Ion channels mediate ion transport across membranes, and play vital roles in processes of matter exchange, energy transfer and signal transduction in living organisms. Recently, structural studies of ion channels have greatly advanced our understanding of their ion selectivity and gating mechanisms. Structural studies of voltage-gated potassium channels elucidate the structural basis for potassium selectivity and voltage-gating mechanism; structural studies of voltage-gated sodium channels reveal their slow and fast inactivation mechanisms; and structural studies of transient receptor potential (TRP) channels provide complex and diverse structures of TRP channels, and their ligand gating mechanisms. In the article we summarize recent progress on ion channel structural biology, and outlook the prospect of ion channel structural biology in the future.
Ion Channel Gating ; physiology ; Ion Channels ; Voltage-Gated Sodium Channels ; chemistry ; metabolism

The history and current situation of cell membrane ion-channel gating mechanism study were reviewed, with an emphasis on the application and the latest developments of kinetic model in gating mechanism study; the problems in present study and ion-channel gating mechanism kinetics model for future investigations were finally discussed.
Cell Membrane ; physiology ; Humans ; Ion Channel Gating ; Kinetics ; Models, Biological ; Research ; trends

BACKGROUNDFew studies have explored the inward sodium current (INa) kinetics of transitional cardiomyocytes. This study aimed to explore the kinetics of transitional cardiomyocytes types alpha and beta.

METHODSThe whole-cell patch clamp technique was used to study the rapid INa of isolated transitional cardiomyocytes in the Koch triangle of rabbit hearts.

RESULTSMaximal amplitude and density of INa in type alpha and type beta was (-1627 +/- 288) pA (alpha), (-35.17 +/- 6.56) pA/pF (beta) and (-3845 +/- 467) pA (alpha), (-65.64 +/- 10.23) pA/pF (beta) (P < 0.05). Steady state activation curves of INa, fitted to a Boltzmann distribution for both types, were sigmoid in shape. Half activation voltage and slope factors did not significantly differ between types at (-43.46 +/- 0.85) mV (alpha), (-41.39 +/- 0.47) mV (beta) or (9.04 +/- 0.66) mV (alpha), (11.08 +/- 0.89) mV (beta). Steady state inactivation curves of INa, fitted to a Boltzmann distribution in both types were inverse "S" shape. Half inactivation voltage and slope factors were (-109.9 +/- 0.62) mV (alpha), (-107.5 +/- 0.49) mV (beta) and (11.78 +/- 0.36) mV (alpha), (11.57 +/- 0.27) mV(beta), (P > 0.05), but time constants of inactivation were significantly different at (1.10 +/- 0.19) mV (alpha) and (2.37 +/- 0.33) ms (beta), (P < 0.05). Time constants of recovery from inactivation of INa for both types were (122.16 +/- 27.43) mV (alpha) and (103.84 +/- 28.97) ms (beta) (P < 0.05).

CONCLUSIONSTransitional cardiomyocytes in rabbit hearts show a heterogeneous, voltage gated and time dependent fast inward sodium current. Types alpha and beta show the features of INa similar to those in slow- and fast-response myocytes, with probably better automaticity and conductivity, respectively.

Animals ; Female ; Ion Channel Gating ; Kinetics ; Male ; Membrane Potentials ; Myocytes, Cardiac ; metabolism ; Rabbits ; Sodium Channels ; physiology

In the retina, pH fluctuations may play an important role in adapting retinal responses to different light intensities and are involved in the fine tuning of visual perception. Acidosis occurs in the subretinal space (SRS) under pathological conditions such as age-related macular degeneration (AMD). Although it is well known that many transporters in the retinal pigment epithelium (RPE) cells can maintain pH homeostasis efficiently, other receptors in RPE may also be involved in sensing acidosis, such as acid-sensing ion channels (ASICs). In this study, we investigated whether ASIC1a was expressed in the RPE cells and whether it was involved in the function of these cells. Real-time RT-PCR and Western blotting were used to analyze the ASIC1a expression in ARPE-19 cells during oxidative stress induced by hydrogen peroxide (H(2)O(2)). Furthermore, inhibition or over-expression of ASIC1a in RPE cells was obtained using inhibitors (amiloride and PCTx1) or by the transfection of cDNA encoding hASIC1a. Cell viability was determined by using the MTT assay. The real-time RT-PCR and Western blotting results showed that both the mRNA and protein of ASIC1a were expressed in RPE cells. Inhibition of ASICs by amiloride in normal RPE cells resulted in cell death, indicating that ASICs play an important physiological role in RPE cells. Furthermore, over-expression of ASIC1a in RPE cells prolonged cell survival under oxidative stress induced by H(2)O(2). In conclusion, ASIC1a is functionally expressed in RPE cells and may play an important role in the physiological function of RPE cells by protecting them from oxidative stress.
Acid Sensing Ion Channels ; metabolism ; Cell Line ; Humans ; Ion Channel Gating ; physiology ; Oxidative Stress ; physiology ; Retinal Pigment Epithelium ; cytology ; metabolism

The different concentration of specific ion species and the electrodiffusion of the ions down their electrochemical gradient generate transmembrane potential. The regulation of membrane potential for the function of numerous membrane proteins such as ion channels, transporters, pumps and enzymes plays primary role in the conversion of extracellular electric stimulation into a sequence of intracellular biochemical signals. Some ion channels regulated by membrane potential are well known, and the membrane non-ion channels protein is also modulated physiologically by membrane potential.
Humans ; Ion Channel Gating ; physiology ; Ion Channels ; metabolism ; Membrane Potentials ; physiology ; Phosphoric Monoester Hydrolases ; metabolism ; Receptors, G-Protein-Coupled ; metabolism

OBJECTIVETo investigate electrophysiology of cardiocytes in ligament of Marshall.

METHODSThe single cardiocytes obtained from ligament of Marshall were direct observed under inverted microscope. The cardiocyte action potential and current density of I(Na), I(Ca), L, I(to), I(K) and I(K1) were researched by whole-cell patch-clamp techniques.

RESULTSThere were two different cardiomyocytes in ligament of Marshall, one was rod shape, the other was short-rectangle shape. The short-rectangle myocyte was short and thick; the rod myocyte was long and thin. The short-rectangle myocyte was more than rod myocyte. The length/width rate of short-rectangle myocyte was less than that of rod myocyte (2.99 +/- 0.95 vs 12.05 +/- 2.41, P < 0.01). The action potential of ligament myocytes was similar to fast responsive cells. The action potential amplitude (APA) and duration (APD) of short-rectangle cells were less than those in rod cells. APA (mV), APD(50) (ms) and APD(90) (ms) were respectively 80.02 +/- 3.68 vs 91.72 +/- 7.56, 69.62 +/- 6.33 vs 83.14 +/- 3.66 and 107.55 +/- 4.25 vs 144.00 +/- 5.15, P < 0.05. The ion current density of I(Na), I(Ca), L, I(to), I(K1) was different between the two kind cells.

CONCLUSIONSThere are two different cardiocytes in ligament of Marshall. The action potential and ion current density of I(Na), I(Ca), L, I(to), I(K1) are different between the two kind cardiocytes.

Action Potentials ; Animals ; Dogs ; Electrophysiology ; Ion Channel Gating ; Ligaments, Articular ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; physiology ; Patch-Clamp Techniques

OBJECTIVETo establish a method of acutely isolating dorsal root ganglion (DRG) neurons for patch clamp study of single-channel.

METHODSDRG neurons of rats were acutely isolated by enzymatic digestion and mechanical blowing.

RESULTSThe acutely isolated DRG cells were easy to form the higher sealing resistance (> 5G Omega), which lowered noise level, so that pA-level single channel currents could be recorded.

CONCLUSIONThe acutely isolated DRG neurons in this study are an ideal for patch-clamp study of single-channel.

Animals ; Cell Separation ; methods ; Female ; Ganglia, Spinal ; cytology ; Ion Channel Gating ; physiology ; Ion Channels ; Male ; Neurons ; cytology ; physiology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley

As a crucial signaling molecule, calcium plays a critical role in many physiological and pathological processes by regulating ion channel activity. Recently, one study resolved the structure of the transient receptor potential melastatin 2 (TRPM2) channel from Nematostella vectensis (nvTRPM2). This identified a calcium-binding site in the S2-S3 loop, while its effect on channel gating remains unclear. Here, we investigated the role of this calcium-binding site in both nvTRPM2 and human TRPM2 (hTRPM2) by mutagenesis and patch-clamp recording. Unlike hTRPM2, nvTRPM2 cannot be activated by calcium alone. Moreover, the inactivation rate of nvTRPM2 was decreased as intracellular calcium concentration was increased. In addition, our results showed that the four key residues in the calcium-binding site of S2-S3 loop have similar effects on the gating processes of nvTRPM2 and hTRPM2. Among them, the mutations at negatively charged residues (glutamate and aspartate) substantially decreased the currents of nvTRPM2 and hTRPM2. This suggests that these sites are essential for calcium-dependent channel gating. For the charge-neutralizing residues (glutamine and asparagine) in the calcium-binding site, our data showed that glutamine mutating to alanine or glutamate did not affect the channel activity, but glutamine mutating to lysine caused loss of function. Asparagine mutating to aspartate still remained functional, while asparagine mutating to alanine or lysine led to little channel activity. These results suggest that the side chain of glutamine has a less contribution to channel gating than does asparagine. However, our data indicated that both glutamine mutating to alanine or glutamate and asparagine mutating to aspartate accelerated the channel inactivation rate, suggesting that the calcium-binding site in the S2-S3 loop is important for calcium-dependent channel inactivation. Taken together, our results uncovered the effect of four key residues in the S2-S3 loop of TRPM2 on the TRPM2 gating process.
Animals ; Asparagine/physiology* ; Binding Sites ; Calcium/metabolism* ; Glutamine/physiology* ; HEK293 Cells ; Humans ; Ion Channel Gating/physiology* ; Sea Anemones ; TRPM Cation Channels/physiology*

The gating mechanism of ClC-1 chloride channel was studied in this paper by heteroexpression of rat wild type ClC-1 gene in Xenopus oocytes and by two-electrode voltage clamping technique. The deactivation gating kinetic parameters were obtained by applying two exponential fitting of the deactivating currents at various extracellular chloride concentrations. It was found that decrease in extracellular chloride concentration increased the fractional amplitude of fast deactivating component, and depressed the fractional amplitude of slow deactivating component accompanied by a decrease in fast and slow deactivating time constants. These results demonstrate that the deactivation kinetic parameters of ClC-1 are largely dependent on the extracellular chloride concentration, which induces changes in channel gating.
Animals ; Chloride Channels ; drug effects ; physiology ; Chlorides ; pharmacology ; Electrophysiology ; Female ; In Vitro Techniques ; Ion Channel Gating ; drug effects ; physiology ; Oocytes ; physiology ; Rats ; Xenopus

This paper was aimed to study conserved motifs of voltage sensing proteins (VSPs) and establish a voltage sensing model. All VSPs were collected from the Uniprot database using a comprehensive keyword search followed by manual curation, and the results indicated that there are only two types of known VSPs, voltage gated ion channels and voltage dependent phosphatases. All the VSPs have a common domain of four helical transmembrane segments (TMS, S1-S4), which constitute the voltage sensing module of the VSPs. The S1 segment was shown to be responsible for membrane targeting and insertion of these proteins, while S2-S4 segments, which can sense membrane potential, for protein properties. Conserved motifs/residues and their functional significance of each TMS were identified using profile-to-profile sequence alignments. Conserved motifs in these four segments are strikingly similar for all VSPs, especially, the conserved motif [RK]-X(2)-R-X(2)-R-X(2)-[RK] was presented in all the S4 segments, with positively charged arginine (R) alternating with two hydrophobic or uncharged residues. Movement of these arginines across the membrane electric field is the core mechanism by which the VSPs detect changes in membrane potential. The negatively charged aspartate (D) in the S3 segment is universally conserved in all the VSPs, suggesting that the aspartate residue may be involved in voltage sensing properties of VSPs as well as the electrostatic interactions with the positively charged residues in the S4 segment, which may enhance the thermodynamic stability of the S4 segments in plasma membrane.
Arginine ; chemistry ; Aspartic Acid ; chemistry ; Cell Membrane ; physiology ; Conserved Sequence ; Ion Channel Gating ; Ion Channels ; chemistry ; Membrane Potentials ; Protein Structure, Tertiary