Functional Modularity of the β-Subunit of Voltage-Gated Ca2+ Channels
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Guanylate kinase
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The S4 transmembrane domain in Shaker (Kv1) voltage-sensitive potassium channels has four basic residues (R1-R4) that are responsible for carrying the majority of gating charge. In Kv4 channels, however, R1 is replaced by a neutral valine at position 287. Among other differences, Kv4 channels display prominent closed state inactivation, a mechanism which is minimal in Shaker. To determine if the absence of R1 is responsible for important variation in gating characteristics between the two channel types, we introduced the V287R mutant into Kv4.3 and analyzed its effects on several voltage sensitive gating transitions. We found that the mutant increased the voltage sensitivity of steady-state activation and altered the kinetics of activation and deactivation processes. Although the kinetics of macroscopic inactivation were minimally affected, the characteristics of closed-state inactivation and recovery from open and closed inactivated states were significantly altered. The absence of R1 can only partially account for differences in the effective voltage sensitivity of gating between Shaker and Kv4.3. These results suggest that the S4 domain serves an important functional role in Kv4 channel activation and deactivation processes, and also those of closed-state inactivation and recovery.
Shaker
Voltage-gated potassium channel
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NMDA receptors are glutamate-activated ion channel complexes central to the functioning of the mammalian nervous system. Opening of the NMDA receptor ion channel pore is initiated by agonist-induced conformational changes in the extracellular ligand-binding domain (LBD) but the dynamic mechanism of this process remains unresolved. We studied how a disulfide bond in the obligatory GluN1 subunit—the sole site of redox modulation in NMDA receptors—controls this activation gating mechanism. This disulfide bond is located in the hinge region of the LBD, and presumably constrains agonist-induced cleft closure of the clamshell-like LBD. Elimination of this bond, by either DTT-mediated reduction or mutagenesis, enhances gating efficiency such that pore opening now occurs with higher frequency and longer duration. The most prominent effect was to shift opening modes to long duration openings reminiscent of a high Po gating mode that the NMDA receptor exhibits under ambient oxidizing conditions. In terms of preopen gating steps, elimination of this bond has effects only on the fast gating step consistent with this step being GluN1-specific and reflecting GluN1 gating movements immediately before channel opening. Overall, our results suggest that the dynamics of the GluN1 LBD have strong effects on late pore opening steps including regulating the duration of pore opening. This redox-mediated gating modulation could be an underlying mechanism of NMDA receptor malfunction in redox-dependent disease states and presents a potential target of pharmacologic action.
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Hodgkin–Huxley model
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The role of sodium channel closed-state fast inactivation in membrane excitability is not well understood. We compared open- and closed-state fast inactivation, and the gating charge immobilized during these transitions, in skeletal muscle channel hNa(V)1.4. A significant fraction of total charge movement and its immobilization occurred in the absence of channel opening. Simulated action potentials in skeletal muscle fibers were attenuated when pre-conditioned by sub-threshold depolarization. Anthopleurin A, a site-3 toxin that inhibits gating charge associated with the movement of DIVS4, was used to assess the role of this voltage sensor in closed-state fast inactivation. Anthopleurin elicited opposing effects on the gating mode, kinetics and charge immobilized during open- versus closed-state fast inactivation. This same toxin produced identical effects on recovery of channel availability and remobilization of gating charge, irrespective of route of entry into fast inactivation. Our findings suggest that depolarization promoting entry into fast inactivation from open versus closed states provides access to the IFMT receptor via different rate-limiting conformational translocations of DIVS4.
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We used single-channel recording and model-based kinetic analyses to quantify the effects of mutations in the extracellular domain (ECD) of the alpha-subunit of mouse muscle-type acetylcholine receptors (AChRs). The crystal structure of an acetylcholine binding protein (AChBP) suggests that the ECD is comprised of a beta-sandwich core that is surrounded by loops. Here we focus on loops 2 and 7, which lie at the interface of the AChR extracellular and transmembrane domains. Side chain substitutions in these loops primarily affect channel gating by either decreasing or increasing the gating equilibrium constant. Many of the mutations to the beta-core prevent the expression of functional AChRs, but of the mutants that did express almost all had wild-type behavior. Rate-equilibrium free energy relationship analyses reveal the presence of two contiguous, distinct synchronously-gating domains in the alpha-subunit ECD that move sequentially during the AChR gating reaction. The transmitter-binding site/loop 5 domain moves first (Phi = 0.93) and is followed by the loop 2/loop 7 domain (Phi = 0.80). These movements precede that of the extracellular linker (Phi = 0.69). We hypothesize that AChR gating occurs as the stepwise movements of such domains that link the low-to-high affinity conformational change in the TBS with the low-to-high conductance conformational change in the pore.
Dynamics
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Mechanosensation, the ability to detect mechanical forces, underlies the senses of hearing, balance, touch, and pain, as well as renal and cardiovascular regulation. Although the sensors are thought to be channels, relatively little is known about eukaryotic mechanosensitive channels or their molecular mechanisms. Thus, because of its tractable nature, a bacterial mechanosensitive channel that serves as an in vivo osmotic "emergency release valve," MscL, has become a paradigm of how a mechanosensitive channel can sense and respond to membrane tension. Here, we have determined the structural rearrangements and interactions between transmembrane domains of MscL that occur upon gating. We utilize an electrostatic repulsion test: If two residues approach upon gating we predicted that substituting like-charges at those sites would inhibit gating. The in vivo growth and viability and in vitro vesicular flux and electrophysiological data all support the hypothesis that residues G26 and I92 directly interact upon gating. The resulting model predicted other interacting residues. One of these sets, V23 and I96, was confirmed to truly interact upon gating by disulfide trapping as well as the electrostatic repulsion test. Together, the data strongly suggest a model for structural transitions and residue-residue proximities that occur upon MscL gating.—Li, Y.,Wray, R., Eaton, C., Blount, P. An open-pore structure of the mechano-sensitive channel MscL derived by determining transmembrane domain interactions upon gating. FASEB J. 23, 2197–2204 (2009)
mechanosensation
Mechanotransduction
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Orai1
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ATP-gated P2X 2 channels undergo permeability changes through a process that is incompletely understood. In the present study, we used fluorescence resonance energy transfer (FRET) and electrophysiology to measure cytosolic gating motions in P2X 2 channels as they enter a state with increased permeability. P2X 2 channels underwent permeability changes with a time course that was similar to decreases in FRET between cyan fluorescent protein and yellow fluorescent protein attached to the cytosolic domain of P2X 2 channels. Wild-type and mutant channels that did not undergo permeability changes also did not show evidence of cytosolic gating motions. Moreover, immobilizing the cytosolic domain by tethering it to the plasma membrane prevented the switch in permeability and impaired the cytosolic gating motions. Both of these phenotypes were restored when the immobilizing tether was cleaved. The data provide a time-resolved measure of state-specific gating motions and suggest how a cytosolic domain may control ion channel permeability.
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