Download Transcription Translation Molecular Structure of Ion Channels

Transcription
Translation
Molecular Structure of Ion Channels
Specialized proteins embedded within the bilayer:
1. Receptors
2. Cell Adhesion Molecules
3. Ion Channels
-control the movement of molecules in and out of the cell.
Proteins: chains of amino acids.
Amino Acids:
-there are only 20 different amino acids (AA).
-each has a central carbon atom.
NH3 (amino)
C
Side Chain
-each has an amino group
-each has an acid group
COOH (acid)
-they differ only in their
side chains.
Lysine:
Glutamic Acid:
COO-
COO+NH
3
C H
+
NH3 C H
CH2
CH2
CH2
CH2
CH2
COO-
CH2
NH3+
-type of protein is determined by the sequence of AAs.
AA
AA
AA
AA
-fold because of differences in charge along the protein.
Protein Synthesis
Nucleic Acids:
-control which proteins to make.
-messenger RNA (mRNA).
DNA:
-found in the nucleus (chromosomes).
-contains the instructions for protein
synthesis.
Soma
mRNA
Translation
Nucleus
Protein
mRNA Transcription
DNA
mRNA (final)
“Splicing”
mRNA
mRNA
Transcription
DNA
Introns
Introns: non coding regions of DNA
Exons: coding regions of DNA
Exons
Studying Ion Channel Structure
How to make a sodium ion channel:
1. Purify the protein from a rich source.
Electric eel
2. Identify short sequences of amino acids within protein
3. Make a single strand of DNA that matches
the sequence.
(Oligonucleotide)
-attach a fluorescent probe.
4. Take samples of mRNA from a source containing
sodium channel mRNA.
5. Make a cDNA library from the mRNAs.
cDNA:
-complementary DNA (single strand).
Reverse Transcriptase
mRNA
cDNA
-the cDNA from the sodium channel protein mRNA will
contain a region that exactly matches (complements) the
the oligonucleotide.
6. Use the oligonucleotide to identify the correct
cDNA.
7. From the isolated cDNA, the amino acid sequence
can be determined.
Sodium Channel: 2000 amino acids long
So what!
What is the structure of
the channel?
-some subunits are hydrophobic (in the membrane).
-connected by hydrophilic loops (outside the membrane)
Hydrophobicity Plot:
-some amino acids are hydrophobic and are
therefore found within the membrane.
The Sodium Channel
Mutant Flies
The Shaker Mutation
Alternative Splicing of Shaker Locus
Shaker Splice Variants
Heterologous Expression
Site-Directed Mutagenesis
Membrane Ion Currents
Conductance Leads to Voltage Change
NA+/K+ Conductance Ratio
Summed Membrane Currents
NA+/K+ G, I, V & E
Voltage Clamp
Depolarization and Currents
Nonlinear Voltage-Dependent Currents
Na+ Ions Produce Inward Current
NA+/K+ Channel Pharmacology