The Journal of General Physiology recently published a paper (Elenes et al., 2009) describing a novel electrophysiological approach to measure the Ca2+-carried current through ligand-gated channels. The method proposed is based on the assumption that all permeant ions compete for a single binding site, which controls permeation and is implemented by fitting single-channel data obtained at different extracellular Ca2+ concentrations ([Ca2+]o) to derive values for the fractional Ca2+ current, i.e., the percentage of the total current carried by Ca2+ ions.

The fractional Ca2+ current values obtained are not too different from some of those found for the rat NR1-NR2A NMDA receptor or the human adult acetylcholine receptor (AChR) using the fluorometric method proposed by Zhou and Neher (1993). In contrast, the values found for the fetal and adult mouse AChR (19.3 and 16.3%) are at odds with the values present in the literature (∼2 and ∼4%, respectively) (Villarroel and Sakmann, 1996; Ragozzino et al., 1998; Fucile et al., 2006). In particular, the fractional Ca2+ current values for the mammalian fetal AChR calculated using different experimental approaches are all comprised between 2.0 and 3.4% (Decker and Dani, 1990; Vernino et al., 1994; Villarroel and Sakmann, 1996; Ragozzino et al., 1998; Fucile et al., 2006).

Of note, the discrepancy is such that either the values of Elenes et al. (2009) or the previously published values are wrong. Here, we point out some issues that may have influenced the estimates by Elenes et al. (2009).

The authors decompose the total current (itotal) flowing through the AChR channel as the sum of a lumped current representing Na+, K+, and Mg2+ ions (iNa+−Mg2+) plus the current carried by Ca2+ ions (iCa2+). From the “one-site” model they derive equations for iNa+−Mg2+ and iCa2+, copied here with their original numbering, for the reader's convenience:

$iNa+−Mg2+=γNa+−Mg2+KDCa2+KDCa2++aCao2+V$
(3)

$iCa2+=γCa2+aCao2+KDCa2++aCao2+V$
(4)

Noticeably, current reversal potential has been omitted in both equations.

Inserting into Eq. 4 the values of KDCa2+ derived by fitting, the authors conclude that with an external solution containing 150 mM NaCl plus 100 mM CaCl2, the inward current is “carried exclusively by Ca2+ ions.” This is a central point in the paper, as it is a key prediction of the model and is used to show that the error introduced neglecting current reversal potential in Eqs. 3 and 4 is small.

This statement is not supported by experimental data presented in the paper. With 100 mM CaCl2 plus 150 mM NaCl in the external solution, the reversal potential of ACh-evoked currents is +18 mV (Elenes et al., 2009). When Ca2+ ions completely replace monovalent ions in the external solution (and therefore are the only inward-flowing ions), the reversal potential of the ACh-evoked current (EACh) undergoes a hyperpolarizing shift (Bregestovski et al., 1979; Lewis, 1979; Adams et al., 1980; Villarroel and Sakmann, 1996). In particular, with 100 mM CaCl2 but no monovalent ions in the external solution, EACh is −22.3 and −10 mV for fetal and adult mouse AChR, respectively (Villarroel and Sakmann, 1996). The large difference of EACh in the presence or absence of extracellular Na+ ions indicates that they do contribute measurably to the inward current, even when [Ca2+]o is 100 mM. Should a high [Ca2+]o prevent the flux of Na+ ions, the latter would not influence EACh any more than other impermeant ions.

Furthermore, to our knowledge, it has never been demonstrated experimentally (for instance, by means of Na+-sensitive fluorescent dyes) that Na+ ions do not flow through AChR channels if [Ca2+]o is high. Ca2+ ions pass through AChR channels, although the unitary channel conductance is reduced in a Ca2+-dependent manner (Bregestovski et al., 1979; Lewis, 1979; Decker and Dani, 1990; Grassi and Degasperi, 2000; Elenes et al., 2009) until it reaches a plateau value at [Ca2+]o of ∼40 mM. At high [Ca2+]o, unitary conductance of AChR channels is independent of the presence of monovalent cations, which has been interpreted as suggesting that at high [Ca2+]o, Ca2+ ions are the main current carriers (Decker and Dani, 1990).

Below, we show how including the reversal potential for iCa2+ and iNa+−Mg2+ (ECa2+ and ENa+−Mg2+, respectively) in Eqs. 3 and 4, and dropping the unsupported assumption that with [Ca2+]o = 100 mM Na+ ions do not contribute to inward current, leads to conclusions that are very different from those presented by Elenes et al. (2009), yet compatible with their experimental data.

Eqs. 3 and 4 must be amended as follows:

$iNa+−Mg2+=γNa+−Mg2+KDCa2+KDCa2++aCao2+(V−ENa+−Mg2+)$
(3a)

$iCa2+=γCa2+aCao2+KDCa2++aCao2+(V−ECa2+)$
(4a)

Under the experimental conditions used by Elenes et al. (2009) ([Na+]o = 150 mM and [Ca2+]o = 100 mM), ENa+−Mg2+ is indeed close to 0 mV, but iCa2+, being a pure Ca2+ current, changes sign at the Ca2+ equilibrium potential. ECa2+ must not be confused with the bi-ionic reversal potential of a current resulting from the steady-state counter-diffusion of extracellular Ca2+ and intracellular K+ ions (such as that measured by the experiment shown by Elenes et al., 2009 in their Fig. 15), as the current carried by K+ ions is included in the term iNa+−Mg2+ in the present model. The slope conductance of iCa2+ and iNa+−Mg2+ can be calculated from Eqs. 3a and 4a (Fig. 1 A; see figure legend for details). Using numerical values given by Elenes et al. (2009), we find that itotal with a slope conductance of 30.6 pS corresponds to a pure iCa2+ with a slope conductance of 7 pS. When the I-V curve is shifted to the left (as done by Elenes et al., 2009), the error introduced is much larger for itotal than for iCa2+ because of their different conductances, thereby causing a marked overestimation of the ratio iCa2+/itotal.

At variance with the original equations, Eqs. 3a and 4a also describe iCa2+ and iNa+−Mg2+ as compatible with the experimental observation that, at high [Ca2+]o, single-channel conductance of itotal varies little regardless of whether monovalent cations are added to external solutions (Decker and Dani, 1990) (Fig. 1 B).

In conclusion, neglecting equilibrium potentials in Eqs. 3 and 4 causes overestimation of the fractional Ca2+ current. We therefore invite the authors to repeat their fits using Eqs. 3a and 4a, as given here. This might lead to estimates for the fractional Ca2+ current in closer agreement with other reports. It might also disclose why the estimates proposed by Elenes et al. (2009) are better for receptors thought to have high fractional Ca2+ current than for those with low ones.

## Acknowledgments

Edward N. Pugh Jr. served as editor.

## References

References
D.J.
,
Dwyer
T.M.
,
Hille
B.
.
1980
.
The permeability of endplate channels to monovalent and divalent metal cations
.
J. Gen. Physiol.
75
:
493
510
.
Bregestovski
P.D.
,
Miledi
R.
,
Parker
I.
.
1979
.
Calcium conductance of acetylcholine-induced endplate channels
.
Nature.
279
:
638
639
.
Decker
E.R.
,
Dani
J.A.
.
1990
.
Calcium permeability of the nicotinic acetylcholine receptor: the single-channel calcium influx is significant
.
J. Neurosci.
10
:
3413
3420
.
Elenes
S.
,
Decker
M.
,
Cymes
G.D.
,
Grosman
C.
.
2009
.
Decremental response to high-frequency trains of acetylcholine pulses but unaltered fractional Ca2+ currents in a panel of “slow-channel syndrome” nicotinic receptor mutants
.
J. Gen. Physiol.
133
:
151
169
.
Fucile
S.
,
Sucapane
A.
,
Grassi
F.
,
Eusebi
F.
,
Engel
A.G.
.
2006
.
The human adult subtype ACh receptor channel has high Ca2+ permeability and predisposes to endplate Ca2+ overloading
.
J. Physiol.
573
:
35
43
.
Grassi
F.
,
Degasperi
V.
.
2000
.
Modulation of fetal and adult acetylcholine receptors by Ca2+ and Mg2+ at developing mouse end-plates
.
Pflugers Arch.
440
:
704
709
.
Lewis
C.A.
1979
.
Ion-concentration dependence of the reversal potential and the single channel conductance of ion channels at the frog neuromuscular junction
.
J. Physiol.
286
:
417
445
.
Ragozzino
D.
,
Barabino
B.
,
Fucile
S.
,
Eusebi
F.
.
1998
.
Ca2+ permeability of mouse and chick nicotinic acetylcholine receptors expressed in transiently transfected human cells
.
J. Physiol.
507
:
749
757
.
Vernino
S.
,
Rogers
M.
,
K.A.
,
Dani
J.A.
.
1994
.
Quantitative measurement of calcium flux through muscle and neuronal nicotinic acetylcholine receptors
.
J. Neurosci.
14
:
5514
5524
.
Villarroel
A.
,
Sakmann
B.
.
1996
.
Calcium permeability increase of endplate channels in rat muscle during postnatal development
.
J. Physiol.
496
:
331
338
.
Zhou
Z.
,
Neher
E.
.
1993
.
Calcium permeability of nicotinic acetylcholine receptor channels in bovine adrenal chromaffin cells
.
Pflugers Arch.
425
:
511
517
.

Abbreviation used in this paper:

• AChR

acetylcholine receptor

.