Inward rectifier potassium (Kir) channels are an important class of potassium channels with the simplest structural architecture of the characterized eukaryotic channels. This review highlights the convergence of structural studies and functional analyses of this large and extensively analysed ion channel family.
Several high-resolution structures of bacterial K+ channels have been solved: KcsA, MthK, KvAP and KirBac1.1. The recent structure of KirBac1.1, which has high sequence homology with eukaryotic Kir channels, offers the opportunity to evaluate the results of functional assays in the context of the relevant channel structure.
The selectivity filter confers selectivity for K+ by coordinating the ions with the backbone carbonyl oxygens of the K+ channel signature sequence. In Kir channels, structural features other than the signature sequence are also important for K+ selectivity.
Inward rectification refers to the preferential flow of ions into the cell. In Kir channels, outward flow of K+ is impeded by a block of the pore by cytoplasmic polyamines and Mg2+. The long cytoplasmic pore that was revealed in the high-resolution structures of KirBac1.1 and the intracellular domain of Kir3.1 explain several of the characteristics of inward rectification that have been identified in functional studies.
Kir channels can change their conformation and thereby reduce ion flow through them, which is referred to as gating. Two locations for the gate have been proposed: the bundle crossing and the selectivity filter. Conformational changes at these two locations might correspond to the functionally observed slow and fast gating, respectively.
Gating of Kir channels is regulated by several cytoplasmic factors, including PtdIns(4,5)P2, arachidonic acid, Na+ and Mg2+ ions, pH, heterotrimeric G proteins, ATP, phosphorylation, oxidation/reduction and interactions with PDZ domains. Mapping the functionally identified sites of interaction between Kir channels and these modulators onto the cytoplasmic structures of Kir3.1 and KirBac1.1 provides insights into the complex and synergic mechanisms of Kir channel modulation.
A challenging area for future explorations concerns the structural rearrangements that are induced by the binding of intracellular modulators and how they translate into opening and closing of the channel.
Inwardly rectifying K+ (Kir) channels have a wide range of functions including the control of neuronal signalling, heart rate, blood flow and insulin release. Because of the physiological importance of these channels, considerable effort has been invested in understanding the structural basis of their physiology. In this review, we use two recent, high-resolution structures as foundations for examining our current understanding of the fundamental functions that are shared by all K+ channels, such as K+ selectivity and channel gating, as well as characteristic features of Kir channel family members, such as inward rectification and their regulation by intracellular factors.
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We would like to thank D. Doyle for the coordinates of KirBac and communication on the structure. We would also like the thank members of the Jan laboratory for stimulating discussions and valuable comments on the review. F.A.H. is a student in the Neuroscience graduate program at UCSF. L.Y.J. is a HHMI investigator. This work was supported by an NIMH grant.
The authors declare no competing financial interests.
Protein Data Bank
Long-chain aliphatic compounds that contain more than one amine group. Putrescine, spermine and spermidine are prime examples. Because of the positive charges on these molecules, polyamines bind electrostatically to proteins, DNA and RNA.
- RCK DOMAINS
(Regulator of K+ conductance domains). A ligand-binding domain found in many ligand-gated K+ channels; in MthK the ligand is thought to be nicotinamide adenine dinucleotide.
Technique in which an alteration is made either at a specific site or randomly in a DNA molecule. Mutated DNA is then reintroduced into a cell and analysed with various techniques to determine which parts of a protein or nucleotide sequence are crucial for its function.
- SALT BRIDGE
Electrostatic interaction between oppositely charged amino-acid side chains in close proximity in a protein.
- RESTING POTENTIAL
The separation of positive and negative charges across the cell membrane results in the membrane potential. The resting potential is the membrane potential at which there is no net current flow across the cell membrane.
- TITRATABLE RESIDUE
An amino acid with a side chain that can bond and release protons within a physiological pH range. Seven of the twenty amino acids are titratable (pKa of the free amino acid is given in parenthesis; this can vary in the protein): aspartate (4.4), glutamate (4.4), histidine (6.5), cysteine (8.5), tyrosine (10), lysine (10) and arginine (12).
- SUBSTITUTED CYSTEINE ACCESSIBILITY METHOD
(SCAM). An approach to the characterization of channel and binding site structures that probes the environment of any residue by mutating it to cysteine and characterizing the reaction of the cysteine with sulphydryl reacting and coordinating reagents.
- SITE-DIRECTED SPIN LABELLING
(SDSL). In SDSL, a nitroxide side chain is introduced by cysteine substitution mutagenesis followed by modification of the unique sulphydryl group with a specific nitroxide reagent. Measurements of the spectral properties of the paramagnetic nitroxide probe with electron paramagnetic resonance (EPR) spectroscopy provide information on its environment in the protein.
- ELECTRON PARAMAGNETIC RESONANCE
(EPR). When an atom with an unpaired electron is placed in a magnetic field, the spin of the unpaired electron can align, either in the same direction or in the opposite direction. EPR is used to measure the absorption of microwave radiation that accompanies the transition between those two states.
- C-TYPE INACTIVATION
Two distinct molecular mechanisms for voltage-gated K+ channel inactivation have been described: N-type, which involves occlusion of the pore by an intracellular domain of the channel, and C-type, which involves a conformational change in the outer pore.
- TEMPERATURE FACTOR
(B-factor, Debye-Waller factor). A measure of atomic vibration as described by the spread of the electron density. A low B-factor indicates low atomic mobility.
- ENERGY WELLS
Discrete sites along the conduction pore of the channel, which are energetically favourable. These sites arise from a delicate balance between interactions with the channel atoms, water in the channel and other ions. Wells are separated by barriers, which hinder diffusion. When the energy wells are low in energy compared with the barriers, the residence time of ions at these positions is long.
(PtdIns(4,5)P2). An anionic phospholipid found at low concentrations in biological membranes. It acts as a membrane-delimited second messenger, regulating the activity of a number of transporters and channels.
- PDZ DOMAIN
A peptide-binding domain that is important for the organization of membrane proteins, particularly at cell–cell junctions, including synapses. They can bind to the carboxy termini of proteins, or can form dimers with other PDZ domains. PDZ domains are named after the proteins in which these sequence motifs were originally identified (PSD95, Discs-large, zona occludens-1).
- RUNDOWN (OR WASHOUT)
Decrease in channel activity over time. Loss of phosphorylation and decrease of the levels of PtdIns(4,5)P2 and ATP have been suggested to cause rundown, but other processes that are not yet understood might occur.
A phosphoprotein that modulates the phototransduction cascade by interacting with the βγ-subunits of the retinal G-protein transducin.
- FLUORESCENCE RESONANCE ENERGY TRANSFER
(FRET). A spectroscopic technique that is based on the transfer of energy from the excited state of a donor moiety to an acceptor. The transfer efficiency depends on the distance between the donor and the acceptor. FRET is often used to estimate distances between macromolecular sites in the 20–100-Å range or to study interactions between macromolecules in vivo.
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Bichet, D., Haass, F. & Jan, L. Merging functional studies with structures of inward-rectifier K+ channels. Nat Rev Neurosci 4, 957–967 (2003). https://doi.org/10.1038/nrn1244
New Structural insights into Kir channel gating from molecular simulations, HDX-MS and functional studies
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