Calcium Entry Channels in Non-Excitable Cells

Juliusz Ashot Kozak, James W. Putney, Jr., Jr. Putney

• Publisher: CRC Press
• Year: 2017
• eBook ISBN: 9781351648639
• Edition: 1
• Topic: Biowissenschaften
• Subtopic: Biologie

Calcium Entry Channels in Non-Excitable Cells focuses on methods of investigating the structure and function of non-voltage gated calcium channels. Each chapter presents important discoveries in calcium entry pathways, specifically dealing with the molecular identification of store-operated calcium channels which were reviewed by earlier volumes in the Methods in Signal Transduction series. Crystallographic and pharmacological approaches to the study of calcium channels of epithelial cells are also discussed. Calcium ion is a messenger in most cell types. Whereas voltage gated calcium channels have been studied extensively, the non-voltage gated calcium entry channel genes have only been identified relatively recently. The book will fill this important niche.

1
Electrophysiological Methods for Recording CRAC and TRPV5/6 Channels

J. Ashot Kozak and Grigori Rychkov

CONTENTS

1.1Introduction

1.2Characteristics of Calcium Entry in Non-Excitable Cells

1.3CRAC Channels

1.3.1CRAC Channels in the Native Environment

1.3.2CRAC Current–Voltage Relation

1.3.3Current Separation

1.3.4Perforated-Patch Recording

1.3.5CRAC Channel Activity with Various Permeating Cations

1.3.6CRAC Single-Channel Conductance

1.3.7Heterologously Expressed Orai/STIM Channels

1.4TRPV5 and TRPV6 Channels

1.4.1Heterologously Expressed TRPV5/6

1.4.2Endogenous TRPV5/6 Channels

1.4.3Single-Channel Conductance

Acknowledgment

References

1.1Introduction

During the past two decades, great advances have been made in the electrophysiological and molecular identification of calcium entry pathways in non-excitable cells. The term “non-excitable” refers to a variety of cell types that are not capable of firing action potentials. Essentially, except for neurons, muscle cells, and some endocrine cells, all other cells in the body are non-excitable. For the most part, they lack the necessary levels of expression of voltage-gated Na⁺ channels (Na_V family) and also voltage-gated Ca²⁺ channels (Ca_V family). Ca²⁺ influx in these cell types is therefore thought to rely on unrelated, voltage-independent channels, such as Orai (CRACM) and TRP family members. In this chapter, we will discuss direct electrophysiological methods used to record the electrical activity of these proteins, focusing on calcium-selective Orai/STIM and TRPV5/TRPV6 channels.

1.2Characteristics of Calcium Entry in Non-excitable Cells

One of the first non-excitable cell types used to investigate the function of non-voltage-gated Ca²⁺ channels was cells of the immune system [1]. In T lymphocytes and mast cells, store-operated calcium entry is the main pathway providing cytoplasmic calcium elevations necessary for key cellular functions, such as antigenic activation, proliferation, and degranulation. Calcium stores inside the cell that were shown to be important for CRAC channel activation and functions are the endoplasmic reticulum (ER) and mitochondria [2–6].

Direct evidence for calcium entry following store depletion was demonstrated using the now classical calcium readdition protoco...

Table of contents

• Cover
• Halftitle Page
• Series Page
• Title Page
• Copyright Page
• Contents
• Series Preface
• Preface
• Contributors
• 1 Electrophysiological Methods for Recording CRAC and TRPV5/6 Channels
• 2 Studies of Structure–Function and Subunit Composition of Orai/STIM Channel
• 3 Signaling ER Store Depletion to Plasma Membrane Orai Channels
• 4 Modulation of Orai1 and STIM1 by Cellular Factors
• 5 CRAC Channels and Ca2+-Dependent Gene Expression
• 6 Function of Orai/Stim Proteins Studied in Transgenic Animal Models
• 7 Assessing the Molecular Nature of the STIM1/Orai1 Coupling Interface Using FRET Approaches
• 8 Optogenetic Approaches to Control Calcium Entry in Non-Excitable Cells
• 9 Regulation of Orai/STIM Channels by pH
• 10 Non-Orai Partners of STIM Proteins
• 11 Store-Independent Orai Channels Regulated by STIM
• 12 Regulation and Role of Store-Operated Ca2+ Entry in Cellular Proliferation
• 13 TRPV5 and TRPV6 Calcium-Selective Channels
• 14 Determining the Crystal Structure of TRPV6
• 15 Identifying TRP Channel Subunit Stoichiometry Using Combined Single Channel Single Molecule Determinations (SC-SMD)
• 16 Pharmacology of Store-Operated Calcium Entry Channels
• Index