What happens when the conductance of a sodium channel increases?

What happens when the conductance of a sodium channel increases?

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My intuition is that, since the concentration of sodium within a cell is higher than the extracellular concentration, when conductance increases, this corresponds to the channel being open and means sodium will enter the cell. Is this correct? And what impact will this have on the membrane potential?

No, not quite. You are on the right lines, but incorrect in thinking that intracellular sodium concentration is higher.

At the resting phase of a neuron, there is less sodium inside the cell and more sodium outside the cell. An ATPase sodium potassium pump is constantly pumping 3 sodium ions outside the cell for every 2 potassium cells that enter the cell.

For voltage gated sodium channels to open, there must be a significant change in voltage, which can be triggered by the binding of neurotransmitter to receptors for example. This triggers the sodium channels open and sodium will flood into the cell due to a high concentration gradient (because there are more sodium outside than inside) but also due to an electrical gradient (the neuron is more negative on the inside; positive sodium ions will be attracted to the inside).

Because sodium is flooding into the cell, this will cause the membrane potential to become more positive (depolarised) and will eventually reach an action potential (30mV). After an action potential, the channels will become de-activated and shut. After a period of hyperpolarisation, the neuron will return to a resting state.


Clam Research Sheds New Light On Red Tide

University of Maine marine scientist Laurie Connell isn't one to brag: she would rather talk about the potential of her current research than the popularity of what she has published in the past. With more than five months at the top of the charts in the highly respected scientific journal Nature, however, discussion of her "clam paper" is nearly unavoidable.

"I was shocked to hear that the paper was so popular. It's had more than 60,000 downloads since November, and it's still going," Connell said with enthusiasm. "I'm not sure what made it so popular, but is does have a very broad appeal."

Connell's report, Sodium channel mutation leads to saxitoxin resistance in clams increases risk of PSP, was the culmination of more than eight years of intensive research by an international team of scientists aimed at achieving a better understanding of a notorious and potentially deadly compound known at saxitoxin. Saxitoxin is the primary culprit in cases of Paralytic Shellfish Poisoning, or PSP, the always dangerous, sometimes-deadly consequence of the coastal phenomenon known as red tide.

Filter feeders like clams accumulate saxitoxin in their tissues as they dine on the algae that carry the poison, passing along a concentrated dose to their mammalian predators. The first research to take a comprehensive look at the affects of saxitoxin on clams, Connell and her team, including retired UMaine researcher Betty Twarog, found that the mollusks suffer many of the same symptoms as human PSP victims.

Well, at least some of them do.

Connell discovered is that not all clams are created equal when it come to fighting off the affects of PSP, and has begun to unravel a microscopic mystery that speaks to the very nature of the nervous system itself.

Thanks to a mutation in their genetic code, red tide resistant clams were able to survive and reproduce despite the presence of saxitoxin, eventually becoming the dominant strain in clam populations that are frequently exposed to red tide.

In fact, Connell and her team of specialists found that the mutant clams were more than 1000 time more resistant to the affects of red tide than their unmutated brethren, a surprising discovery that has significant implications in both clam management and medical research.

Because of its power over the nerve impulse, saxitoxin has been used extensively by medical researchers to study the function of the nervous system and its associated diseases. Connell's comprehensive approach opens new doors to future research by connecting sodium channel function to specific control sites in the organism's DNA.

The discovery that some clam populations were genetically much more resistant to red tide poisoning than others could open up new directions for managing the soft-shell clam fishery.

"The ability of individual populations to resist the affects of saxitoxin could be used to determine how long clam beds would have to remain closed after a red tide event," said Connell. "Genetically resistant clams are able to continue feeding much longer, accumulating more toxins in their tissues which take longer to purge. Knowledge of the genetic susceptibility of clams to red tide could help managers make better decisions on what clams to use in seeding programs, how long to close clam beds, and other issues."

The project's implications don't stop there. Connell's discoveries have been of interest to marine ecologists, public health officials, bioengineers, fishermen: the list goes on and on. The significance of the research in such a broad range of disciplines certainly speaks to its popularity in Nature.

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Author summary

Action potentials are electrical waves that propagate through long axons and are at the core of neuronal communication. The information they convey is encoded in their precise timing, an essential feature for learning, memory, and motor control. This timing must be maintained even though axons are exposed to various outside influences on their long journey to other brain areas and peripheral muscles. We studied how three axon types that show distinct morphological and physiological properties, but interact in a single time-sensitive behavior, respond to changes in ambient temperature. Combining imaging, electrophysiology, and modeling, we determine the mechanisms that allow axons to maintain action potential timing. We show that near temperature-insensitivity of action potential velocities supports temperature-robust timing over long-distances, but that this does not require the underlying ion channel properties to be temperature-insensitive. Indeed, ion channel properties could vary substantially in their temperature responses as long as two Sodium channel parameters—the activation gate time constant and the maximum conductance—were coordinated. Thus, even temperature-sensitive ion channels support temperature-robust action potential timing.

Citation: DeMaegd ML, Stein W (2020) Temperature-robust activity patterns arise from coordinated axonal Sodium channel properties. PLoS Comput Biol 16(7): e1008057.

Editor: Joseph Ayers, Northeastern University, UNITED STATES

Received: November 14, 2019 Accepted: June 15, 2020 Published: July 27, 2020

Copyright: © 2020 DeMaegd, Stein. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files. All model files are available from the ModelDB database (accession number 260972).

Funding: This work was supported by funding from NSF 1755098 to WS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.


Oocyte electrophysiology

Oocytes expressing rat NaV1.2 were prepared and two-electrode voltage clamped essentially as previously described (Fiedler et al. 2008 Zhang et al. 2007). Oocytes were harvested from Xenopus frogs, following a protocol approved by the University of Utah's Institutional Animal Care and Use Committee that conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Briefly, oocytes were bathed in ND96 and sodium currents (INa) were recorded by holding the membrane potential at � mV and stepping to � mV for 50 ms every 20 s. The oocytes were exposed to toxin in a small (30 μl) static bath to conserve material. Recordings were done at room temperature (�ଌ).


μ-Conopeptides were synthesized as previously described (Zhang et al. 2007). TTX was obtained from Alomone Labs (Jerusalem, Israel), STX from either Calbiochem (San Diego, CA) or the National Research Council Canada (Halifax, Nova Scotia, Canada), and GTX2/3 from the National Research Council Canada. In experiments involving high concentrations, STX and GTX2/3 were lyophilized then dissolved in ND96. All stocks were stored at �ଌ in a nonfrost-free freezer until use.

Data analysis

Off-rate constants (koff values) were determined from single-exponential fits of peak INa following toxin washout observed rate constants (kobs values) were obtained by single exponential fits of INa following toxin addition and on-rate constants (kon values) were obtained from slopes of kobs versus [toxin], all as reported previously (West et al. 2002). In particular instances (see the text immediately following), the time course of the recovery of INa following toxin washout was biphasic and the recovery was fit to a double-exponential curve as previously reported (Zhang et al. 2009).

Numerical simulation of the reaction scheme in Fig. 1C

To model the time-dependent changes in the concentrations of the various components, the reaction scheme in Fig. 1C was simulated by reiteration of the following formulas in a for-loop using the graphical programming language LabVIEW (National Instruments, Austin, TX)

Pseudofirst-order kinetics was assumed i.e., the concentration of alkaloid, denoted by [$] (representing STX, GTX2/3, or TTX), and that of μ-conopeptide, denoted by [P], remained constant. When concentrations were expressed in micromoles (μM) and time in minutes, a time increment Δt of 10 𢄤 proved sufficiently small to accommodate the concentrations and rate constants used for the simulations used in this study. The rate constants are as denoted in Fig. 1C and their values were obtained by measuring the kinetics of block and recovery of INa following exposure to, and washout of, alkaloid and peptide, each alone or in combinations, as follows: kRA and kAR were the kon and koff values, respectively, of the reaction of NaV with peptide alone kBR and kRB were the kon and koff values, respectively, of the reaction of NaV with alkaloid alone and kAC was determined from the kon value for the block of residual current of the peptide·NaV complex at saturating [peptide]. There was no direct means to determine kCA, but its value could be calculated, since the product of the rate constants for the four clockwise steps equals that for the four counterclockwise steps that is, kCA = [kRA*kAC*kCB*kBR]/[kRB*kBC*kAR]. When all of the NaV was converted into a ternary complex by use of saturating concentrations of toxins, the time course for functional recovery after toxin washout could be fit by a single-exponential curve with a rate constant koff, which was smaller than kBR (and larger than kAR), indicating that kCB was rate-liming during toxin washout therefore kCB could be approximated by koff. A more accurate value of kCB could be estimated from the experimentally determined values for six of the rate constants, kRA, kAR, kAC, kRB, kBR, and kBC (see next paragraph for experimental determination of kBC), and calculating the seventh, kCA, as described earlier, while the value of kCB was varied until the mean squared error between the simulated and experimental time courses of recovery from block reached a minimum. Values of kCA and kCB determined in this fashion are presented in Table 4 .

Table 4.

Equilibrium constants (nM) of reactions in Fig. 1 C

A. Kdvalues of peptides for channel alone versus channel saturated with peptide
Channel Alone Channel Saturated With Alkaloid
PeptideNone Kd (kBR/kRB)KIIIA Kd (kCA/kAC)KIIIA[K7A] Kd (kCA/kAC)
    TTX38 ± 6730 ± 80045 ± 22
    STX8 ± 26 ± 3
    GTX2/35 ± 17 ± 4
B. Kdvalues of peptides for channel alone versus channel saturated with alkaloid
Channel Alone Channel Saturated With Alkaloid
AlkaloidNone Kd (kAR/kRA)TTX Kd (kCB/kBC)STX Kd (kCB/kBC)GTX2/3 Kd (kCB/kBC)
KIIIA5 ± 5105 ± 254 (1�) × 10 3 † 290 ± 30
KIIIA[K7A]115 ± 40138 ± 3087 ± 18156 ± 62

Equilibrium Kd values, mean ± SD, were calculated from koff/kon ratios (notations of corresponding rate constants in Fig. 1 C are indicated in parentheses) values for these rate constants were obtained from Tables 1 – 3 , except for those of kCB and kCA, which were obtained by numerical simulation, as described in methods . Values of kCB, in min 𢄡 , were (for indicated peptide𠄺lkaloid combinations): 0.021 (KIIIA–TTX) 0.35 (KIIIA–STX) 0.029 (KIIIA–GTX2/3) 0.022 (KIIIA[K7A]–TTX) 0.013 (KIIIA[K7A]–STX) and 0.014 (KIIIA[K7A]–GTX2/3) SDs of koff values for corresponding ternary complexes in Table 2 served as proxies for SDs for kCB values and these were used in calculating SDs for corresponding kCA and Kd values. Mean values of kCA ± SD, in min 𢄡 , were (for indicated peptide𠄺lkaloid combinations): 0.0022 ± 0.0024 (KIIIA–TTX) 0.025 ± 0.012 (KIIIA[K7A]–TTX) 0.0038 ± 0.0018 (KIIIA[K7A]–STX) and 0.004 ± 0.0021 (KIIIA[K7A]–GTX2/3).

The value of kBC was estimated from experiments in which alkaloid was added before peptide and exploiting the large disparity between kCB and kBR (except in one instance, involving KIIIA and STX see following paragraph), which produced a double-exponential time course for the recovery of INa following toxin washout. The coefficients of the double-exponential fits provided the koff values and spans of the fast and slow phases. In every case, the fast koff had a value close (within a factor of 𢏂) to kBR (i.e., the rate constant for the recovery from block of the binary alkaloid·NaV complex) and the slow koff had a value close (within a factor of 𢏂) to kCB (i.e., the rate constant for recovery from block of the ternary complex formed with saturating concentrations of both alkaoid and peptide applied for sufficient time to convert all of the NaV into a ternary complex). These results supported the notion that the span of the slow phase was likely to provide a good estimate of the fraction of the channels that were in the ternary peptide୺lkaloid·NaV complex at the start of toxin washout (Zhang et al. 2009). It was assumed that preceding toxin washout, the fraction of ternary complex formed (FTCF) as a function of time followed a single-exponential time course and therefore the estimated value for the observe rate constant kobs was calculated from the equation kobs = [ln (FTCF)]/time. For the sake of simplicity, data were obtained only at a single time point. This kobs was used with the equation kobs = kon*[peptide] + koff and, since koff (i.e., ∼kCB) was invariably about an order of magnitude smaller than kobs, the apparent kon (i.e., kBC) was calculated from kobs/[peptide].

A procedure different from that described earlier had to be used to estimate kBC in the case of STX and KIIIA because the difference between kCB and kBR was only fourfold and the experimental time course of recovery of INa following toxin washout could not be fit by a double-exponential curve better than a single-exponential one. Instead, the experiment was simulated using different values for kBC and the value producing simulated results that corresponded best with the experimental data was used to approximate kBC. The remaining rate constants used in this approximation are specified in results .

Molecular modeling and docking of NaV1.2, μ-conotoxin KIIIA, and the alkaloids TTX and STX

A model of rat NaV1.2 was constructed using as a template the model of rat NaV1.4 proposed by Lipkind and Fozzard (2000), which was composed of 12 parts from the four domains. Each domain was represented by three structural fragments: S5-segment, P-loop, and S6-segment. Sequence analysis of these 12 parts from NaV1.2 and NaV1.4 shows 91.0% identity (data not shown). We found every structural fragment for each domain of NaV1.2 that corresponded to Lipkind and Fozzard's model of NaV1.4. Our model of NaV1.2 was built by introducing modifications of specific residues using PyMOL (DeLano Scientific, Palo Alto, CA) the residues of the NaV1.4 model were replaced by corresponding backbone-dependent rotamer residues of the NaV1.2 using the PyMOL mutagenesis function. The conopeptide KIIIA was constructed with coordinates from Khoo et al. (2009) and the coordinates of TTX and STX were from Tikhonov and Zhorov (2005).

Automated docking of TTX, STX, and KIIIA to the model of NaV1.2 was performed using the AutoDock4.2 software package (Morris et al. 2009). Flexible torsions of ligands were assigned using AUTOTORS and a grid box defined by 60 × 60 × 60 grid points and spacing of 0.375 Å with AUTOGRID. Docking simulations were performed using a Lamarckian genetic algorithm (Morris et al. 1998), with the following parameters: population size = 300, mutation rate = 0.02, crossover rate = 0.8. The numbers of dockings per simulation were 50 and 256 for conopeptide and alkaloid, respectively. The number of dockings per simulation for conopeptide was lower than that for alkaloid due to the larger values of flexible torsions and corresponding longer simulation times. The maximum number of energy evaluations was set to 250,000 and the maximum number of generations was 27,000. One cluster-representative structure was selected from each of the docking runs. Clusters were examined for quantity of conformations and those with the greatest number were chosen for further analysis. A secondary criterion for selecting a cluster-representative structure was the value of binding energy.

Rationale for examining the effect of doubling the AMPA conductance

A doubling of the AMPA component of the synaptic current evoked by glutamate is an example of plasticity that has been observed in dopamine neurons in response to the administration of drugs of abuse, both in vivo (Zhang et al. 1997) and in vitro (Borgland et al. 2004). The ratio of the peak excitatory postsynaptic current (EPSC) evoked by the stimulation of AMPA and N-methyl- d -aspartate (NMDA) receptors can be measured by blocking all other synaptic currents pharmaceutically, voltage clamping the cell to +40 mV, then stimulating the slice. Under these conditions, the ratio of the peak AMPA EPSC to the peak NMDA EPSC can be increased from 0.38 to 0.75 by a single injection of cocaine (Ungless et al. 2001). An increase in the AMPA component was responsible for this change. The AMPA/NMDA ratio was positively correlated with locomotor activity, and Jones and Bonci (2005) speculated that an increase in the synaptic AMPA response could result in an enhancement of the response of dopamine neurons to reward related stimuli. In addition, iontophoretic administration of AMPA has been shown to increase both the frequency and the fraction of spikes fired in bursts in chloral hydrate anesthetized rats (Christoffersen and Meltzer 1995).

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Functional expression of ENaC/Deg-like channels in canine chondrocytes

We began by showing that our isolated canine chondrocytes expressed positive immunoreactivity for all three subunits of ENaC (α-ENaC, β-ENaC and γ-ENaC Figure 1A–F). Next, in potassium-free solutions (‘Inside-out patch’: Tables 1 and 2), we observed a small ion channel in 60% of patches with a slope conductance of 9 ± 1 pS (n = 5) (Figure 1G, I). This reversed near to the equilibrium potential for sodium calculated for these conditions (−4.9 mV) (Figure 1I), and the open probability (Po) was significantly reduced by the ENaC/Deg blockers, 10 μM amiloride (Figure 1I) and 100 nM benzamil (inhibition of 96 ± 2%, n = 5 and 56 ± 2%, n = 3, respectively P ≤ 0.05 in both cases, anova ). The kinetics of this low conductance channel was typical for ENaC channels. To quantify this, we performed a kinetic analysis. Since ENaC channels have an unusually high permeability for Li + (Canessa et al., 1994 Schild et al., 1997 Kellenberger et al., 1999 ), we optimized conditions by recording events under cell-attached patch mode with Li + included in the pipette as the charge carrier (‘Lithium solution’: Table 1). Under this configuration, and with membrane potential of −80 mV, the unitary currents were <1 pA inward, again typical of ENaC/Deg channels. Data was idealized and model fitted as described in the methods. Initially, we found events could not be satisfactorily fitted with a simple two-state model (closed–open) but was much better fitted with two open and two closed states (Figure 2). Best fit rate constants are given in full in Table 3. These rate constants were used to calculate the PDFs, mean open and mean closed times in Figure 2.

Immunohistochemistry and patch-clamp recording reveal the presence of ENaC protein expression and a benzamil and amiloride sensitive cation conductance. Immunofluorescence staining of αENaC (A), βENaC (B) and γENaC (C) in primary cultures of canine articular chondrocytes (first passage cells, 100× objective). Positive immunoreactivity for all subunits was observed although strongest for α- and β-ENaC. The concentration of the primary antibodies was 1 μg·mL −1 (dilution 1:200). The secondary antibody was a goat polyclonal to rabbit IgG (Fc-specific, affinity purified, pre-adsorbed) conjugated to DyLight® 488 (Abcam ab98462). After extensive washes in PBS-T, the nuclei were counterstained with propidium iodide (red fluorescence). Green staining shows the ENaC protein, orange staining is the nuclei and yellow staining is the overlap of the two. (D–F) The negative control was treated identically, but with the omission of primary IgG and shows the red fluorescent nuclear staining with very little background green fluorescence (scale bars for A–F: 10 μm). (G–J) Inside-out patch clamp recordings from canine chondrocytes using inside-out solutions: Tables 1 and 2. (G) Traces of inside-out low conductance single channel activity at the given membrane potentials. The scale bar horizontal line is 100 ms vertical line is 500 fA. (H) All-points amplitude histogram for the low conductance channel at −40 mV. (I) Single channel current–voltage curve for the low conductance channel. Vrev was −1 ± 5 mV (n = 5), slope conductance 9 ± 1 pS (n = 5). (J) Open probability (Po) versus time, calculated over successive 0.4 s windows before and during the addition of the ENaC channel inhibitor, amiloride (10 μM). Low conductance single channel Po was reduced by 96 ± 2% (n = 5).

Kinetic analysis of the low conductance cation channel. Kinetic analysis of the ENaC/Deg-like low conductance was performed under cell-attached patch conditions. Li + was included in the patch-pipette and served as the charge carrier (Solutions: Table 1). (A) Upper panel, the SKM idealized record from this trace (see Methods). Lower panel, raw single channel trace at −80 mV, baseline corrected. (B) An example patch closed-time distribution superimposed with a bi-exponential PDF calculated from panel D: (τ1, area): 5.9 ms, 0.83, (τ2, area): 94.5 ms, 0.17. Mean: 21.0 ms. (C) An example open-time distribution, again fit with a bi-exponential PDF calculated from panel D: (τ1, area): 3.1 ms, 0.5, (τ2, area): 26.5 ms, 0.46. Mean: 13.8 ms. (D) Schematic of our best fit kinetic model, full data in text (Table 3). Throughout this figure, red is used to denote closed states, and blue denotes open state.

From To K (s −1 ) SEM
State 1 State 2 Mean
Closed (2) Open (3) 183.4 21.8
Open (3) Closed (2) 203.8 5.1
Closed (2) Closed (1) 19.3 3.0
Closed (1) Closed (2) 14.6 3.7
Open (3) Open (4) 41.7 17.0
Open (4) Open (3) 38.6 9.8

We next investigated whether constitutive activity of these channels influenced the cellular membrane potential (Vm). We used standard physiological saline solutions (extracellular: Table 1, intracellular: Table 2) and measured Vm in current clamp mode. Control Vm was −13.2 ± 3.8 mV, similar to previously reported resting membrane potentials in chondrocytes (Wright et al., 1992 , Lewis et al., 2011a ). Application of 10 μM amiloride caused the Vm to became significantly more negative ( Figure 3A, 9.5 ± 0.8 mV, n = 5). Addition of 100 nM benzamil (Figure 3B), an even more specific inhibitor of ENaC (Kellenberger and Schild, 2002 Alexander et al., 2011 ), also significantly changed Vm (7.5 ± 1.7 mV, n = 5 hyperpolarization, P ≤ 0.05 anova ). These effects were reversible and dose-related (Figure 3A–C).

Whole-cell patch clamp shows the presence of a sodium conductance characteristic of ENaC. (A and B) Whole-cell current clamp recordings of a canine chondrocyte in ‘physiological saline solutions’ (Tables 1 and 2, including 145 mM external NaCl). Application of either amiloride (A) or benzamil (B) reversibly hyperpolarize the membrane. Mean values given in the text. (C) Comparison of the effect of amiloride and benzamil on membrane potential in a number of experiments such as those shown in panels A and B. Data are fitted with Equation 3, where R is the change in membrane potential (dVm). Hill slope (h) was constrained to unity, m for amiloride was −13 ± 1.2 mV and pD2 = 5.5 ± 0.1, for benzamil m was −8.2 ± 0.3 mV and pD2 = 8.4 ± 0.07 (benzamil from eight cells, amiloride each point, three to five cells). (D) Representative whole-cell voltage ramps in ‘ENaC permeability’ solutions (Tables 1 and 2). The current traces shown illustrate a recording in control and then 1 μM benzamil solution. The resulting difference currents for each combination of solutions is shown in panel F. (E) Representative continuous whole-cell voltage clamp recordings as a chondrocyte is superfused with 1 μM benzamil. The small constitutive inward current is blocked resulting in an increase in outward current corresponding to a mean total whole-cell conductance of 1.5 ± 0.4 nS (n = 9). (F) Mean benzamil difference currents with 22 (49), 66 (13), 110 (6) and 150 (7) mM intracellular sodium (calculated Na equilibrium potential in mV) are shown (n = 14). (G) Mean Vrev plotted against intracellular sodium concentration. The smooth line represents a fit to Equation 2 with P (PNa/PK) of 24.

To determine the sodium permeability of the benzamil-sensitive current (relative to K + ), we switched to whole-cell voltage clamp. With the standard physiological solutions and at a holding potential of −15 mV, application of 1 μM benzamil blocked a small constitutive inward current and resulted in an outward current deflection (mean conductance 1.5 ± 0.4 nS, n = 9, Figure 3). Whole-cell voltage ramps were then run in the presence and absence of benzamil, using four different intracellular sodium concentrations optimized for recording ENaC currents (22, 66, 110 and 150 mM). Subtraction of the whole-cell currents in the presence of benzamil from those recorded in vehicle gave the benzamil difference currents shown in Figure 3F. Difference currents at four different intracellular sodium concentrations were obtained (Figure 3F, G), and the reversal potentials for these followed the calculated changes in Na + equilibrium potential (Figure 3G).

Finally, we investigated whether ENaC/Deg channel activity contributed to canine chondrocyte RVI. In cell-attached patch experiments (protocols as above), with Li + as the charge carrier (‘Lithium solution’: Table 1), we found that increasing bath/extracellular osmotic pressure with addition of 180 mM sucrose activated ENaC channels (Figure 4), significantly increasing the open probability of the ENaC/Deg-like channel (from 0.27 ± 0.05 to 0.54 ± 0.07, n = 10, P ≤ 0.05, paired t-test). In volume recording experiments, we found hypertonic challenge first led to shrinkage, but that within 20 min, volume recovered in approximately 50% of cells. Such volume recovery is termed RVI, and only cells that exhibited RVI under control conditions were used in the following experiments. In cells exhibiting RVI, volume recovered to 92 ± 4%, n = 8 (Figure 5B). The ability of these cells to undergo RVI was maintained when the extracellular Na + was completely replaced by Li + (Figure 5D), suggesting a Li + -permeable channel is involved. In Li + solutions, cells exhibited slightly less shrinkage this was still statistically significant shrinkage to 74 ± 3%, P < 0.001 and within 20 min of maximum shrinkage returned to 94 ± 2% of starting volume. RVI was inhibited by benzamil with a PD2 of 7.5 (see Figure 5 for details).

The ENaC/Deg-like channel is activated by exposure to hypertonic solutions. (A) Cells were initially equilibrated in 309mOsm and then switched to 489mOsm as indicated by the bar. Osmolarity was increased with addition of sucrose. An ensemble average of single channel activity is shown (n = 9 patches) the mean increase in current was 4.67 ± 1.7 pA (n = 8, P ≤ 0.05), and this increase was not seen in the presence of 1 μm benzamil. (B) Representative section taken from one of the 309mOsm records averaged in panel A. (C) Representative section taken from one of the 489mOsm records averaged in panel A.

RVI is retained in lithium, but sensitive to low concentrations of benzamil. (A) Confocal images showing changes of cell volume with time. The top panels show the chondrocyte from above (X–Y plane), and the lower panels show the reconstructed cell view from the side (Z–X plane). In order to capture relatively rapid volume changes, scan resolution was set to a low value (31 Z layers per time slice). These images were necessary to verify that cells were approximately spherical (please see Methods: Equation 1). 60× objective scale bar 20 μm. (B) Under control conditions, when cells are incubated in 309mOsm physiological saline (Table 1) then exposed to hypertonic solutions (489mOsm), they first shrink passively as water leaves the cell via osmosis (marked ‘I’). They then swell through the process of RVI, eventually returning to the starting volume (RVI indicated by ‘II’). Osmolarity was increased with addition of sucrose. Not all cells underwent RVI. We use a two-exposure protocol. Only those cells that exhibited RVI on the first exposure to hypertonic challenge were then used for these experiments. The second exposure either contained benzamil or further control vehicle solution, mean of eight cells. (C) The same protocol as that used in panel B, but with the presence of 100 nM benzamil RVI is inhibited. Mean of five cells. (D) RVI protocol with extracellular Na + replaced by Li + . For (B) to (D) solid filled markers indicate data points and the solid line (and y-axis on the right) indicates the osmolarity. (E) Concentration inhibition curve for inhibition of RVI by benzamil. Five cells per concentration. Data are represented as fractional recovery from the theoretical maximum shrinkage (0.63 = 309/489) and fit with Equation 3, where R is fractional recovery, m was 0.84 ± 0.2, h is 3.5 ± 0.9 and pD2 =7.5 ± 0.01. (F) Schematic of our working model for RVI, with phases ‘I’ and ‘II’ indicated.


Acknowledgments: Supported by a grant from the Belgian Science Policy (IAP VII/19) and by the 𠇏onds Léon Fredericq”. G.D. is a Marie-Curie COFUND postdoctoral fellow at the University of Liege. Co-funded by the European Union. J.D. is supported by the F.R.S.-FNRS (Belgian Fund for Scientific Research). The authors acknowledge constructive discussions with Prof. Vincent Seutin (Universiy of Liège), Prof. Pierre Maquet (University of Liège), Dr. Timothy O’Leary (Brandeis University), and Prof. Eve Marder (Brandeis University).

Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels

Corresponding author Dale J. Benos: Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294-0005, USA. Email: [email protected] Search for more papers by this author

Department of Physiology, Dartmouth Medical School, Hanover, NH 03755-3830, USA

Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294-0005, USA

Corresponding author Dale J. Benos: Department of Physiology and Biophysics, University of Alabama at Birmingham, 1918 University Boulevard, Birmingham, AL 35294-0005, USA. Email: [email protected] Search for more papers by this author

Department of Physiology, Dartmouth Medical School, Hanover, NH 03755-3830, USA


Application of recombinant DNA technology and electrophysiology to the study of amiloride-sensitive Na + channels has resulted in an enormous increase in the understanding of the structure-function relationships of these channels. Moreover, this knowledge has permitted the elucidation of the physiological roles of these ion channels in cellular processes as diverse as transepithelial salt and water movement, taste perception, volume regulation, nociception, neuronal function, mechanosensation, and even defaecation. Although members of this ever-growing superfamily of ion channels (the Deg/ENaC superfamily) share little amino acid identity, they are all organized similarly, namely, two short N- and C-termini, two short membrane-spanning segments, and a very large extracellular loop domain. In this brief Topical Review, we discuss the structural features of each domain of this Deg/ENaC superfamily and, using ENaC as a model, show how each domain relates to overall channel function.

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High-Level Expression and Functional Reconstitution of Shaker K+ Channels

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