Background

Cells and organelles depend on electrochemical gradients across their membranes, which are maintained through ATP-driven, membrane-embedded transporter proteins. Transport of ions across cellular membranes can be primary or coupled to drive the uptake or export of other solutes. Perturbations of the highly regulated transport systems are the cause to many diseases and can be the reason for failure of pharmacotherapy (Padan and Landau, 2016; Clausen et al., 2017; Picci et al., 2022). Given the pivotal role of these transporters in cell homeostasis and health, a thorough understanding of their organization, functioning, and dynamics is highly desirable. A powerful tool to visualize the activity of ion transporters in living cells and reconstituted membrane systems are fluorescent compounds based on synthetic molecules. Although a variety of ion-targeting probes are commercially available, many have limitations in studies with membrane transporters. For example, water-soluble dyes such as pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid) and fluorescamine (4-phenylspiro-[furan-2(3H),1-phthalan]-3,3′-dion) have been used to monitor the intravesicular pH values in studies of H⁺-translocating proteins (S. Li et al., 2011; Ohlsson et al., 2012; M. Li et al., 2015; Berg et al., 2017). However, their modest brightness (pyranine) and high bleaching rates (fluorescamine) make them difficult to use for pH measurements in vivo and in vitro. Rhodamine-based dyes provide an alternative due to their improved brightness and photostability; however, for reconstituted systems, problems with insufficient encapsulation during protein reconstitution and leakage out of vesicles were reported (Schubert, 2003; Leiding et al., 2009). More recently, membrane-bound pH sensors based on lipid-conjugated fluorophores have been utilized (Kemmer et al., 2015; Veshaguri et al., 2016; Schwamborn et al., 2017; Gerdes et al., 2018; Kühnel et al., 2019). These lipid-conjugated pH sensors efficiently co-reconstitute with membrane proteins into large liposomes, thereby avoiding loss during reconstitution and allowing studies in reconstituted systems down to the single vesicle level (Kemmer et al., 2015; Veshaguri et al., 2016; Kühnel et al., 2019). Lipid-conjugated pH sensors were also instrumental to study the activity of lipases on native substrate systems (Bohr et al., 2020). Furthermore, short-chain lipid-conjugated pH sensors enable monitoring of pH changes from neutral to acidic conditions in the endocytic pathway of living cells (Kühnel et al., 2019).

Here, we describe the synthesis of lipid-conjugated pH sensors from amine-reactive pHrodo esters and phosphatidylethanolamines. The method described here enables fast preparation of the sensor and can be used as a template to couple other amine-reactive fluorophores to phosphatidylethanolamines.

Things to consider before starting

1. Choice of the fluorophore

   The fluorophore must contain an amine-reactive functional group such as NHS- or STP-ester, to allow for coupling with the ethanolamine head group of the lipid. Furthermore, the fluorophore characteristics must be suitable for the later use of labelled lipids, e.g., pH range to induce a change in fluorescence emission. In Table 1, a number of reactive fluorophores are listed that we coupled effectively to phosphatidylethanolamines by using the protocol described here.

2. Choice of lipid

   The lipid headgroup must contain an ethanolamine group to allow for the coupling reaction with amine-reactive dyes. The lipid chains can vary. Herein, dioleoyl- and dipalmitoylphosphatidylethanolamine (DOPE and DPPE, respectively) were employed. The usage of other lipids might lead to decreased yields, as the reaction conditions are optimized for DOPE and DPPE.

3. Choice of analysis method

   A method to verify the successful formation of the labelled lipid is e.g., high resolution mass spectroscopy (MS), since small amounts of substance can be reliably detected. Two potential methods are MALDI-TOF MS (matrix-assisted laser desorption/ionization with time-of-flight analysis MS) and ESI MS (electrospray ionization MS).
   

   
   Table 1. Fluorophores that have been coupled effectively to phosphatidylethanolamine (PE)

   Fluorophore	Excitation (nm)	Emission (nm)	pH range#	pKa#
   pHrodo Red NHS-ester	566	588	2.7–7.0	4.6
   pHrodo Green STP-ester	505	543	2.7–7.0	4.6
   SNARF-1 NHS ester	488–530	585/650†	6–11	9.3
   Alexa FluorTM 488 NHS-ester	494	517	insensitive	---
   TAMRA NHS-ester	541	567	insensitive	---

   #Values upon coupling to DOPE (Kemmer et al., 2015; Kühnel et al., 2019).

   †Values upon coupling to DOPE. Note that the emission spectrum of SNARF-1 undergoes a pH-dependent wavelength shift, thus allowing the ratio of the fluorescence intensities from the dye at two emission wavelengths to be used for more accurate determinations of pH.

   All catalog numbers provided below shall serve as guide; alternative sources can be used as well.