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Piezo proteins have been proposed as the long-sought-after mechanosensitive (MS) cation channels in mammals that play critical roles in various mechanotransduction processes, such as touch sensation and vascular development. However, their ion-conducting pore and ion permeation mechanisms have remained undefined. Here we identify domains and specific residues in Piezo1 that control the essential pore properties, including unitary conductance, ion selectivity and pore blockage, pinpointing the ion-conducting pathway. By uncovering the bona fide ion-conducting pore, these findings not only provide definitive proof that Piezo proteins are genuine pore-forming subunits of MS cation channels, but also shed light on elucidating the ion permeation and gating mechanisms of this prototypic class of mammalian MS cation channels.

Voltage-gated sodium channels (Nav) are important targets for treating various diseases. Crystal structure of the bacterial voltage-gated sodium channel NavMs in the open conformation has been obtained by Ulmschneider et al recently. We used this structure in our simulation work in order to study channel stability, sodium ion coordination as well as the ion permeation mechanism. We have employed free energy techniques to calculate the potential of mean force (PMF) for ion movement through the NavMs channel.

It is widely known that ions and water molecules alternately permeate (Pattern A) through the selectivity filter of the K+ channels. However, microscopic mechanisms underlying the exclusive use of the Pattern A remain unsolved. Here we performed the molecular dynamics simulation of ion permeation through the Kv1.2 channel at several different K+ concentrations. We found that the main permeation pattern is, as is expected, Pattern A at all concentrations, while the other kinds of pattern also emerge. At low concentrations, Pattern B, where an ion is transported accompanied with two water molecules, is observed. This behavior is consistent with the recent experiment measuring the streaming potential. On the other hand, Pattern C, where ions are transported without intervening water molecules, is also seen at high concentrations. Pattern C has been reported by some theoretical studies. At the physiological concentration, Pattern B and C, as well as Pattern A, are nearly equally used. In order to clarify why Pattern B and C emerge, the positions of the ions in the selectivity filter were analyzed. In the case of Pattern B, when a next incoming ion approaches the channel, ions are located at the downstream of the selectivity filter, thus two water molecules, having been occupied the selectivity filter, are franked by ions. Once an incoming ion enters into the selectivity filter that bears ions at the upper stream, no water molecule is inserted between the ions, and Pattern C emerges. Thus, the macroscopic observation such as the water-ion coupling ratio reflects differences in the microscopic ion distributions in the selectivity filter at the instance of an upcoming ion enters.

The BK-type potassium channels contains a K+-selective ion permeation pathway that opens and closes in response to changes of transmembrane voltage or intracellular Ca2+ concentration. Previous studies using large probes such as cytosolic blockers and MTS reagents have suggested that, unlike the voltage-gated potassium (Kv) channels, the BK ion permeation gate may not reside at the cytosolic entrance of the pore. However, this issue remains incompletely resolved. Here we utilize Cd2+, a probe of comparable sizes to K+, to further test state-dependent access to the BK inner cavity. We introduced cysteines at A316, a BK pore lining position inside the proposed Kv cytosolic gate, and determined Cd2+-Cys coordinating rate at this position in both and closed states. The result shows that the state-dependence of coordination between Cd2+ and A316C is moderate, with coordinating rate less than 10 times faster in the open state. In addition, the coordinating rate of A316C with Cd2+ in a closed BK channel is comparable to that of an inner cavity cysteine (I361C) with Cd2+ in an open CNG channel. We also examined Cd2+-Cys coordination in the YVP motif (V319C) near the cytosolic entrance of the pore. The counterpart region in Kv channel forms a tight steric exclusion when channel is closed. As a result, the homologous site of V319 in Kv channel (V474) is not accessible to Cd2+ in closed state. In BK channel, Cd2+ coordinates with V319C in both open and closed states, with open-state coordinating rate less than 1-fold faster than that of closed-state coordinating rate. These results suggest that the BK ion permeation gate is not located at its cytosolic entrance. Instead, the selectivity filter is the only possible region in BK to control ion flow.

Single-channel electrophysiology provides for high-resolution measurements of the dynamics of single macromolecules, the channel proteins (Neher and Sakmann, 1976). Among the measurements that have proven fruitful have been ability to monitor, in almost real time, the interactions between the channel and a blocker, or modifier, of ion permeation (Neher and Steinbach, 1978; Prod’hom et al., 1987; Hemsley and Busath, 1991). The emphasis usually has been on the channel, but different blockers/modifiers may have different characteristic “fingerprints” (Hemsley and Busath, 1991), which provides for possibilities in terms of using the characteristics of the blocking events to determine the chemical identity of the blocking species.

In the ion permeation pathway of the K+ channel, the narrow selectivity filter is a critical region where ions and water molecules permeate with a single-file fashion. Theoretical studies have been concentrated on the permeation process in the selectivity filter, whereas the role of the central cavity, a common architecture that all the known K+ channels have, is not systematically examined yet. Here we investigated ion permeation through the Kv1.2 channel by the molecular dynamics simulation and analyzed the role of the central cavity. It is found that the central cavity attracts the K+ ions when the ion concentration in the bulk is below the physiological range. Accordingly, the concentration in the central cavity is higher than that in the bulk, increasing the entry chance of ions into the selectivity filter. Thus, the central cavity plays a role to make ion permeation rapid. On the other hand, the ion concentration in the central cavity is found to be saturated at high concentration in the bulk, and the conductance is saturated. These results indicate that the concentration of ions in the central cavity generates the Michaelis-Menten-type behavior of the conductance-concentration curve in the Kv1.2 channel. In contrast, it is known that the saturation of the conductance is not observed in the KcsA channel. We found that the ion concentration in the central cavity increases progressively as the bulk concentration increased up to 2 M, which is in agreement with the apparent non-saturating concentration-conductance curve in the previous studies.

Potassium channels can conduct passively K+ ions with rates of up to ∼ 108 ions per second at physiological conditions, and they are selective to these species by a factor of 104 over Na+ ions. Ion conduction has been proposed to involve transitions between two main states, with two or three K+ ions occupying the selectivity filter separated by an intervening water molecule. The largest free energy barrier of such a process was reported to be of the order of 2-3kcal mol−1. Here, we present an alternative mechanism for conduction of K+ in K+ channels where site vacancies are involved, and we propose that coexistence of several ion permeation mechanisms is energetically possible. Conduction can be described as a more anarchic phenomenon than previously characterized by the concerted translocations of K+-water-K+. Experiments also suggest that local structural changes in the selectivity filter may act as the a gate referred to as C-type inactivation. An extensive computational study on KirBac, is presented which supports the existence of a physical gate or constriction in the selectivity filter of K+ channels. Our computations identify a new selectivity filter structure, which is likely associated with C-type inactivation.

TRPM3 was identified as a Ca2+-permeable, non-selective cation channel (PCa/PNa of 1.57) that showed a small but constitutively single channel conductance of ∼65 pS for Ca2+ and around ∼130 pS for Cs+. Like the structure of all TRP channels, TRPM3 channels contain six transmembrane spanning regions with a pore-forming reentrant loop between the fifth (S5) and the sixth (S6) transmembrane domain. It is generally accepted that, physical and chemical activating stimuli, lead to gating of a single, central cation-conducting pore formed by S5, S6 and the interconnecting pore loop. For TRPM3, stimulation by heat or chemical compounds like pregnenolone sulphate (PS) and nifedipine, will open the central pore and induces outwardly rectifying currents in TRPM3-expressing cells. In contrast to this view, evidence is shown for the existence of an alternative ion permeation pathway in TRPM3, distinct form the central pore, that can be specifically activated by the combined application of PS and clotrimazole (Clt). Recently, we have identified CIM0216, a single compound that is able to open both ion permeation pathways of TRPM3 by itself. The combined application of CIM0216 and La3+, a blocker of the central pore, has allowed us to further investigate the biophysical properties of the alternative ion permeation pathway in TRPM3.

Connexins form the intercellular channels composing gap junctions in vertebrates and providing both the electronic coupling and the exchange of ions and small molecules between adjacent cells. A gap junction channel consists of two opposed hemichannels that were recently shown to be functional when embedded in a nonjunctional membrane. There, the hemichannels mediate release of messengers controlling several important physiological processes including cellular proliferation and tissue remodeling. Moreover, an increasing number of evidence has placed the hemichannels as potential regulators of homeostatic imbalance present in diverse neurodegenerative diseases.Cx26 is a representative of the gap junction channels with a known structure. Here we resort to molecular modeling methods in order to study process of ion permeation through its hemichannel. Based on obtained trajectories we estimated ion flux and current in a range of voltages applied experimentally for native Cx26 and its two known mutants: D50N and G45E. We have analyzed current-voltage dependencies for the native channel and demonstrated an opposing effect of the two mutations. Finally we studied the process of ion permeation when Ca2+ is present in the system. We have shown, for the first time in silico, possible modulation of the Cx26 current-voltage dependence by Ca2+ ions.