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A patch-clamp study on human sperm Cl- channel reassembled into giant liposome

Jun-Ping BAI, Yu-Liang SHI

Key Laboratory of Neurobiology, Institute of Physiology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai  200031, China


Asian J Androl  2001 Sep; 3: 185-191

Keywords:  Cl- channel; giant liposome; patch clamp; human sperm membrane; ion channel reassembly
Abstract

Aim: To record the single-channel currents and characterize the electrophysiological properties of the Cl- channels in human sperm membrane. Methods: The membrane proteins extracted from the human sperm were reassembled into liposome bilayer, and the liposomes were fused into giant liposomes with a diameter more than 10 m by dehydration-rehydration procedure. The giant liposomes were used to study the Cl- channel activities by patch-clamp technique. Results: By patch clamping the giant liposome in an asymmetric NMDG (N-methyl-D-glucamine)-Cl (bath 100// pipette 200 mmol/L) solution system, three kinds of single-channel events with unit conductances of (74.18.3) pS, (117.05.7) pS and (144.74.5) pS, respectively, were detected. Their activities were voltage-dependent and all were blocked by SITS (4-acetamido-4-isothiocyanato-stilbene-2, 2-disulfonic acid) in a concentration-dependent manner. By constructing the open and close dwell time distribution histograms and then fitting them with exponential function, two time constants were obtained in both the open and the close states. The burst activity and conductance substate of the channels were observed. Conclusion: There exist three kinds of Cl- channels with different conductance in human sperm membrane at least.

1 Introduction

Maturation, capacitation and acrosome reaction (AR) of human sperm are associated with ionic flux through the membrane. There are growing evidences that the ion channels of the sperm plasma membrane play a key role in initiating gamete interaction and in mediating ionic fluxes in response to some hormones and bioactive substances[1]. To characterize the ion channel is a basic step in clarifying the mechanism involved in sperm maturation, capacitation and AR. In recent years, by combining bilayer-reconstitution of channel proteins with patch-clamp recording technology, the Ca2+ channel and several monovalent cation channels have been characterized successfully from human sperm membrane[2-5].

Due to certain reasons, researches on Cl- channel had been ignored until not long ago when the channel of the neuron attracted popular attention. In regard to the role of Cl- in mammalian sperm, it was reported that Cl- is essential in AR induced by zona pellucida (ZP) or progesterone, that AR was inhibited by Cl- channel blockers or in Cl- -free medium[6,7], and that progesterone-induced Cl- efflux was observed during AR[8]. Due to the small size of sperm, there were only a few studies dealing with the characterization of Cl- channel in mammalian or human sperm membrane[3-5]. Moreover, it is difficult to obtain sufficient data for detailed analysis due to a low success rate of the methods used, either the planar bilayer reconstitution[3,4] or the patch-clamping intact sperm approach[5]. Therefore, we employ giant liposome reconstituted with channel proteins, i. e., reassembling the proteins into liposomes and fusing the liposomes into giant liposomes by dehydration-rehydration procedure[9,10], for the patch clamp study on Cl- channel in human sperm membrane.
2 Materials and methods

2.1 Preparation of human sperm membrane protein

Human semen was collected from 20 healthy donors, aged 25 to 45 years, by masturbation. The membrane protein was prepared according to the method described by Yan et al[11]. In brief, spermatozoa were separated from the seminal plasma by centrifugation at 3,000g for 5 min. The sperm pellet was washed 5 times by 0.01mol/L phosphate buffered saline (PBS, pH7.4). Pellet spermatozoa were collected and resuspended in the Chaps (3-(3-Cholamidopropyl)-dimethylammonio-1-propanesulfonate)-containing solution- and stirred at 4 for 12h to extract membrane proteins. The insoluble material was removed by centrifugation at 22,400g for 40 min. The protein concentration of the supernatant (1-3 mg/mL) was measured by the Coomassie Brilliant Blue method and the extract was stored at -70 .

2.2 Reassembling sperm channel into bilayer of giant liposome

The procedure is a modification of that described by Keller et al[12]. Briefly,100 mg lecithin from soybean (type II, Sigma) in 1 mL of distilled water was intermittently stirred with Vortex for 30 min, and then suspended by sonication in the Branson Sonifer at 40 w for 5-10  min under nitrogen protection. The suspension (0.25 mL) was mixed with the extracted membrane protein preparation (~1 mL) and the dialysis solution (NaN3-free, ~1.25 mL). Then, the mixture was dialyzed against 400-600 volume of the dialysis solution for 70h. The dialyzed sample was centrifuged at 160,000g for 1h, and the pellet was resuspended with 200 L of 10 mmol/L Hepes (N-2-Hydroxyethyl piperazine-N-2-ethanesulfonic acid buffer containing 5% ethylene glycol, pH 7.4). The resuspended sample was deposited on a clean glass slide in 15  L aliquot and submitted to partial dehydration (3-6 h) in a desiccator containing anhydrous CaCl2, and then stored in refrigerator. Before use, the sample was rehydrated by using 15  L of 100 mmol/L NMDG-Cl solution on a slide, which was then placed in a closed Petri dish with a wet paper pad on the bottom. Twelve hours later, giant liposomes could be observed. The procedure for the preparation of proteinfree giant liposomes was the same as mentioned above, but without adding the extracted membrane protein. All the manipulations were done at 4 .

2.3 Patch clamping giant liposome and recording channel activity

An aliquot of the giant liposomes preparation (~10 L) was mixed with 200  L DEAE-Sephadex A-50 suspension (3 mg/ mL in bath solution) in a small dish and incubated at room temperature for 15-30  min. This treatment anchored the liposomes to the gel beads at the bottom of dish, making it easy to get giga-seal. After the remainder was removed by washing 3 times with the bath solution, single-channel recording was performed by using standard patch clamp technique.

The resistance of the pipette filled with the solution was 9-12 M. The current was defined as positive when cations flowed out the pipette into the bath. The signal was amplified through EPC-7 patch clamp amplifier  (List Medical Electronics, Germany), monitored on an oscilloscope, simultaneously digitized by AD/DA interface (Digidata 1200, Axon Instrument Co., USA) and stored in a computer for off-line analysis with the pClamp 6.02 software (Axon Instrument Co., USA). Recorded data were digitized at a sampling interval of 150 s and filtered at 3 kHz. The distribution histogram of current amplitudes was fitted with Gaussian distribution function and the mean current was measured[13]. The channel open or closedwell time histogram was constituted and fitted by a sum of exponential probability density function. AxoScope 1.01, SigmaPlot 2.0 and CorelDraw 8.0 were used to plot the figures. The results were presented as meanSD.

2.4 Solutions and reagents

The solution to extract proteins was composed of (mmol/L): Chaps 37, Hepes 10, NaCl 500, EDTA 1, PMSF (phenylmenthylsulfonyl fluoride) 0.2, pepstatine A 0.001, and ethylene glycol 5%, pH 7.4; the dialysis solution was composed of  (mmol/L): NaCl 100, Hepes 10, and NaN3 0.001%, pH 7.4. The NMDG-Cl solutions filling the recording pipette and bath were buffered with 1 mmol/L Hepes-Tris, pH 7.4. The concentration of the symmetric solution system was bath 200//pipette 200 mmol/L and that of the asymmetric system, bath 100//pipette 200 mmol/L. Chaps, Hepes, EDTA, PMSF, pepstatine A, and SITS were purchased from the Sigma Co. (USA), and sephadex A50, from the Pharmacia Co. (UK). All other reagents are of the analytical grade.

3 Results

As described in our pervious work[10], it is easy to form gigaohm seal between the patch pipette and the giant liposome prepared with the dehydraton-rehydraton method, and to obtain an excised patch for single-channel recording. In the present study, 60 inside-out patches were excised from more than 120 giant liposomes in a NMDG-Cl solution system, in which all the permeable cations were substituted with impermeable NMDG, and Cl- was the only permeable ion left. Most patches were stable and maintained for hours. The majority was single-channel event, however, multi-channel opening in a patch was also observed. No channel activity was found in the patches isolated from the protein-free giant liposome.

3.1 Unit conductance

In a bath 100//pipette 200 mmol/L NMDG-Cl solution system, a series of recording of the channel event at a range of holding potential of 100mV was obtained from 28 patches. By constructing the amplitude distribution histogram of channel event and then fitting it with the assumption of Gaussian, we measured the mean channel current, plotted the I-V relationship and thus obtained the unit conductance. The results showed that there were three kinds of channels with different conductance of 74.18.3 pS (n=11), 117.05.7 pS (n=8) and 144.74.5 pS (n=9). The I-V curves for the three channels were straight lines. The intersection of the curves with the voltage axis, namely, the reversal potentials (Vrev), were close to the theoretical value of Cl- electrode potential. They were 19.74.1 mV (74 pS), 17.34.0 mV (117 pS) and 18.32.9 mV (144 pS), respectively, in the asymmetric solution. In the symmetric solution (200 mmol/L), the I-V curves crossed the origin of the coordinate. Fig 1 showed the I-V curve and the Vrev of a 114pS channel in the asymmetrical solution and those after switching to the symmetrical, indicating that the recorded channel currents are carried by Cl-.

Fig 1. The activity of human sperm Cl- channels reassembled into giant liposome A. Original recordings in an asymmetrical NMDG-Cl solution system (bath100// pipette 200 mmol/L) at different voltages; B. All-point current amplitude histogram  at -50  mV; C. Single-channel current   (I)-voltage (V) curve with slope conductance of 114 pS in asymmetrical  or symmetrical solution of 200 mmol/L. The results were obtained from a patch.

3.2 Inhibition of channel activity by Cl- channel blocker

SITS is an irreversible blocker of the Cl- channel. In order to further identify the Cl- permeability of the above mentioned channels, we observed the effect of SITS on the channel after the recording became stable. It was found that 200 mol/L SITS in the bath solution completely blocked the channel activity. The effect was concentration-dependent, indicating a partial inhibition at 5-50mol/L and no visible effect at SITS concentrations below 5 mol/L (Fig 2). The inhibitory effect could be observed at all of the three channels.

Fig 2. Concentration-dependent inhibition of SITS on human sperm Cl- channel. Unit conductance is 74 pS in bath 100// pipette 200 mmol/L NMDG-Cl solution.

3.3 Voltage-dependence of the channels

By comparing the channel activity at various holding potentials, it was observed that within a certain extent, the channel open was voltage-dependent, as indicated by the following facts: 1) the channel open probability (Po) showed a maximum at -100 mV and was decreased upon depolarization; 2) the channel open frequency was higher at negative potentials, and was obviously lower at positive potentials (Fig 1 and Fig 3). The same phenomena were found at the three channels. Fig 3 showed a multi-channel activity in a patch, in which fast flicking openings of the three channels at -100 mV were observed and the number of open channels decreased at 50 mV and 100 mV. Meanwhile, the Po and the open frequency of the channel were reduced with the increase in the holding potential.

Fig 3. Voltage-dependence of human sperm Cl- channel A. Original recordings from a three-channel patch; B. Po-voltage relationship. The unit conductance of channel is 117 pS in bath 100// pipette 200 mmol/L NMDG-Cl solution. The results were obtained from a patch.

3.4 Open and close time constants

To estimate the related time constants, the open and close dwell times of the three kinds of channels were measured and then the open and close time distribution histograms were constructed. The distribution of the dwell time was best fitted by two exponential functions, namely, having two time constants each both for the open and the close state. These implied that the channels have at least two open states and two close states[14]. Fig 4 showed the fitted result of the 74 pS channel. The two open time constants were 0.35 ms and 1.17 msand the close time constants, 0.52 ms and 2.24 ms.

Fig 4. Open and close time constants of human sperm Cl- channel.  A: Original recordings; BC: Distribution of the open and close times of the channel recorded at least 30 s. The unit conductance is 74 pS in bath 100// pipette 200 mmol/L NMDG-Cl solution.

3.5 Substate and burst activity 

The subconductance state level between the close and the full open states has been reported in Cl- channels[15]. In this work, a substate was frequently observed at the three kinds of channels (Fig 5 A-B). In contrast to the result of multi-channel activities (Fig 6 A), an analysis of the original recording (Fig 5 B) showed that the probability values are not consistent with binomial distribution(Fig 6 B). Thus, it canbe reasonably excluded that the two open levels are due to the presence of two independent channels[16]. Generally, the conductance of the substate was 50-70% of that of a full open channel, and the substate was mainly transferred from a close state. Burst activity was frequently found from the channel event recording, especially at a negative potential (Fig 5C).

Fig 5. The subconductance state (A-B) and cluster opening (C) of human sperm membrane Cl- channels (bath 100// pipette 200 mmol/L NMDG-Cl) with unit conductances 74 pS(A), 117 pS(B) and 117 pS(C), respectively. They were obtained from three different patches.

Fig 6. Amplitude histograms (left) and open probabilities (P) (right) of Cl- channels at different conductance levels. A was drawn from the data shown in Fig 3, their P at four conductance levels can be best fitted by binomial distribution (correlation coefficient=0.989), showing that there were three independent channels in a patch. B was from the recording of Fig 5B. The distribution of P is different from that expected from binomial distribution of two independent channels (correlation coefficient=0.308), indicating that there exist a substate between close and full open state in the Cl- channel.

4 Discussion

Up to now, there is no systematic data describing the electrophysiological properties of Cl- channels in human sperm membrane. In this work, channel proteins of human sperm membrane were isolated and reconstituted into giant liposomes, and three kinds of Cl- channels with different conductance were observed by patch clamping technique.

In the literature, the values of Cl- channel conductance were reportedly to have a very broad distribution from 0.5 pS to 1300 pS in 150 mmol/L symmetrical Cl- solution[15,17]. The three channels in the sperm membrane recorded in this work should be belong to the medium-conductance Cl- channel. As judged from the unit conductance, they are different from those recorded in planar lipid bilayer[4]. The difference might result from the usage of different detergents (Triton -100 or Chaps) in extracting proteins. As a characteristic of the Cl- channel, subconductance has been observed in various tissues, e.g., torpedo electric organ, mollusk neuron, mammalian glial cell and lymphocytes[18,19]. In the present work the substate was also found in the Cl- channels of human sperm membranes.

Theoretically, during the liposome reconstitution process, the orientation of channel reassembled into a bilayer is random, however, we believe that it may be identical under a similar reassembling condition. This is supported by our previous work on the rectifier Na+ channel[10] and the present work on Cl- channel, both indicating that the ion channels with the same conductance have similar I-V relationship and reversal potential in different patches. If the channel orientation were different, an opposite direction of the rectification characteristics and reversal potential could be found in certain patches. In the inside-out patches of giant liposome, the Na+ channel reassembled with the same procedure was blocked by tetrodotoxin (TTX), a blocker only acting extracellularly on the Na+ channel, introduced into the bath[10]. This fact indicated that the orientation of the reassembled channel in giant liposome is just opposite to that in the natural sperm membrane. Thus the holding potential mentioned in this work should be equivalent to the intracellular potential, and a range of -50 mV-100 mV was close to the resting potential of the intact sperm. That the Cl- channels possess high Po and open frequency at the range of the potential, show that, the channels are fully open at the resting potential of the sperm.

According to the gating mode, the Cl- channel is classified into the voltage- and the ligand-gated channels. The background Cl- channel is a kind of voltage-gated channel and due to its extensive existence, activation in a broad voltage range and no inactivation in a long period of time, it is one of the most adequately studied channels. The background Cl- channel was classified into three types according to their voltage-dependence, i.e., hyperpolarization-activated, depolarization-activated and fully open at the level of resting potential[15,19-21]. Based on the channel behavior and the above deduction on the channel orientation in giant liposome, we suggest that the human sperm Cl- channels mentioned in this work belong to the third type. The voltage regulation of the open frequency at the Cl- channels was also observed in the brown adipose tissue mitochondria[22].

Similar to our previous work[10], the starting material for the reconstitution was not completely purified and identified. However, we found under electronmicroscope that the spermatozoa still retained their intact outline after protein extraction, and only a portion of the membrane was solubilized. The results are similar to those obtained with the boar sperm[23]. The authors believe that the channel activities described in the present work are derived from sperm plasma membrane proteins.

Patch clamp studies on intact mouse sperm have recently been reported[5], however, the data obtained were not sufficient for elucidating the channel's electrophysiological properties. Therefore, giant liposome reconstitution is still an available method to characterize the ion channels at the human sperm membrane. We believe that with the further advancement of the techniques and the combined use of the two methods, additional characterization on the types, features and functions of the ion channels at the human sperm membrane would be obtained.

Acknowledgements

We thank Miss WP Wang for her technical assistance. This work was supported by a grant from the National Natural Science Foundation of China (39870197).

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Correspondence to: Dr. Yu-Liang SHI, Shanghai Institute of Physiology, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.
Tel: +86-21-6437 0080 ext 154    Fax: +86-21-6433 2445
E-mail: ylshi@server.shcnc.ac.cn
Received 2001-06-08    Accepted 2001-08-13