Research laboratory of Lei Wang, PhD
Chemical Biology Laboratory

Ion channels

Ion channels: molecular mechanism and optical control

Voltage-gated ion channels are activated by changes in membrane potential and play critical roles in propagating neuronal signals. Crystal structures of ion channels have revolutionized our understanding, yet lack of data on certain channel conformations prevents a complete comprehension of channel dynamics. To understand brain function, selective manipulation of channels and neurons in vivo would be tremendously useful. We designed unnatural amino acids to probe ion channels in mammalian cells and neurons to understand the dynamic channel activation and inactivation. We incorporated unnatural amino acids with extended side chains into the K+ channel Kv1.4 in HEK293 cells, and found that the diameter of the inactivation peptide is essential for fast channel inactivation. This finding, unobtainable with conventional mutagenesis, supports the hypothesis that the inactivation peptide extends through a side portal to exert inactivation, in contrast to the classic ball-and-chain model (Nat. Neurosci, 10, 1063-1072, 2007).

Uaa O-methyl-L-tyrosine

Figure 5.1: Uaa O-methyl-L-tyrosine extends the side chain length of tyrsoine by 0.9 angstrom, which impedes the inactivation peptide of Kv1.4 from threading through its side portal, abolishing the fast inactivation of the ion channel

To understand how voltage-gated ion channels sense membrane potential, we incorporated an environmentally sensitive fluorescent unnatural amino acid into the voltage sensitive domain (VSD) in neurons. Upon membrane depolarization, we observed fluorescence increase or decrease in a position-dependent manner (Stem Cells 29, 1231-1240, 2011). We are using such information to explain the voltage gating mechanism of the VSD.

fluorescent unnatural amino acid

Figure 5.2: A fluorescent unnatural amino acid optically reports the conformational change of the VSD in neurons differentiated from neural stem cells

Optical control of protein function provides excellent spatial-temporal resolution for studying proteins in situ. Although light-sensitive exogenous proteins and ligands have been used to manipulate neuronal activity, a method for optical control of neuronal proteins using Uaas in vivo is lacking. We genetically incorporated of a photoreactive Uaa into the pore of an inwardly rectifying potassium channel Kir2.1. The Uaa occluded the pore, rendering the channel nonconducting, and, on brief light illumination, was released to permit outward K+ current. Expression of this photoinducible inwardly rectifying potassium (PIRK) channel in rat hippocampal neurons created a light-activatable PIRK switch for suppressing neuronal firing. We also expanded the genetic code of mammals to express PIRK channels in embryonic mouse neocortex in vivo and demonstrated a light-activated PIRK current in cortical neurons. These principles could be generally expanded to other proteins expressed in the brain to enable optical regulation (Neuron, 80, 358-370, 2013).

photoactivation

Figure 5.3: Photoactivation of Kir2.1 channel in embryonic mouse neocortex