Photomodulation of the ASIC1a acidic pocket destabilizes the open state.

Acid-sensing ion channels (ASICs) are important players in detecting extracellular acidification throughout the brain and body. ASICs have large extracellular domains containing two regions replete with acidic residues: the acidic pocket, and the palm domain. In the resting state, the acidic pocket is in an expanded conformation but collapses in low pH conditions as the acidic side chains are neutralized. Thus, extracellular acidification has been hypothesized to collapse the acidic pocket that, in turn, ultimately drives channel activation. However, several observations run counter to this idea. To explore how collapse or mobility of the acidic pocket is linked to channel gating, we employed two distinct tools. First, we incorporated the photocrosslinkable noncanonical amino acids (ncAAs) 4-azido-L-phenylalanine (AzF) or 4-benzoyl-L-phenylalanine (BzF) into several positions in the acidic pocket. At both E315 and Y318, AzF incorporation followed by UV irradiation led to right shifts in pH response curves and accelerations of desensitization and deactivation, consistent with restrictions of acidic pocket mobility destabilizing the open state. Second, we reasoned that because Cl- ions are found in the open and desensitized structures but absent in the resting state structures, Cl- substitution would provide insight into how stability of the pocket is linked to gating. Anion substitution resulted in faster deactivation and desensitization, consistent with the acidic pocket regulating the stability of the open state. Taken together, our data support a model where acidic pocket collapse is not essential for channel activation. Rather, collapse of the acidic pocket influences the stability of the open state of the pore.

Molecular Investigation of Chicken Acid-Sensing Ion Channel 1 ß11-12 Linker Isomerization and Channel Kinetics.

Structures of the trimeric acid-sensing ion channel have been solved in the resting, toxin-bound open and desensitized states. Within the extracellular domain, there is little difference between the toxin-bound open state and the desensitized state. The main exception is that a loop connecting the 11th and 12th ß-strand, just two amino acid residues long, undergoes a significant and functionally critical re-orientation or flipping between the open and desensitized conformations. Here we investigate how specific interactions within the surrounding area influence linker stability in the "flipped" desensitized state using all-atom molecular dynamics simulations. An inherent challenge is bringing the relatively slow channel desensitization and recovery processes (in the milliseconds to seconds) within the time window of all-atom simulations (hundreds of nanoseconds). To accelerate channel behavior, we first identified the channel mutations at either the Leu414 or Asn415 position with the fastest recovery kinetics followed by molecular dynamics simulations of these mutants in a deprotonated state, accelerating recovery. By mutating one residue in the loop and examining the evolution of interactions in the neighbor, we identified a novel electrostatic interaction and validated prior important interactions. Subsequent functional analysis corroborates these findings, shedding light on the molecular factors controlling proton-mediated transitions between functional states of the channel. Together, these data suggest that the flipped loop in the desensitized state is stabilized by interactions from surrounding regions keeping both L414 and N415 in place. Interestingly, very few mutations in the loop allow for equivalent channel kinetics and desensitized state stability. The high degree of sequence conservation in this region therefore indicates that the stability of the ASIC desensitized state is under strong selective pressure and underlines the physiological importance of desensitization.

Mutation of a conserved glutamine residue does not abolish desensitization of acid-sensing ion channel 1.

Desensitization is a common feature of ligand-gated ion channels, although the molecular cause varies widely between channel types. Mutations that greatly reduce or nearly abolish desensitization have been described for many ligand-gated ion channels, including glutamate, GABA, glycine, and nicotinic receptors, but not for acid-sensing ion channels (ASICs) until recently. Mutating Gln276 to a glycine (Q276G) in human ASIC1a was reported to mostly abolish desensitization at both the macroscopic and the single channel levels, potentially providing a valuable tool for subsequent studies. However, we find that in both human and chicken ASIC1, the effect of Q276G is modest. In chicken ASIC1, the equivalent Q277G slightly reduces desensitization when using pH 6.5 as a stimulus but desensitizes, essentially like wild-type, when using more acidic pH values. In addition, steady-state desensitization is intact, albeit right-shifted, and recovery from desensitization is accelerated. Molecular dynamics simulations indicate that the Gln277 side chain participates in a hydrogen bond network that might stabilize the desensitized conformation. Consistent with this, destabilizing this network with the Q277N or Q277L mutations largely mimics the Q277G phenotype. In human ASIC1a, the Q276G mutation also reduces desensitization, but not to the extent reported previously. Interestingly, the kinetic consequences of Q276G depend on the human variant used. In the common G212 variant, Q276G slows desensitization, while in the rare D212 variant desensitization accelerates. Our data reveal that while the Q/G mutation does not abolish or substantially impair desensitization as previously reported, it does point to unexpected differences between chicken and human ASICs and the need for careful scrutiny before using this mutation in future studies.

Coupling structure with function in acid-sensing ion channels: challenges in pursuit of proton sensors.

Acid-sensing ion channels (ASICs) are a class of trimeric cation-selective ion channels activated by changes in pH within the physiological range. They are widely expressed in the central and peripheral nervous systems where they participate in a range of physiological and pathophysiological situations such as learning and memory, pain sensation, fear and anxiety, substance abuse and cell death. ASICs are localized to cell bodies and dendrites, including the postsynaptic density, and within the last 5 years several examples of proton-evoked ASIC excitatory postsynaptic currents have emerged. Thus, ASICs have become bona fide neurotransmitter-gated ion channels, activated by the smallest neurotransmitter possible: protons. Here we review how protons are thought to drive the conformational changes associated with ASIC activation and desensitization. In particular, we weigh the evidence for and against the so-called 'acidic pocket' being a vital proton sensor and discuss the emerging role of the ß11-12 linker as a desensitization switch or 'molecular clutch'. We also examine how proton-induced conformational changes pose unique challenges to classical molecular dynamics simulations, as well as some possible solutions. Given the emergence of new methodologies and structures, the coming years will probably see many advances in the study of acid-sensing ion channels.

ß11-12 linker isomerization governs acid-sensing ion channel desensitization and recovery.

Acid-sensing ion channels (ASICs) are neuronal sodium-selective channels activated by reductions in extracellular pH. Structures of the three presumptive functional states, high-pH resting, low-pH desensitized, and toxin-stabilized open, have all been solved for chicken ASIC1. These structures, along with prior functional data, suggest that the isomerization or flipping of the ß11-12 linker in the extracellular, ligand-binding domain is an integral component of the desensitization process. To test this, we combined fast perfusion electrophysiology, molecular dynamics simulations and state-dependent non-canonical amino acid cross-linking. We find that both desensitization and recovery can be accelerated by orders of magnitude by mutating resides in this linker or the surrounding region. Furthermore, desensitization can be suppressed by trapping the linker in the resting state, indicating that isomerization of the ß11-12 linker is not merely a consequence of, but a necessity for the desensitization process in ASICs.