Activation of membrane receptors by a neurotransmitter conjugate designed for surface attachment

Abstract

The derivatization of surfaces with bioactive molecules is a research area of growing importance for cell and tissue engineering. Tetherable molecules used in such applications must contain an anchoring moiety as well as the biofunctional group, typically along with a spacer to prevent steric clashes between the target molecule and the tethering surface. Post-synaptic membrane receptors at chemical synapses in neural tissue mediate signaling to the post-synaptic neuron and are activated by the binding of diffusible neurotransmitter molecules released by the pre-synaptic neuron. However, little attention has been directed at developing neurotransmitter analogs that might retain functionality when tethered to a surface that could be interfaced with post-synaptic receptor proteins. Muscimol (5-aminomethyl-3-hydroxyisoxazole), an analog of GABA (γ-aminobutryic acid), is a known potent agonist of GABA_A and GABA_C post-synaptic receptors found in retina and other central nervous system tissue. The present paper reports experiments testing the electrophysiological activity of “muscimol-biotin” on cloned GABA receptors expressed in Xenopus oocytes. This compound, which is potentially suitable for tethering at avidin-coated surfaces, consists of muscimol conjugated through an N-acyl linkage to a 6-aminohexanoyl chain that is distally terminated by biotin. We find that muscimol-biotin, as well as a structurally similar compound (muscimol-BODIPY^®) containing a bulky fluorophore at the distal end of the aminohexanoyl chain, exhibits substantial agonist activity at GABA_A and GABA_C receptors. Muscimol-biotin and other similarly biotinylated neurotransmitter analogs, in combination with surface functionalization using avidin-biotin technology, may be useful in applications involving the controlled activation of neuronal post-synaptic receptors by surface-attached molecules.

Introduction

Recent advances in the fields of biomaterials and micro-/nanotechnology have raised interest in developing neuroprosthetic devices that can interact with nerve cells under disease conditions to achieve controlled physiological stimulation of these cells. Approaches currently under investigation include, for example, the electrical stimulation of retinal neurons by devices implanted within the eye [1], [2], [3]. Another approach, distinct from that of direct electrical stimulation, builds on the fact that signal transmission at chemical synapses in neural tissue involves the activation of membrane receptor proteins on the post-synaptic neuron by chemical neurotransmitter released by the pre-synaptic cell. This latter approach envisions nerve cell stimulation through the controlled presentation of a receptor-active molecule (neurotransmitter or analog) to post-synaptic receptors and a resulting controlled stimulation of the post-synaptic neuron. Studies aimed at interfacing neurotransmitter-releasing devices with nerve tissue also have been reported [4], [5], but this “chemical” approach to nerve cell stimulation remains largely unexplored. Within this latter approach, the availability of neurotransmitter analogs capable of being tethered to a supporting surface could be useful in the development of micro-/nanostructures as neuroprosthetics [e.g., 6] as well as in advancing fundamental understanding of intercellular signaling in neural tissue.

The long-term goal of the research reported in this paper is to develop molecular platforms containing surface-tethered neurotransmitter analogs that can interact in controlled and specific fashion with native post-synaptic receptors. As the focus of this work we have chosen types A and C receptors for the neurotransmitter γ-aminobutyric acid (GABA). GABA is the major inhibitory neurotransmitter in CNS tissue, and GABA_A and GABA_C receptors, which gate transmembrane chloride channels, occur in high abundance in neurons such as bipolar cells of the retina [7], [8], [9], [10]. The ability to modulate GABA_A or GABA_C receptor activity in retinal bipolar cells thus could be useful, for example, in the development of neuroprosthetic structures for application as a therapy in retinal degenerative disease.

A key initial step toward developing surfaces with the desired receptor-modulating property is the identification of neurotransmitter analogs that both exhibit physiological activity and are potentially suitable for surface attachment. A logical approach to achieving tetherability of a known receptor-active compound is to derivatize this compound with a spacer chain (to prevent steric interference between the target receptor and the ultimate tethering surface) that is distally terminated by a functional group capable of mediating surface attachment. However, unlike bioactive molecules of relatively high molecular weight (e.g., peptides), neurotransmitters in retina and other CNS tissue are small amino acids (e.g., glutamate and GABA) that possess few sites for potential conjugation with a tethering moiety. Furthermore, even simple alteration of a small-molecule receptor ligand (e.g., derivatization of a carboxylic or methyl group) can dramatically reduce the ligand's affinity for its target receptor [11], [12]. Thus, chain-derivatization of amino acid neurotransmitters and analogs is unlikely as a general rule to preserve substantial biofunctionality of the receptor-active parent compound.

Muscimol (5-aminomethyl-3-hydroxyisoxazole) (Fig. 1) is a well-studied agonist of GABA_A and GABA_C receptors and exhibits a potency comparable with that of GABA [11], [12], [13]. Recently, two studies have presented evidence that N-acyl derivatives of muscimol can bind to GABA receptors. First, Wang et al. [14] have shown that muscimol linked through a 6-aminohexanoyl (6-aminocaproyl) chain to a BODIPY^® fluorophore (here termed “muscimol-BODIPY”; Fig. 1) exhibits localization to GABA_A receptors in retinal neurons. Second, Meissner and Häberlein [15] have found that muscimol conjugated directly (i.e., without an alkyl chain) to the fluorophore Alexa Fluor 532 shows high affinity for GABA_A receptors of rat hippocampal neurons. Overall, however, only a few N-acylated muscimol derivatives have been described in the literature [14], [15], [16], and none of these studies have investigated the electrophysiological activities of these derivatives.

In light of the above-summarized results obtained with N-acyl muscimol derivatives, and of the well established suitability of avidin-biotin technology for micropatterning and surface functionalization [6], [17], [18], [19], we reasoned that an N-acyl derivative of muscimol containing an aminohexanoyl chain terminated by a biotin group (“muscimol-biotin”; Fig. 1) would be of interest to investigate as a candidate agonist at GABA_A and GABA_C receptors. Here we report the synthesis of muscimol-biotin and the testing of its electrophysiological activity at cloned GABA_A and GABA_C receptors expressed in Xenopus oocytes. The Xenopus oocyte provides a useful model system for the study of GABA receptor activity, as the oocyte membrane lacks endogenous GABA receptors [20]. We find that muscimol-biotin, as well as muscimol-BODIPY, exhibits substantial agonist activity at both GABA_A and GABA_C receptors. These findings represent, to our knowledge, the first demonstration of electrophysiological activity by N-acyl derivatives of muscimol. Preliminary results of this study have been reported [21], [22].