Recovery from acidosis is a robust trigger for loss of force in murine hypokalemic periodic paralysis

Periodic paralysis is an ion channelopathy of skeletal muscle in which recurrent episodes of weakness or paralysis are caused by sustained depolarization of the resting potential and thus reduction of fiber excitability. Episodes are often triggered by environmental stresses, such as changes in extracellular K+, cooling, or exercise. Rest after vigorous exercise is the most common trigger for weakness in periodic paralysis, but the mechanism is unknown. Here, we use knock-in mutant mouse models of hypokalemic periodic paralysis (HypoKPP; NaV1.4-R669H or CaV1.1-R528H) and hyperkalemic periodic paralysis (HyperKPP; NaV1.4-M1592V) to investigate whether the coupling between pH and susceptibility to loss of muscle force is a possible contributor to exercise-induced weakness. In both mouse models, acidosis (pH 6.7 in 25% CO2) is mildly protective, but a return to pH 7.4 (5% CO2) unexpectedly elicits a robust loss of force in HypoKPP but not HyperKPP muscle. Prolonged exposure to low pH (tens of minutes) is required to cause susceptibility to post-acidosis loss of force, and the force decrement can be prevented by maneuvers that impede Cl− entry. Based on these data, we propose a mechanism for post-acidosis loss of force wherein the reduced Cl− conductance in acidosis leads to a slow accumulation of myoplasmic Cl−. A rapid recovery of both pH and Cl− conductance, in the context of increased [Cl]in/[Cl]out, favors the anomalously depolarized state of the bistable resting potential in HypoKPP muscle, which reduces fiber excitability. This mechanism is consistent with the delayed onset of exercise-induced weakness that occurs with rest after vigorous activity.

The dual role of chloride in synaptic vesicle glutamate transport

The transport of glutamate into synaptic vesicles exhibits an unusual form of regulation by Cl- as well as an associated Cl- conductance. To distinguish direct effects of Cl- on the transporter from indirect effects via the driving force Δψ, we used whole endosome recording and report the first currents due to glutamate flux by the vesicular glutamate transporters (VGLUTs). Chloride allosterically activates the VGLUTs from both sides of the membrane, and we find that neutralization of an arginine in transmembrane domain four suffices for the lumenal activation. The dose dependence suggests that Cl- permeates through a channel and glutamate through a transporter. Competition between the anions nonetheless indicates that they use a similar permeation pathway. By controlling both ionic gradients and Δψ, endosome recording isolates different steps in the process of synaptic vesicle filling, suggesting distinct roles for Cl- in both allosteric activation and permeation.

Dual regulation of the native ClC-K2 chloride channel in the distal nephron by voltage and pH

ClC-K2, a member of the ClC family of Cl− channels and transporters, forms the major basolateral Cl− conductance in distal nephron epithelial cells and therefore plays a central role in renal Cl− absorption. However, its regulation remains largely unknown because of the fact that recombinant ClC-K2 has not yet been studied at the single-channel level. In the present study, we investigate the effects of voltage, pH, Cl−, and Ca2+ on native ClC-K2 in the basolateral membrane of intercalated cells from the mouse connecting tubule. The ∼10-pS channel shows a steep voltage dependence such that channel activity increases with membrane depolarization. Intracellular pH (pHi) and extracellular pH (pHo) differentially modulate the voltage dependence curve: alkaline pHi flattens the curve by causing an increase in activity at negative voltages, whereas alkaline pHo shifts the curve toward negative voltages. In addition, pHi, pHo, and extracellular Ca2+ strongly increase activity, mainly because of an increase in the number of active channels with a comparatively minor effect on channel open probability. Furthermore, voltage alters both the number of active channels and their open probability, whereas intracellular Cl− has little influence. We propose that changes in the number of active channels correspond to them entering or leaving an inactivated state, whereas modulation of open probability corresponds to common gating by these channels. We suggest that pH, through the combined effects of pHi and pHo on ClC-K2, might be a key regulator of NaCl absorption and Cl−/HCO3− exchange in type B intercalated cells.

Tuning the ion selectivity of glutamate transporter–associated uncoupled conductances

The concentration of glutamate within a glutamatergic synapse is tightly regulated by excitatory amino acid transporters (EAATs). In addition to their primary role in clearing extracellular glutamate, the EAATs also possess a thermodynamically uncoupled Cl− conductance. This conductance is activated by the binding of substrate and Na+, but the direction of Cl− flux is independent of the rate or direction of substrate transport; thus, the two processes are thermodynamically uncoupled. A recent molecular dynamics study of the archaeal EAAT homologue GltPh (an aspartate transporter from Pyrococcus horikoshii) identified an aqueous pore at the interface of the transport and trimerization domains, through which anions could permeate, and it was suggested that an arginine residue at the most restricted part of this pathway might play a role in determining anion selectivity. In this study, we mutate this arginine to a histidine in the human glutamate transporter EAAT1 and investigate the role of the protonation state of this residue on anion selectivity and transporter function. Our results demonstrate that a positive charge at this position is crucial for determining anion versus cation selectivity of the uncoupled conductance of EAAT1. In addition, because the nature of this residue influences the turnover rate of EAAT1, we reveal an intrinsic link between the elevator movement of the transport domain and the Cl− channel.