Bio2005 Minli Lectures I

Ion channels and receptors: structure, mechanisms and regulation

Min Li, Ph.D. ([email protected]) Department of Neuroscience High Throughput Biology (HIT) Center PDF copy of Lecture Slides (www.molecularinteraction.org)

Specific goals (how to get the most of the lectures)… • Understand the basic concepts • Appreciate importance and richness of biological questions and the classic experiments

Outline Background • General biology of membrane proteins • Receptors, channels and transporters • Ionic gradients in animal cells

Molecular biology of ion channels • • • •

Architecture and functional domains of ion channels Classification of ion channels Regulation and gating of ion channels Methods for studying ion channels

Cell biology of ion channels • Subunit assembly of ion channels • Membrane trafficking and regulation • Molecular coupling of ion channels and signaling proteins

Ion channels, human diseases and therapeutics • CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) • KCNQ potassium channels (epilepsy) • hERG (cardiac safety pharmacology)

Ion channels vs. receptors Ion Channels: Passively allow ions to flow one side of membrane to the other

Receptors: Interact with ligands to transmit signals

Membrane proteins in prokaryotes and eukaryotes…

Wallin and von Heijne, 1998

Number of proteins

Membrane proteins in prokaryotes and eukaryotes…

#1

#2 #3

Number of transmembrane segments

Membrane proteins in prokaryotes and eukaryotes…

Wallin and von Heijne, 1998

Plasticity…

Subcellular distribution of proteins …

Nature Biotechnology 19, 242 - 247 (2001)

Composition of cellular membranes

Molecular composition of the plasma membrane … Overall: Lipids ~49%, proteins ~49% and carbohydrates~1% •

Lipids –

Phospholipids - amphipathic molecule- polar head groupnonpolar fatty acid side chains. These molecules provide the basic structure of membranes which is a bilayer (or two layers of lipids with hydrophobic side chains facing inward). The outer face is called extroplasmic and inner face is called cytoplasmic.

–

Glycolipids - found almost exclusively in the outer face of the

–

plasma membrane, consisting of covalent linkage of a sugar molecule to a lipid. Common examples: galactocerebroside and gangliosides are prominent components of the plasma membrane of cells in the CNS. Cholesterol- regulates membrane fluidity.

Molecular composition of the plasma membrane … Overall: Lipids ~49%, proteins ~49% and carbohydrates~1%

•

Proteins –

Integral membrane proteins- proteins that are embedded in the lipid bilayer

–

Peripheral proteins- proteins attached to one face or another of membrane through non-covalent interaction between lipids, integral membrane proteins or carbohydrates.

–

Functions- Proteins carry out most of the functions of membrane. They are channels, receptors, enzymes etc.

Molecular composition of the plasma membrane …

Overall: Lipids ~49%, proteins ~49% and carbohydrates~1%

Carbohydrates Covalently attached to proteins and lipids primarily on the exoplasmic face of the membrane. Some times observed with EM as a hazy structure called a glycocalyx.

The fluid mosaic model •

The fluid mosaic model of lipid bilayer membranes, codified by Singer and Nicolson in 1972, describes the essential features of the biological membrane. It is a two-dimensional fluid, or liquid crystal, in which the hydrophobic integral components such as lipids and membrane proteins are constrained within the plane of the membrane, but are free to diffuse laterally. Singer SJ, Nicolson GL. Science. 1972 Feb 18;175(23):720-31

Membrane-bound proteins … • Integral or transmembrane proteins – they have hydrophobic regions that permits sections of the protein reside with the membrane. The proteins have a cytoplasmic domain, a transmembrane domain and an extracellular domain.

• Peripheral membrane proteins – they are tethered to the membrane by other structures (not peptides) such as lipid modifications.

Plasma membrane functions… 1. Semi-permeable barrier: The lipid bilayer accounts for the basic barrier functions of the plasma membrane. • •

permeable to water, O2 and small hydrophobic molecules such as steroids, ethanol impermeable highly charged molecules charged ions such as Na, K.

2. Regulate transport in and out of cell. 3. Communication 4. Adhesion

How might a cell form electrical gradient?

Chemical basis of a living organism Proteins – 60% Lipids – 16% Metabolites – 10% Glycans – 6% Nucleic acids – 5% Inorganic ions – 3%

The ionic basis of membrane potential

The ionic basis of membrane potential

Ionic gradients in a typical animal cells

Classification of ion channels Based on gating – Voltage-gated, ligand-gated, mechano-sensitive channels Based on permeability – Potassium, sodium, calcium and chloride channels Based on ligand pharmacology IP3 receptors, glutamate receptors, etc

Classification of ion channels Based on gating – Voltage-gated, ligand-gated, mechano-sensitive channels Based on permeability – Potassium, sodium, calcium and chloride channels Based on ligand pharmacology IP3 receptors, glutamate receptors, etc

Plasma membrane acts as a circuit

Electrophysiology - Voltage-clamp

Electrophysiological Recording

K+

Identification of ion channels via selective pharmacological block

(tetrodotoxin)

(tetraethylamonium)

Different ion channel genes in Drosophila

Initial cloning of genes encoding ion channels

Nature (1989)

Expression cloning of genes encoding ion channels Isolate mRNA

Fractionate mRNAs by size

Inject individual fractions into oocytes

Identify the fraction(s) with positive response

Make cDNA from the positive fraction

Re-fractionate and clone the one cDNA responsible for the response

Formation of heteromeric complexes

Nature (1990)

Functional role of ion channels …

Credit: Drs. C. Peters & D. Isbrandt

Voltage-gated ion channels: Action potential

Voltage-gated ion channels – in diseases Genomic amplification and oncogenic properties of the KCNK9 potassium channel gene Mu et al Cancer Cell, Vol 3, 297-302, March 2003 “ …..

KCNK9 encodes a potassium channel that is amplified from 3-fold to 10-fold in 10% of breast tumors and overexpressed from 5-fold to over 100-fold in 44% of breast tumors……”

Voltage-gated ion channels - therapeutics

Voltage-gated potassium channels

•Domain organization •Inactivation particle/ball •Pore region •Assembly domain (T1) •Composition and stoichiometry

Voltage-gated sodium channels

Voltage-gated calcium channels

Assays to test activities of ion channels

• • • •

Electrophysiology Ionic flux Voltage-sensitive dye Fluorescence

Single channel and macroscopic recording

Channel activity and gating I

Channel activity and gating II

Inactivation: mechanisms and domains

Summary •

I. Classification and structure – – – – – – – – – –

•

II. Motifs and functional domains – – –

•

Ion permeation pathway: Voltage sensor: Mechanism of inactivation: “ball and chain” model.

III. Accessory subunits and regulatory proteins – –

•

Significance pore-forming subunits auxiliary subunits gating ion selectivity: Voltage-gated K+ channel Voltage-gated Na+ channel Voltage-gated Ca2+ channel Conformational States: closed. opening inactivated Structure

Classification Regulation & stoichiometry

IV. Subunit assembly and trafficking – – –

Subunit assembly Trafficking Heteromultimeric channels

Formation of heteromeric complexes

Nature 345:530 (1990)

Formation of heteromeric complexes

Nature 345:530 (1990)

Formation of heteromeric complexes

Inactivation: mechanisms and domains

How would you go about testing one or more inactivation particles are needed?

hERG potassium channel

What if the inactivation is faster than the activation?

If inaction is fast!

Stoichiometric assembly – Many channels are multimeric complexes with specific stoichiometry, e.g. (alpha)4(beta)4. How would a cell know an assembled complex is in a correct stoichiometry?