Voltage Sensor In Voltage-gated Channels Research Articles

A Novel Voltage Sensor in the Orthosteric Binding Site of the M2 Muscarinic Receptor

G protein-coupled receptors (GPCRs) mediate many signal transduction processes in the body. The discovery that these receptors are voltage-sensitive has changed our understanding of their behavior. The M2 muscarinic acetylcholine receptor (M2R) was found to exhibit depolarization-induced charge movement-associated currents, implying that this prototypical GPCR possesses a voltage sensor. However, the typical domain that serves as a voltage sensor in voltage-gated channels is not present in GPCRs, making the search for the voltage sensor in the latter challenging. Here, we examine the M2R and describe a voltage sensor that is comprised of tyrosine residues. This voltage sensor is crucial for the voltage dependence of agonist binding to the receptor. The tyrosine-based voltage sensor discovered here constitutes a noncanonical by which membrane proteins may sense voltage.

Can ClC-2 Chloride Channel be Activated by Hyperpolarization Alone in Cells Dialyzed with Non-Permeant Anions?

Open and closing of ion channels is driven by conformational changes triggered by either an intrinsic voltage sensor in voltage-gated channels or by ligand binding in ligand-gated channels. However, ClC-2, a two pore Cl− channel is activated by hyperpolarisations despite of lacking of an intrinsic voltage sensor. The structural information available to date strongly suggests that in the closed conformation each pore of ClC-2 is occluded by the negatively charged side chain of a glutamate residue. Although the gating mechanism is unknown, there is evidence which indicates that intracellular anions and extracellular protons regulate gating. The present work was aimed to determine the contribution of pore occupancy caused by intracellular anions in gating the mouse ClC-2 channel. In our experiments, ClC-2 was readily activated by hyperpolarisations when permeant anions such Cl−, SCN−, Br− and I− were present on either side of the membrane. However, ClC-2 was not activated in cells dialyzed with acetate (0 Cl−) and exposed to [H+]O=10−7.3 M. In contrast, the channels were opened by increasing [Cl−]i at [H+]O=10−10 M_a condition unlikely to protonate the glutamate's side chain. Importantly, voltage gating occurred when F− or glutamate were present in the cytosolic side. Since these two anions are non-permeant we propose that they must enter the pore and interact with the glutamate side chain from the inside in order to induce opening. Thus, we propose that a strong hyperpolarisation drives the intracellular anions into the permeation pathway and the subsequent electrostatic interaction between the anions and the negatively charged side chain opens the pore. Our data support the hypothesis that voltage-dependent gating in mouse ClC-2 requires intracellular anions. Supported by grant 219949 from CONACyT.

Critical role of conserved proline residues in the transmembrane segment 4 voltage sensor function and in the gating of L-type calcium channels.

The fourth transmembrane segment (S4) has been shown to function as a voltage sensor in voltage-gated channels. On membrane depolarization, a stretch of S4 moves outward and initiates a number of conformational changes that ultimately lead to channel opening. Conserved proline residues are in the middle of the S4 of motifs I and III in voltage-dependent Ca2+ channels. Because proline often introduces a "kink" into a helical structure of proteins, these residues might have an intrinsic function in the voltage sensor. Here, we report that the removal of S4 prolines results in a dramatic shortening of channel open time whereas the introduction of extra prolines to the corresponding positions in motif IIS4 and IVS4 lengthens channel open time. The number of S4s with a proline residue showed a clear positive correlation with the mean open time of the channel. The mean open time was >11-fold longer for a channel mutagenized to have prolines in all four S4s compared with a channel that had no prolines in the S4 region. Additionally, prolines in the S4s slowed activation kinetics and shifted the voltage dependence of activation and inactivation in a hyperpolarized direction. Our results strongly suggest that proline residues in the S4s are critical for stabilizing the open state of the channel. Moreover, it is suggested that motif IS4 and IIIS4 contribute to the channel opening more efficiently than motif IIS4 and IVS4.

SURFACE CHARGES AND ION CHANNEL FUNCTION

Like other proteins, ion channels are charged. The amino acid sequence contains acidic and basic amino acids that are positively or negatively charged at physiologic pH. Other charges are added by posttranslational events such as the addition of sialic acid during complex carbohydrate trimming and phosphorylation. At least some of the charges appear to be essential for ion channel function as part of ligand and permeant ion binding sites and as the voltage sensor of voltage-gated channels (e.g. 35). Surface charges also contribute to channel function in more subtle ways. Any charge on or near the surface of a channel will polarize the surrounding environment, thus establishing an electrostatic potential, i.e. the surface potential, between the channel and aqueous solution (e.g. 54). The surface potential will alter the surrounding ionic atmosphere and change the con­ centrations of all charged solutes (permeant ions, toxins, or ligands). Op­ positely charged solutes (counterions) will be attracted to the charge and