionotropic glutamate receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA).^7–10 Additionally, it expresses T-type and L-type voltage-gated Ca²⁺ channels (VGCC) without voltage-gated Na⁺ channels. These distinct characteristics generate a gradual potential for synaptic transmission rather than an action potential.^11–14

To understand the precise visual processing and transmission in RBCs, their inhibitory and excitatory mechanisms need to be elucidated. Therefore, inhibitory synaptic inputs to RBCs from horizontal and amacrine cells have been described in detail.¹⁵ Horizontal cells release the γ-aminobutyric acid (GABA) onto the dendrites of the bipolar cells in the outer plexiform layer (OPL) for feedforward inhibition and formation of the receptive field.^16,17 The A17 amacrine cells release GABA to the axon terminal of the RBCs, located in the innermost layer of the inner plexiform layer (IPL), as a reciprocal feedback inhibition to control the pre-synaptic glutamate release from RBCs.^18–21 Additionally, GABAergic amacrine cells synapse into RBCs, which are modulated by the dopamine pathway.²² However, information regarding the intrinsic inhibitory properties of RBCs, such as K⁺ and Cl⁻ channels which are controlled by membrane potential or intracellular Ca²⁺, is limited. Although various types of K⁺ channels and Cl⁻ channels were characterized in the nervous system as important regulators for neural excitation,^23–25 only a few were discussed in RBCs.²⁶ For example, voltage-gated K⁺ channels, voltage-gated Cl⁻ channels, and Ca²⁺-activated Cl⁻ channel, anoctamin 1 (ANO1) were identified in the RBCs using electrophysiology and immunohistochemistry. However, their role in excitability has not yet been described.^27–31 Nevertheless, those identified channels cannot sufficiently explain the prominent K⁺ current in RBC, and their functional role in excitability and synaptic transmission in RBCs has not been revealed. Thus, more information on K⁺ channels is required to understand the mechanisms controlling RBCs.

Ca²⁺-activated K⁺ channels are classified into three types based on their conductance, defined as large (BK), intermediate (IK), and small (SK). Those are critical for modulating the excitatory post-synaptic potential, action potential, after-hyperpolarization, and neurotransmitter release in neurons.³² The expression and function of Ca²⁺-activated K⁺ channels have been studied in retinal neurons. The BK channels have been found in the photoreceptors, on-bipolar cells, horizontal cells, amacrine cells, and ganglion cells in various species.^33–38 Additionally, the localization of SK channels in horizontal cells, amacrine cells, and ganglion cells has been reported in rodent retinas.^35,39–41 These studies showed that BK and SK are mainly involved in the depolarization, spiking activity, and spontaneous oscillations of retinal neurons. However, they are not expressed in RBCs.^31,42 Meanwhile, IK expression in the retina has not been widely explored, except for a previous report showing IK localization in the salamander photoreceptors.³⁵ Therefore, we assume that IK can be expressed in RBCs as an inhibitory regulator coupled with VGCC to control Ca²⁺-dependent excitation.

To test our hypothesis, we used the patch-clamp method and recorded the K⁺ current of RBCs in the mouse retina. We isolated the Ca²⁺-dependent component of the K⁺ current in the mouse rod bipolar cell, and revealed that the IK channel is the origin of the Ca²⁺-dependent K⁺ current. Additionally, we revealed that IK mediated the repolarization processes of RBCs to induce the fast and transient synaptic transmission form RBCs to AII amacrine cells, therefore IK has an important role for visual signal transmission.

Materials and Methods

Ethical Standards

All animal experiments followed the regulations of the Catholic Ethics Committee of the Catholic University of Korea, based on the guidelines of the National Institute of Health (NIH) for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). The animal experimental protocol was approved by the Institutional Animal Care and Use Committee and Department of Laboratory Animals of the College of Medicine, The Catholic University of Korea (Approval Number: CUMC 2020-0245-06).

Retinal Slice Preparation

C57BL/6 mice (6-7 weeks old) were used for patch-clamp experiments. The mice were kept under a 12-hour light/dark cycle in a climate-controlled laboratory. As described in our previous study,³¹ acute retinal slices were prepared to perform patch-clamp method. After anesthetization by intraperitoneal injection of zolazepam (20 mg/kg) and xylazine (7.5 mg/kg), the eyes were enucleated, and the mice were sacrificed by an overdose of zolazepam. The anterior compartments of the eye, including the cornea and lens, were immediately removed in the iced extracellular solution (NaCl 126, KCl 2.5, CaCl₂ 2.4, MgCl₂ 1.2, NaH₂PO₄ 1.2, NaHCO₃ 18, and glucose 11, in mm) oxygenated with 95% O₂-5% CO₂ gas, and the retina was gently dissected from the sclera. Retina was embedded in 1.5% agar with extracellular solution and sectioned into 200 μm thickness using a vibratome (Campden instrument, Loughborough, England). The prepared retinal sections were incubated in the oxygenated extracellular solution at room temperature for 1 h (24°C-27°C).

Visualization of the Rod Bipolar Cell and AII Amacrine Cell

The retinal slices were observed using a BX51 microscope (Olympus, Tokyo, Japan). The cell bodies of rod bipolar cells are located at the top of the INL, adjacent to the OPL. After achieving the whole-cell state, rod bipolar cells were confirmed by their morphological characteristics through the intracellular diffusion of the Alexa 488 dye, which showed a single projecting axon to the innermost layer of the IPL, with two or three axon terminals. The cell bodies of the AII amacrine cells were located at the border between the INL and IPL with a thick apical dendrite projecting into the IPL. AII amacrine cells were confirmed by their morphological characteristics after perfusion with Alexa 488 dye, whose processes were bistratified in the IPL by lobular appendages in sublamina a and stratifying dendrites in stratum five.^43,44

Electrophysiology

A recording solution containing 125 NaCl, 4 KCl, 1 MgCl₂, 2.5 CaCl₂, 10 HEPES, and 10 Glucose (in mm) was prepared, and its pH was adjusted to 7.4 with NaOH. A pipette solution containing 30 KCl, 100 K-gluconate, 1 MgCl₂, 0.5 EGTA, 5 ATP-Na, 3 ATP-Mg, and 1 GTP-Na (in mm) was prepared, and its pH was adjusted to 7.3 with KOH. Recording was performed in room temperature, 24°C-27°C. A MultiClamp 700 B amplifier and Axon Digidata 1440A (Molecular Devices, San Jose, CA, USA) were used for voltage- and current-clamp recording. Pipette resistance was 10 to 12 MΩ in the extracellular solution when it was filled with pipette solutions. Pipette offset was compensated by the MultiClamp 700 B panel. The recording pipettes were tightly attached to the cell membrane to achieve a gigaohm seal. After the giga-ohm seal state, the whole-cell state was achieved using a brief application of negative pressure. The pipette resistance and whole-cell capacitance were electronically compensated by the MultiClamp 700 B panel, and leak subtraction was not performed. The average series resistance was 14.5 ± 1.59 (mean ± standard deviation, n = 8). The stimulation protocols in the voltage-clamp and current-clamp methods were designed using pClamp 10 software (Molecular Devices).

Drug Application

All inhibitors were applied through the bath-application system. Mibefradil (T-type Ca²⁺-channel), nifedipine (L-type Ca²⁺-channel) iberiotoxin (BK), apamin (SK), clotrimazole (IK), and TRAM34 (IK) were used to inhibit the currents from the rod bipolar cells. All inhibitors were purchased from the Tocris Bioscience (Bristol, England).

Immunohistochemistry

For the immunohistochemistry, eyecups were fixed with 4% PFA for 1 h, and they were kept in 30% sucrose in PB 0.1 M overnight to prepare frozen blocks using the OCT compound. The frozen tissues were sectioned by 8 μm thickness using the LEICA cryostat (LEICA, Wetzlar, Germany). Retinal sections were washed with 0.01 M PBS and boiled with 10 mm citrate buffer for antigen retrieval. After then, sections were blocked by a cocktail of 10% normal donkey serum, 2% bovine serum albumin, and 0.2% Triton X-100 for 1 h, and those were incubated with primary antibodies for 18 h at 4°C. The sections were washed in PBS and incubated with a secondary antibody for 2 h at room temperature. Anti-KCa1.1 (1:100; APC-151, Alomone, Jerusalem, Israel), Anti-KCa2.1 (1:100; APC-039 Alomone), Anti-KCa2.2 (1:100; APC-028, Alomone), Anti-KCa2.3 (1:100; APC-025, Alomone), Anti-KCa3.1 (1:100; ALM-051, Alomone), Anti-PKCα (1:1000; sc-208, Santa Cruz, Dallas, Texas, U.S.A.), Cy3 (1:1500; Jackson Immunoresearch, West Grove, PA, USA), and Alexa 488-conjugated antibodies (1:500; Molecular Probes, Eugene, OR, USA) were used for primary and secondary antibodies. All images were captured using a Zeiss LSM 900 confocal microscope (Carl Zeiss Co. Ltd, Oberkochen, Germany).

Statistics and Reproducibility

The amplitude and decay time of the patch-clamp results were measured using the Clampfit software (Molecular Devices). Student’s t-test performed statistical analyses between two groups, and more than three groups were analyzed by one-way ANOVA using the Prism 8.0 software (GraphPad, San Diego, CA, USA) to determine statistical significance, with a P-value of < 0.05. Bar graphs were displayed by mean ± S.E.M.

Results

Isolation of the Ca²⁺-activated K⁺ Currents in the Rod Bipolar Cell

To perform patch-clamp recording from RBCs in retinal vertical slices, we selected the cell bodies of the RBCs at the top of the inner nuclear layer (INL), and their morphological characteristics were visualized by intracellular perfusion of Alexa 488 dye after the whole-cell state (Figure 1A-B). RBCs were stimulated using a step-voltage protocol that stimulates the cells for 2000 ms from a −70 mV holding potential to the target voltage, ranging from −50 to 30 mV at 10 mV intervals. Therefore, the voltage-dependent outward currents were recorded. These outward currents mainly consisted of K⁺ because the Cl⁻-induced outward currents were less than 100 pA at 30 mV, as shown in our previous reports.³¹ In the current traces, we defined a fast component (red arrow), indicating the peak amplitudes between 0 and 200 ms during stimulation, and a sustained component (green arrow), indicating the amplitudes at 25 ms before the end of stimulation (Figure 1F). Both components of K⁺ currents conspicuously increased from −20 mV, and thus, we hypothesized that they are related to the Ca²⁺ influx from VGCC, reaching a peak around −20 mV in the RBCs.¹¹ To confirm the Ca²⁺dependence of the K⁺ current, we measured K⁺ currents at 30 mV, and 5 mm BAPTA was added to the intracellular solution. After a 5-min intracellular approach of BAPTA, the sustained component was significantly reduced to 62% (Figure 1C-E, n = 5 for control and BAPTA, P < 0.05), whereas the fast component was not affected by BAPTA (Figure 1C-E, n = 5 for control and BAPTA, P > 0.05). To evaluate whether the K⁺ current is induced by Ca²⁺ influx through the VGCC, we applied 15 μm mibefradil and 15 μm nifedipine, respectively or concurrently, through the bath application system for 3 min and performed step-voltage stimulation. As shown in Figure 1F-I, a single application of mibefradil or nifedipine partially inhibited K⁺ currents, whereas the combination of the two inhibitors synergistically inhibited K⁺ currents. In the I-V relationship, the fast components of the K⁺ currents were weakly reduced by a single or concurrent application of mibefradil and nifedipine, while the sustained components were strongly reduced by a single or concurrent application of mibefradil and nifedipine. In particular, mibefradil was found to be more effective than nifedipine was. In details, 35% of the peak amplitudes of the fast components at 30 mV were reduced by mibefradil, nifedipine, and a cocktail of the two inhibitors (n = 7, P > 0.05 between the inhibitor groups), while 77% of the sustained components at 30 mV were reduced by mibefradil (with or without nifedipine), and 48% were reduced by nifedipine alone (n = 7, P < 0.05, Figure 1J and K). These results suggest that the Ca²⁺-sensitive K⁺ current mainly consists of sustained components of the K⁺ currents and is more strongly related to the mibefradil-sensitive Ca²⁺ channel than the nifedipine-sensitive Ca²⁺ channel is. Based on the effects of the Ca²⁺ channel inhibitors and BAPTA, we determined that the sustained K⁺ current originated from Ca²⁺-activated K⁺ channels.

Expression of the Ca²⁺-activated K⁺ Channels in the Rod Bipolar Cell

To identify the specific types of the Ca²⁺-activated K⁺ channel expressed in the RBCs, we performed double-labeling immunofluorescence experiments with anti-BK, SK, or IK channel antibodies and anti-PKCα antibody, a specific marker for the rod bipolar cell.^45–47 As shown in Figure 2A, BK was mainly detected in the inner segment (IS), OPL, INL, and ganglion cell layer (GCL). SK1 was primarily detected in the proximal portions of the INL and GCL (red in Figure 2B). Additionally, both SK2 (red in Figure 2C) and SK3 (red in Figure 2D) were detected in the IPL and GCL. However, the immuno-reactivity of anti-BK and SK1-3 antibodies was not co-labeled with the anti-PKCα antibody, which labeled rod bipolar cells (Figure 2A-D). Immunoreactivity of the anti-IK antibody (Alomone) was detected in the IS, OPL, distal and proximal portions of the INL, IPL, and GCL (Figure 2E). IK immuno-reactivity in the upper portion of the INL was overlapped with PKCα immuno-reactivity, indicating expression of the IK in the cell bodies of the RBCs (Figure 2E). Taken together, these results suggest that Ca²⁺-activated K⁺ currents in RBCs, as demonstrated in Figure 1, originate from the IK.

Pharmacological Isolation of the IK Current in the Rod Bipolar Cell

To confirm whether IK is the origin of Ca²⁺-activated K⁺ currents in rod bipolar cells, we pharmacologically isolated IK currents using the patch-clamp method. We tested the pharmacological effects of iberiotoxin, apamin, and clotrimazole, which are specific BK, SK, and IK channel inhibitors, respectively.^32,48 Retinal slices were pre-incubated for 10 min with iberiotoxin (500 nm) or apamin (500 nm), and the step-voltage protocol was applied to the rod bipolar cells to measure changes in K⁺ currents. Compared with the control traces, the K⁺ currents were slightly affected by iberiotoxin and apamin (Figure 3A-C). In the I-V relationship, the peak amplitudes of the fast and sustained components were minimally affected by iberiotoxin and apamin (n = 6 in each group, Figure 3D-E). At 30 mV, 6.5% and 8.8% of the fast components were reduced. In addition, 16% and 12.6% of the sustained components were reduced by iberiotoxin and apamin, respectively. We also analyzed the decay time between the 100% and 75% amplitudes to analyze current deactivation. The decay times were not significantly altered by iberiotoxin or apamin (P > 0.05, Figure 3F). These results suggest that functional BK and SK channels are not expressed in RBCs.

Next, we used clotrimazole and TRAM34, which are known IK-specific inhibitors. Clotrimazole (15 μm) was pre-treated by bath application for 10 min before achieving whole-cell state, and TRAM34 (1 μm) was applied through the pipette solution for 10 min as described in previous studies.^49,50 First, we demonstrated control traces, which were recorded at 0 min after achieving the whole-cell state and 10 min after achieving the whole-cell state (Figure 4A-B). Current traces changed minimally after 10-min intervals. Meanwhile, pre-application of clotrimazole effectively inhibited K⁺ currents more than the control current at 0 min did after the whole-cell state (Figure 4C). The internal application of TRAM34 significantly reduced K⁺ currents at 10 min after the whole-cell state more than the control traces at 10 min after the whole-cell state did (Figure 4D-E). Almost all residual current was inhibited by the addition of 5 μm 4-AP, which is a general voltage-gated K⁺ channel inhibitor (Figure 4F). In the I-V relationship, both IK inhibitors successfully inhibited sustained components rather than fast components (Figure 4G-H). In details, at 30 mV, 22.6% and 10.5% of the fast component were reduced, while 44.2% and 37.5% of the sustained component were reduced by clotrimazole and TRAM34, respectively (n = 8, P < 0.05, multiple comparison test). The consistent inhibitory effects of both IK inhibitors strongly suggest that the IK channel induces slow and long-lasting outward K⁺ currents in rod bipolar cells. A significant decrease in the decay time between the 100% and 75% amplitudes by IK inhibitors also suggests that the IK channel mediates the sustained component of the K⁺ current (Figure 4I). Through the inhibitory effect of the IK inhibitors, we clearly demonstrated that IK is a major source of K⁺ current in the RBCs, specifically inducing sustained components.

IK Channel Contributes to Sustained Repolarization After Peak Membrane Potential in the Rod Bipolar Cell

To understand the functional role of the IK channel during rod bipolar cell excitation, we used a current-clamp method and evaluated the effect of an IK inhibitor during depolarization. The basal membrane potential was fixed to −70 mV, and 65 pA was injected for 200 ms to achieve sufficient depolarization of the rod bipolar cell. In the control traces, the injected current depolarized the membrane potential, which showed a rapid rise to a single excitatory peak and sustained subsequent repolarization (Figure 5A). A rapid single excitatory peak was induced by the VGCCs (Supplementary Figure 1), which was detected in the previous paper.⁵¹ After the bath application of the 15 μm clotrimazole, rapid depolarization to the single peak from the baseline was not significantly changed compared to control traces, while the repolarization after the single peak was significantly reduced (Figure 5B). For a detailed analysis, we measured four parameters during the excitation process of the RBCs, including the peak and plateau membrane potentials from the baseline (−70 mV), after-hyperpolarization, and the recovery time from the end of the current injection to the baseline (Figure 5A-B). The peak membrane potential and after-hyperpolarization were not statistically different between the control and clotrimazole-treated groups (n = 14, P > 0.05, Figure 5C and E). Whereas the plateau membrane potential was significantly increased by clotrimazole (n = 14, P < 0.05, Figure 5D), and the recovery time from the end of the current injection to baseline was significantly increased by clotrimazole (n = 14, P < 0.05, Figure 5F). The results for the 4 parameters suggest that IK has little effect on the initial onset of depolarization and mainly controls sustained repolarization, and that the results were temporally matched with the sustained components in the voltage-clamp mode.

IK Channel Elongates the EPSP Duration in Synaptic Transmission Between Rod Bipolar Cell and AII Amacrine Cell

Inhibition of repolarization by clotrimazole strongly suggests that IK may be involved in synaptic transmission between RBCs and AII amacrine cells. To elucidate this, we recorded pairs of RBCs and AII amacrine cells to record the post-synaptic current from the AII amacrine cells when the RBCs were stimulated. The cell body of the AII amacrine cells was located at the border of the INL and IPL and had a round- or oval-shaped cell body with an apical single dendrite. After 488 dye perfusion, a narrow field and bistratified morphology throughout the IPL were visualized.⁵² RBCs were selected based on the width of the dendritic field in AII amacrine cells (Supplementary Figure 2). First, we stimulated the rod bipolar cell with a 65 pA current injection to achieve sufficient depolarization and recorded the synaptic current of the AII amacrine cells with or without clotrimazole. If the pairs of the RBCs and AII amacrine cells was selected, fast activation and decay of the synaptic current synchronized with peak excitation of the rod bipolar cells were recorded from the AII amacrine cells.⁵³ With the application of clotrimazole, repolarization of the rod bipolar cells diminished, and the post-synaptic currents from AII amacrine cells showed elongation of the decay process to the baseline (Figure 6A, red arrow). To obtain detailed information, we analyzed four parameters of the post-synaptic current: onset time (base to peak), peak amplitudes, decay time (peak to baseline), and area under the curve (AUC; Figure 6A’). The onset time was significantly reduced, but the difference was only 0.002 s (n = 11, P < 0.05; Figure 6B). The difference in peak amplitudes was not statistically significant (n = 11, P > 0.05; Figure 6C), whereas the decay time and area of the current trace were significantly increased by clotrimazole (n = 11, P > 0.05, Figure 6D and E).

Injection of the 65 pA current could induce strong depolarization of the RBCs, which sufficiently activates the IK channel and induces neurotransmission. Thus, it is beneficial to detect the role of IK channels in the repolarization process. However, a 65 pA current injection could be outside the physiological range compared to the light response of rod bipolar cells.^54,55 Therefore, we stimulated RBCs with a 25 pA current injection to understand the role of the IK current in the relatively weak excitation state. The control traces in Figure 6F show rapid onset and repolarization in RBCs and the synaptic current of the AII amacrine cells by 25 pA current injection, and these traces were similar to those of the 65 pA current injection. With the application of clotrimazole, the second peak (red arrow) of the membrane potential occurred in the rod bipolar cells during the repolarization process after the initial peak, indicating re-depolarization. A re-evoked synaptic current was also detected in AII amacrine cells, which was synchronized with the second peak membrane potential of the rod bipolar cells (red dashed line in the inset of Figure 6F).

For precise quantitative analysis, we manually counted the number of redepolarized membrane potentials in each RBC. In the control, 14% (1/7) of RBCs showed a second peak, whereas 80% (8/10) of the rod bipolar cells showed a second peak after clotrimazole treatment (Figure 6G, left). And only 14% (1/7) of RBCs showed re-release of glutamate, inducing a second synaptic current in AII amacrine cells, whereas 40% (4/10) of the RBCs showed re-release of glutamate (Figure 6G, right). And “the total number of the second peak/total number of the cell” in the control and clotrimazole-treated group showed an average of 0.1 and 0.9, respectively (Figure 6H). These results strongly suggest that the IK channel suppresses unintended excitation and synaptic transmission in RBCs.

IK Channel Contributes to Faster Recovery for Consecutive Synaptic Transmissions

In the above results, we demonstrated that IK channel induces the repolarization of RBCs, which mediates transient synaptic transmission from RBCs to AII amacrine cells. These results were obtained after a single stimulation of RBCs. Thus, we were curious whether the IK channel could affect the consecutive response of rod bipolar cells by repetitive stimulation because cells could prepare for the next depolarization during repolarization. To evaluate this, we designed a two-pulse protocol that stimulates RBCs twice at 10, 60, 110, 160, 210, 260, and 310 ms intervals and recorded the membrane potential of the RBCs with the synaptic current of AII amacrine cells. Traces of the two-pulse protocol are presented in Figure 7A (control) and 7B (clotrimazole). We noticed that the recovery of the peak membrane potential of the RBCs was slightly altered by clotrimazole. Meanwhile, post-synaptic currents in AII amacrine cells, induced by the second stimulation of rod bipolar cells, were strongly reduced by clotrimazole application (Figure 7A-B, red arrows). For quantitative analysis, we followed the time change of the peak membrane potential of the RBCs and the post-synaptic current of the AII amacrine cells in the two-pulse protocol (n = 4). We normalized the second membrane potential and second post-synaptic current using the first membrane potential and first post-synaptic current, as shown in Figure 7C (membrane potential) and 7D (post-synaptic current). In the control, the second membrane potential was reduced to 86% by a second pulse with a 10-ms interval, and it recovered to more than 97% after a 110-ms interval. Recovery of the membrane potential showed little difference between the control and clotrimazole application groups (P > 0.05 at each time point). However, unlike the membrane potential of RBCs, the post-synaptic currents from AII amacrine cells were significantly altered by clotrimazole. In the control, amplitude of the post-synaptic currents induced by second stimulation at 10 ms was average 11% compared to first stimulation, and it recovered to 27% at 110 ms. With the clotrimazole, amplitude of the post-synaptic currents induced by second stimulation at 10 ms was average 1% compared to first stimulation, and it recovered to 5% at 110 ms, which showed statistically lower recovery than the control did (P < 0.05, multiple t-test). These results showed that activation of the IK channel had little effect on the peak membrane potential of rod bipolar cells, while it strongly reduced synaptic transmission by repetitive stimulation, which means that IK is important for synaptic function.

Discussion

Evaluation of K⁺ channels in neurons is essential for understanding how they control neuronal excitation and synaptic transmission.^56,57 Voltage, Ca²⁺, or other intracellular factors can modulate the gating of various K⁺ channels, which modulate neuronal excitation differently than inhibitory synaptic input like GABA because of different acting times, cellular locations, and gating mechanisms. Thus, various types of K⁺ channels, including voltage-gated K⁺ channel, Ca²⁺-activated K⁺ channel, A-type K⁺ channel, inward rectifying K⁺ channels, have been extensively studied in CNS neurons.⁵⁸ However, although the retina belongs to the CNS, K⁺ channels in retinal neurons are not well understood. Thus, the characterization of K⁺ channels in the main excitatory retinal neurons is needed. A RBCs are a single-on-type bipolar cell in the scotopic pathway, sharing the same excitatory mechanism as a cone-on-bipolar cells, and it conveys the scotopic signal to the photopic pathway via AII amacrine cells.⁵⁹ Thus, the configuration of the K⁺ channel in the RBCs could help understand the on-cone bipolar cell excitatory mechanism, which is essential for understanding visual transmission.

In this study, RBCs showed prominent voltage-dependent outward currents (500-600 pA) in the step-voltage protocol. Additionally, the K⁺ current abruptly increased from −20 mV (Figure 1C), which is similar to the I-V relationship of VGCCs in RBCs,¹¹ which is around −20 mV (Figure 1C). Thus, we hypothesized that Ca²⁺-activated K⁺ channels are the major components of K⁺ currents. VGCC inward currents could be masked by K⁺ outward currents because the amplitude of VGCC is too small compared to K⁺ current, thus we presented VGCC currents in a Cs⁺-based internal solution in our previous paper,³¹ consistent with other papers.^11,51,60 The consistent inhibitory effects of the VGCC inhibitors (mibefradil and nifedipine) and BAPTA on the sustained component of K⁺ currents directly showed Ca²⁺-activated K⁺ currents. The immunohistochemistry results showing IK expression in rod bipolar cells supported our hypothesis (Figures 1-2). The inhibitory effect of BAPTA and EGTA could infer the distance between the Ca²⁺ source and Ca²⁺ targets. For example, in the bipolar cell terminals, the distance between the VGCC and ribbon in micro- or nanoscale could be discriminated by BAPTA and EGTA,⁶¹ and synaptic transmission from bipolar cell could be controlled by Ca²⁺ buffering like 5 mm BAPTA,⁶² also in the cones.⁶³ In our results, 5 mm BAPTA intracellular application could not completely inhibit Ca²⁺-activated K⁺ currents activated by VGCC. Those results suggest that IK and VGCC in the cell bodies of the RBCs could be close in nanodomain scale, thus IK finely controls VGCC-induced cell excitability.

We verified the inhibitory effect of IK-specific inhibitors (clotrimazole and TRAM34) on K⁺ currents in RBCs, whereas BK and SK channel inhibitors were not effective (Figures 3-4). Thus, we could conclude that the IK channel is the source of the Ca²⁺-activated K⁺ currents in RBCs, based on the negative results of the BK and SK channels of rodent retinal rod bipolar cells in previous studies,^31,42 and the pharmacological definitions indicating that IK is sensitive to IK inhibitors, unlike BK and SK inhibitors.^48,64,65

Previous studies on the IK channel in the CNS mainly focused on non-neuronal cells, including microglia, astrocytes, and endothelial cells.^32,66–69 Descriptions of IK in CNS neurons have only been reported in a few studies on pyramidal neurons and Purkinje cells. These studies revealed that IK induces after-hyperpolarization.^49,70,71 However, the role of the IK channels in neural excitation remains unclear. For example, there have been conflicting results regarding after-hyperpolarization in pyramidal neurons,⁷² and the role of IK in synaptic transmission has not been elucidated. This indicates that the detailed role of the IK channel may differ in each type of CNS neuron. In the retina, only Pelucchi et al. revealed that IK is localized in the rod photoreceptors of the salamander retina.³⁵ However, this report could not explain the gating mechanism of IK and its functional role because they only isolated the IK current at a membrane potential higher than the physiological range. Therefore, we focused on the gating factor of the IK channel and its contribution to the depolarization process and synaptic transmission in RBCs. To confirm the reliability of the investigation, we measured input resistance in the current-clamp recording. The input resistance of the RBCs was 1.19 ± 0.09 GΩ at peak membrane potential, and it changed to 1.22 ± 0.07 GΩ after clotrimazole treatment (P > 0.05, n = 14). While, the input resistance of the RBCs was 0.94 ± 0.09 GΩ at plateau membrane potential, and it significantly changed to 1.10 ± 0.02 GΩ after clotrimazole treatment (P < 0.05, n = 14, Supplementary Figure 3). Those input resistance values are consistence with the previous paper,⁵¹ and significant increase of input resistance in the plateau potential could be due to inhibition of IK channel. We analyzed four parameters during the depolarization of rod bipolar cells: peak membrane potential, plateau potential, after-hyperpolarization, and decay time in the current-clamp mode. Among them, the plateau potential and decay time were significantly increased by IK inhibitor, indicating that IK is important for repolarization from immediately after peak depolarization to the baseline (Figure 5). The repolarization effect of IK overlapped with that of ANO1 observed in our previous studies.^30,31 Although both IK and ANO1 induce Ca²⁺-activated inhibitory signals, IK may have different roles. IK is more sensitive to mibefradil (Figure 1), whereas ANO1 is similarly affected by mibefradil and nifedipine.³¹ This means that T-type Ca²⁺ channels are very close to IK as a Ca²⁺ source, which is a distinctive feature of the IK channel in rod bipolar cells compared to BK and SK, which form complexes with VGCCs.^73,74

Generally, T-type Ca²⁺ channels are low-voltage-activated Ca²⁺ channels that induce low-threshold exocytosis in neurons.^75,76 This has also been reported in retinal rod bipolar cells.^12,77,78 These properties of T-type Ca²⁺ channels may be important for sensing and conveying low-intensity light in the scotopic pathway. In our study, the IK channel was linked to T-type Ca²⁺ channels and induced repolarization. Thus, IK is an essential inhibitory regulator of T-type Ca²⁺ channel-induced low-threshold depolarization in RBCs, which might result in precise synaptic transmission under dim-light conditions.

To understand the specific role of IK during synaptic transmission, we used a dual patch-clamp method that records pairs of RBCs and AII amacrine cells. With the 65 pA current injection into RBCs, we obtained a stable post-synaptic current in the AII amacrine cells. In this protocol, we found that the inhibition of IK in RBCs elongated the post-synaptic current in AII amacrine cells, while peak amplitudes were not significantly changed (Figure 6A-E). Elongation of the post-synaptic current in AII amacrine cells, evoked by RBC stimulation, is consistence with the sustained components described by Kim et al. in the previous paper.⁷⁹ They described that the readily releasable pool (RRP) in the RBC could be increased after eye open, thus sustained component is elongated. Our results suggest that the IK is important for the termination of the synaptic currents to save the RRP. That means age might be not the only factor that contributes to sustained components of the synaptic current in AII amacrine cells, because the expression pattern of the K⁺ channel and its activity also could be changed by age, and it could influence the RRP. This indicates that the IK channel plays a role in inhibiting excessive synaptic transmission from RBCs to AII amacrine cells.

Notably, the effect of the IK channel on synaptic transmission between RBCs and AII amacrine cells differed from that of the BK channel in the photoreceptor terminals in a previous study.⁸⁰ They reported that a sufficient efflux of K⁺ by BK in the axon terminal of the rod could increase the extracellular K⁺ concentration and promote synaptic release. These conflicting results may be due to the conductance of K⁺ efflux and the expression site of Ca²⁺-activated K⁺ channels. Because IK channels in RBCs have smaller conductance than BK channels have and are mainly expressed in the cell body, the IK channel cannot sufficiently increase the extracellular K⁺ concentration compared to the BK channel in the photoreceptor axon terminals.

To evaluate the function of IK in a more physiological range, we reduced the amplitude of the current injection (25 pA) to RBCs to analyze the role of IK in rod bipolar cell excitability. Low amplitudes of current injection reflect low-threshold VGCC activation, which might be helpful for mimicking dim light stimulation in the rod pathway. With the 25 pA current injection with IK inhibition, RBCs showed a double-peak pattern during depolarization, which was not detected in the control state (Figure 6). Because activation of the VGCC is a unique source of the fast and transient peak of the membrane potential in the RBCs (Supplementary Figure 1), the double peak suggests the re-activation of VGCCs. The re-activation of the VGCCs could be interpreted in two ways: the re-opening of the VGCCs after deactivation and the activation of the remaining VGCC, which was not recruited for the first peak. Although we could not know the exact reason for the double-peak, it is certain that the second depolarization occurred by insufficient repolarization due to IK inhibition. After fast depolarization and repolarization, the membrane voltage decreased to below the threshold of the VGCCs; thus, the VGCCs could remain in a resting state. However, due to IK channel inhibition, insufficient repolarization maintains the membrane voltage of the rod bipolar cells over the threshold of the VGCC; thus, VGCCs could be in a re-activation state. This indicates that the IK channel inhibits the over-excitation of rod bipolar cells for precise synaptic transmission.

Repolarization is a critical step in neural excitation because the gating of the K⁺ channel sufficiently reduces the membrane potential to close the voltage-gated ion channels; thus, excitatory voltage-gated cation channels can prepare for the next excitation.^81,82 In this study, we revealed that the IK channel plays a role in the repolarization of RBCs, suggesting that the IK channel plays a role in the consecutive excitation of RBCs and synaptic transmission. To evaluate this, we performed a two-pulse protocol and analyzed the secondary membrane potential of RBCs and the synaptic currents of AII amacrine cells. Interestingly, the IK channel had little effect on the recovery of the peak membrane potential of the second stimulation, whereas inhibition of the IK channel reduced the secondary synaptic current (Figure 7). These results indicate that the IK channel suppresses excessive synaptic transmission in a single stimulation, which is important for preparing the next excitation of the RBCs and the AII amacrine cells pathway. Those results could be interpreted that IK inhibition suppresses the repolarization and elongates the sustained components of the post-synaptic current in AII amacrine cells, thus RRP could be consumed. Therefore, consecutive synaptic transmission less occurred after IK inhibition. That means proper repolarization is important to maintain a RRP of vesicles which has a role in computing temporal contrast in retinal rod bipolar cells.^83,84

In summary, we demonstrated the distinct characteristics of Ca²⁺-activated K⁺ channels in RBCs. The IK channel is activated by a low-threshold Ca²⁺ channel, which induces a sustained component of K⁺ currents and affects the repolarization process. Due to sustained K⁺ currents, the IK channel specifically modulates the repolarization process rather than the peak membrane potential of rod bipolar cells. This may be due to the distinctive role of the IK channel compared to the voltage-gated K⁺ channels in RBCs. In synapses, IK plays a complex role. First, it suppresses excessive prolongation of the synaptic current. Second, IK suppress the unintended re-activation of Ca²⁺ channels in RBCs during the repolarization state. Third, IK is essential for consecutive activation of RBC via the AII amacrine cell pathway.

We isolated a unique type of K⁺ channel, IK, from rod bipolar cells. There is little known about the role of the IK channel in the CNS, including retinal neurons, and the inhibitory mechanism of rod bipolar cells is not well known. Therefore, we expect that our results will expand our understanding of the role of IK in the neural system as well as visual transmission. However, we still need further evaluation of IK channels in the retina because our immunoreactivity showed IK expression in amacrine cells, and IK expression in the other bipolar cell subtypes is unknown. Moreover, understanding the role of IK in macroscopic retinal function is required to completely understand the contribution of IK to vision.

Author Contributions

Yong Soo Park (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft), Ki-Wug Sung (Investigation, Methodology, Project administration, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing), and In-Beom Kim (Funding acquisition, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing).

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) of Korea, funded by the Ministry of Education (grant number 2021R1I1A1A01049783 [SSP]) and by the Basic Science Research Program of the National Research Foundation (NRF) of Korea, funded by the Ministry of Education, Science, and Technology (NRF-2022R1A2C2006178 [I-BK]).

Conflict of Interest

None declared.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References