Determining the Role of Plasma Membrane Cholesterol in Modulating ClC Channel Function Open Access

Hedden, Cameron (Spring 2021)

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Transmembrane ion channels facilitate the movement of charged species across plasma membranes in various cell types from all organisms, and channelopathies that impair or eliminate this ion conductance can result in lethal diseases like Cystic Fibrosis (CF). In recent years, new drugs have been synthesized that attempt to mitigate this defective ion conductance in CF, but there is mounting evidence that suggests that alterations of the plasma membrane impact the ability of these drugs to enhance patient outcomes. The plasma membrane in which ion channels exist and function is an incredibly complex mosaic of lipids, proteins and carbohydrates, yet how changes in this environment affect channel properties is not well understood. The present study sought to determine how depletion of plasma membrane cholesterol would impact the function of ion channels within the ClC family of voltage-gated transmembrane proteins, which are expressed widely throughout the body and are thought to be implicated in many neurological pathologies. Numerous cholesterol binding domains within the primary sequences of five ClC ion channels from two species were identified. Analysis of several primary amino acid sequences and computational modeling led to the identification of 10 potential cholesterol binding domains within human ClC-1 that were likely implicated in ClC protein-cholesterol interactions based on localization to the transmembrane domains of the protein. Of these 10 potential binding domains, sequence homology identified four domains that exhibited 100% conservation in all five of the ClCs studied. Two electrode voltage clamp (TEVC) studies of rabbit ClC-2 further supported an underlying dependence on membrane cholesterol for effective conductance of current across the plasma membrane. These results suggest an important role for plasma membrane cholesterol for effective function in ClC ion channels, and further suggest that depletion of plasma membrane cholesterol limits the ability of ClC ion channels to conduct current. With these results in mind, future work should focus on using site-directed mutagenesis to confirm the importance of the identified cholesterol binding domains and replicating TEVC studies with these mutant ClCs.

Table of Contents

Abstract 1

Introduction 2

Cystic Fibrosis and What it Taught Us 2

The Advent of Highly Effective Modulator Therapy in CF 4

The Plasma Membrane: Location is Everything 6

A New Protein with the Same Affliction 7

Methods 10

Searching the Primary Sequences for Potential Cholesterol Binding Sites 10

Mapping CRAC/CARC Domains onto Human ClC-1 11

Multiple Sequence Alignment 11

Cholesterol Docking Simulations 12

Constructs and Cell Maintenance 13

Two Electrode Voltage Clamp 13

Current/Voltage Analysis 15

Results 16

Identifying CRAC and CARC Domains in ClC Ion Channels 16

Mapping CRAC/CARC Domains onto Human ClC-1 17

Multiple Sequence Alignment 17

Docking Cholesterol on Human ClC-1 18

Effects of MꞵCD on Rabbit ClC-2 19

Discussion 22

Conclusion 26

Tables and Figures 27

Table 1. CRAC Domains in ClC-1 (Human) 27

Table 2. CARC Domains in ClC-1 (Human) 28

Table 3. CRAC Domains in ClC-2 (Human) 29

Table 4. CARC Domains in ClC-2 (Human) 30

Table 5. CRAC Domains in ClC-Ka (Human) 31

Table 6. CARC Domains in ClC-Ka (Human) 32

Table 7. CRAC Domains in ClC-Kb (Human) 33

Table 8. CARC Domains in ClC-Kb (Human) 34

Table 9. CRAC Domains in ClC-2 (Rabbit) 35

Table 10. CARC Domains in ClC-2 (Rabbit) 36

Figure 1. Scanning primary amino acid sequences for CRAC and CARC domains using R. 37

Figure 2. Solved Cryo-EM structure of human ClC-1. 38

Figure 3. Representative configuration file for cholesterol docking studies. 39

Figure 4. Mapped CRAC domains onto the solved Cryo-EM structure of human ClC-1. 40

Figure 5. Mapped CARC domains onto the solved Cryo-EM structure of human ClC-1. 41

Figure 6. Multiple sequence alignment of five ClC proteins. 42

Figure 7. Representative docking positions of cholesterol at potential CRAC binding sites. 43

Figure 8. Representative docking positions of cholesterol at potential CARC binding sites. 44

Figure 9. Circuit diagram of Two Electrode Voltage Clamp with a Xenopus oocyte. 45

Figure 10. Current-voltage relationship of oocytes from four treatment groups. 46

Figure 11. Whole cell net transmembrane current elicited at -160 mV for four treatment groups. 47

References 48

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