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Electrophysiological Response of Cultured Trabecular Meshwork Cells to Synthetic Ion Channels

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Electrophysiological Response of Cultured Trabecular Meshwork Cells to Synthetic Ion Channels View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Chemistry & Biology, Vol. 10, 35–43, January, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S1074-5521(02)00307-1 Electrophysiological Response of Cultured Trabecular Meshwork Cells to Synthetic Ion Channels Pawel Fidzinski,1 Andrea Knoll,2 Rita Rosenthal,1 ion channels as potential drugs to modulate cellular Anna Schrey,2 Andrea Vescovi,2 Ulrich Koert,2,4 functions or to restore lacking ion channel activity. Michael Wiederholt,1 and Olaf Strauß3,* We have chosen gramicidin A (gA) [5, 6] as a lead 1Institut fu¨ r Klinische Physiologie structure for ion channel design (Figure 1) [11, 13–17]. Universita¨ tsklinikum Benjamin Franklin gA, a bacterial antibiotic, acts in planar lipid bilayers as Freie Universita¨ t Berlin a cation channel with a weak Eisenmann I selectivity Hindenburgdamm 30 [5]. Its active conformation, a ␤-helical head-to-head 12200 Berlin dimer, has a length in the range of the lipophilic part of 2 Institut fu¨ r Chemie der Humboldt Universita¨t zu Berlin the phospholipid bilayer. Due to specific interactions Hessische Strasse 1-2 of the gA side chains with both the membrane interior 10115 Berlin and the membrane-water interface, this dimer is able to 3 Universita¨ tsklinikum Hamburg-Eppendorf establish a membrane-spanning pore [6]. Klinik und Poliklinik fu¨ r Augenheilkunde Our channel design is based on the covalent linkage Bereich Experimentelle Ophthalmologie [11, 17–19] of the gA motif and the incorporation of Martinistrasse 52 synthetic building blocks [13, 16, 20, 21], such as the 20246 Hamburg cis tetrahydrofurane amino acid (H-THF-OH) [22], with Germany the goal to tune the channel function. Thus, in synthetic THF-gram the hydrophobic valine-rich motif (residues 1–8) is replaced by a tetra-THF sequence [13]. Since Summary monovalent cations show a high affinity to ether and polyether functionalities, the tetrahydrofurane moiety is The response of living cells of the trabecular mesh- supposed to interact with monovalent cations and influ- work to synthetic ion channels is described. The THF- ence the overall ion-conductance properties. Both N gramicidin hybrids THF-gram and THF-gram-TBDPS termini in THF-gram are covalently linked by a C4-diacid as well as a linked gA-TBDPS and gramicidin A were moiety in order to increase the efficiency of pore forma- applied to cultured ocular trabecular meshwork cells. tion [11, 18, 19]. The introduction of a lipophilic capping THF-gram application (minimal concentration, 10Ϫ8 M; of the hydrophilic gramicidin C terminus using the saturation, 10Ϫ7 M) led to an additional conductance t-Butyldiphenylsilyl (TBDPS) group leads to THF-gram- which displayed characteristics of weak Eisenman-I- TBDPS. The syntheses of THF-gram and THF-gram- selective cation channels, no cell destruction, an TBDPS were carried out by segment coupling in solution asymmetric change of the inward/outward currents, as decribed earlier [13, 23]. All synthetic ion channels -and higher current densities using Cs؉ as charge car- were purified by RP-HPLC (Figures 2A and 2C) and char -rier compared to Na؉ and K؉. Linked-gA-TBDPS acterized by 1H-NMR and MS. In addition, a linked gA showed at 10Ϫ12 M increases of the membrane conduc- TBDPS was synthesized to explore the effect of the tance comparable to gA at 10Ϫ7 M and a much faster covalent linkage in more detail [23]. response of the cells. Thus, THF-gramicidin hybrids In this study, trabecular meshwork cells of the eye form a basis for the use of synthetic ion channels in were chosen as the biological target for the implantation biological systems, which eventually may lead to new of the synthetic ion channels [24]. The trabecular mesh- therapeutic approaches. work is a smooth muscle-like tissue which is involved in the regulation of the intraocular pressure, a major risk Introduction factor in the etiology of glaucoma [25, 26]. Our goal was to study the response of the living cells to the different With the development of the patch-clamp technique, synthetic ion channels, e.g., the effect on the native it became more and more clear that ion channels are membrane conductance or the response time. The func- involved in the regulation of nearly all cell functions [1, tional analysis of the synthetic ion channels in planar 2]. Several severe hereditary diseases which are caused lipid bilayers was addressed first. Second, the response by mutations in ion channel genes (channelopathies) of the trabecular meshwork cells to the synthetic ion show the medicinal relevance of ion channels [1]. In the channels was studied by patch-clamp techniques. recent past, substantial progress was made to correlate structure and function of biological ion channels [3, 4]. Additional understanding of ion channel function came Results from studies of synthetic ion channels in artificial mem- branes [5–11]. The investigation of synthetic ion chan- Single-Channel Recordings in Artificial nels in living cells is a nearly unexplored field [12]. This Lipid Bilayers challenging task holds the potential to develop synthetic Conductance experiments with THF-gram were per- formed in artificial phospholipid bilayers of the black- *Correspondence: [email protected] lipid type prepared from soybean lecithin and n-decane. 4 Current address: Fachbereich Chemie, Philipps-Universita¨ t Mar- In these recordings, THF-gram showed characteristic burg, 35032 Marburg, Germany. single-channel events (Figure 2B). Several different sub- Chemistry & Biology 36 Figure 1. Structures of gA, THF-Gram, THF-Gram-TBDPS, and Linked gA-TBDPS conductance levels were observed, indicating more Naϩ in a range of 2.6: 1 for the conductance and 10: 1 ϩ ϩ than one-channel active conformation. The amplitude for the permeability between NH4 and Na (see Supple- histogram for Kϩ currents of THF-gram shows that the mental Data). THF-gram-TBDPS showed a channel be- two highest level (2.3 and 2.5 pA) contribute the most to havior similar to THF-gram. The channel-opening times the overall membrane conductance (see Supplemental for THF-gram and for THF-gram-TBDPS were shorter Data). Thus, these levels were used for further evalua- compared to linked-gA-TBDPS (see below). Several tion. Concerning ion selectivity, THF-gram showed a conductance levels (Figure 2D) and a weak ion selectiv- ϩ ϩ ϩ ϩ ϩ ϩ ϩ weak ion preference in the order NH4 Ͼ Cs Ͼ K Ͼ ity NH4 Ͼ Cs Ͼ K Ͼ Na were observed for THF- Figure 2. HPL-Chromatograms and Single-Channel Recordings in Artificial Bilayers (BLM) of Synthetic Ion Channels (A) HPL-chromatogram of THF-gram. Rainin Dynamax C-8 column: A, H2O; B, MeOH; 85%–100% B over 25 min. (B) Single-channel current trace of THF-gram. (C) HPL-chromatogram of THF-gram-TBDPS. Macherey-Nagel Nucleosil 100-5 C-8 column: A, H2O; B, MeOH; 85%–100% B over 25 min. (D) Single-channel current trace of THF-gram-TBDPS. Single-channel currents were recorded in soybean lecithin membranes: 1 M KCl; 100 mV; 20ЊC; c ϭ 10Ϫ8 M. Cell Response to Synthetic Ion Channels 37 92.2 Ϯ 9% of the values measured before application of DMSO (n ϭ 3), and the reversal potential was changed by ϩ1 Ϯ 1.5 mV (n ϭ 3). After 10 min application of 0.5% methanol to the bath solution, the current density was 97.3 Ϯ 11.3% of the values measured before application of methanol (n ϭ 3), and the reversal potential was changed by Ϫ0.53 Ϯ 2.8 mV (n ϭ 3). The values observed after application of methanol or DMSO were not signifi- cantly different from the control values. Effects of THF-Gram and THF-Gram-TBDPS on Trabecular Meshwork Cells Application of 10Ϫ6 M THF-gram led to an increase in the membrane conductance of trabecular meshwork cells (Figure 4). This was observed in 80% of all investigated cells. 103 Ϯ 26 s (n ϭ 7) after beginning of the compound application, the hyperpolarization-induced inward cur- rents and the depolarization-induced outward currents started to increase. Usually, after 10–15 min in the pres- ence of the substance the current amplitudes reached maximal values. At that point, the overall density of membrane currents was increased by 5.8 Ϯ 2.0 pApFϪ1 (n ϭ 7; Figure 5A) when using Kϩ/Naϩ as charge carriers Figure 3. Comparison of Single-Channel Properties from gA and (Ringer solution as bath solution and standard pipette Linked-gA-TBDPS solution). Under these conditions, the reversal potential Typical single-channel currents in BLM soybean lecithin membranes of the membrane currents was shifted by ϩ12.1 Ϯ 3.4 in 1 M KCl at a membrane potential of 100 mV in the presence of gA ϭ (10Ϫ12 M; [A]) and linked-gA-TBDPS (10Ϫ14 M; [B]). For an interesting mV (n 10) toward more positive values (Figure 5B). explanation for the flickering observed with linked-gA-TBDPS, see [31]. The effect of THF-gram was concentration dependent between 10Ϫ8 M and 10Ϫ6 M. An asymmetric change of the outward/inward current was observed (Figure 5C). gram-TBDPS. However, with THF-gram-TBDPS the val- The increase in the outward current density (by 4.7 Ϯ ues for the highest conductance levels were about 1.6 pApFϪ1;nϭ 7; 10Ϫ6 M of THF-gram) was larger than 10%–40% lower for the different cations and the perme- the increase in the inward current density (by 1.1 Ϯ 0.4 ability ratios less pronounced than for THF-gram. In fur- pApFϪ1;nϭ 7; p ϭ 0.049).
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