We have shown in previous studies that Reissner's membrane in mouse and gerbil absorbs Na+
from the cochlear lumen by electrogenic transepithelial transport, which was apparently mediated by apical ENaC, basolateral Na+
-ATPase, and basolateral K+
]. This Na+
absorption was blocked by amiloride and benzamil. The most commonly-observed target of these drugs is ENaC, comprised of the three subunits α-, β- and γ-ENaC. We addressed the question of cation selectivity of this pathway in Reissner's membrane epithelial cells with 5 series of patch clamp experiments and selective candidate gene expression analysis.
Benzamil-sensitive currents under whole-cell patch clamp
We first tested whether benzamil-sensitive currents, which was earlier observed as transepithelial currents with the current-density vibrating probe [5
], could be detected under whole-cell patch clamp conditions (Series 1). Indeed, benzamil (1 μM) reduced the inward current when the pipette and bath solutions (P1, B1) approximated the physiological situation (ignoring differences in apical cation and intracellular Cl-
composition) (Additional file 1
: Fig. S1 and Fig. S3; Table ).
Inward and outward wholecell patch clamp currents, conductances and reversal voltage under established cationic conditions.
Indeed, benzamil (1 μM) reduced the inward current when the KCl-rich pipette solution (P1) mimicked the presumed intracellular composition (although Cl-
was higher than often observed) and when the NaCl-rich bath (B1) mimicked the basolateral (perilymph) composition, (Additional file 1
: Fig. S1 and Fig. S3; Table ). Benzamil was used throughout these experiments at a concentration (1 μM) that yielded a complete inhibition of the transepithelial current [5
] and significantly inhibited the inward whole-cell current (at -100 mV; I-100
) by 48.5%; from -1305 ± 277 pA to -671 ± 146 pA (n = 7). These whole-cell data are consistent with the transepithelial measurements of Na+
absorption by Reissner's membrane.
Expression of benzamil-sensitive cation channels in RM
We utilized gene array and RT-PCR to partially address the question of the participation of benzamil-sensitive nonselective cation (NSC) channels. Several isoforms of acid-sensitive ion channels (ASIC) and cyclic-nucleotide gated (CNG) channels were listed in our gene array of mouse Reissner's membrane (GEO GSE6196; Table ) [6
]. ASIC1a, 2a, 2b, 3 and 4 were listed; ASIC1a and 3 yielded a call of "Present", but ASIC1a was tested by RT-PCR and determined to be "Absent" (Table ). ASIC3 was not determined by RT-PCR; however, ASIC3 is stimulated, rather than inhibited, by amiloride at neutral pH [7
] and therefore would not be expected to contribute to the currents measured in the present study. CNGA1, 2, 4 and CNGB3 were listed in the gene array and all received a call of "Absent". CNGA3 and CNGB1 were not listed in the gene array and were not tested by RT-PCR. As mentioned above, ENaC can be a NSC channel under some subunit combinations and the α-,β- and γ -ENaC subunits were all expressed in RM (Table ).
Transcript analysis of amiloride-sensitive channel genes in Reissner's membrane.
Na+ and K+ conductance of benzamil-sensitive current
Whole-cell patch clamp currents were measured (Series 2) under conditions where the only major permeant ions were either Na+
(Figure , ). Cl-
was replaced by methanesulfonate (Table ). In symmetrical Na+
-rich bath and pipette solutions (B2, P2), the current-voltage (I-V) relationship was nearly linear, with a slight inward rectification at large negative voltage. The slight inward rectification observed at large negative voltage in the first series of symmetrical Na+
experiments was at least partially due to the time dependence of the response. This series utilized 300 ms voltage steps and the second linear series utilized 200 ms steps. The strong rectification of the benzamil-sensitive current, however, may at least partially be due to the known voltage sensitivity of inhibition of ENaC by benzamil [8
]. Benzamil significantly inhibited the inward whole-cell current (carried mostly by bath Na+
at -100 mV) by 87.5%; from I-100
= -2044 ± 552 pA to -255 ± 57 pA (n = 6). Some experiments displayed a slow rundown in channel activity, as has been observed by others [8
]. A representative experiment is shown in Figure and a summary of similar experiments is shown in Figure and Table .
Figure 1 Representative whole-cell patch clamp currents with only Na+ or K+ as the major permeant ions. The compositions of the bath perfusates are indicated by the horizontal bars and the colors of the bars are identical to the colors in the corresponding graphs. (more ...)
Figure 2 Summary of whole-cell patch clamp currents with only Na+ or K+ as the major permeant ions. A) Bar graph of the conductances and B) currents at -100 mV in symmetrical Na+-rich solutions (B2, P2) and K+-rich solutions (B3, P3). C) Current-voltage (I-V) (more ...)
Bath and pipette solutions (mM)
In symmetrical K+-rich bath and pipette solutions (B3, P3), the current and conductance at -100 mV were significantly smaller than in Na+ (Figure , ; Table ). Benzamil had no significant effect on the inward whole-cell current (carried mostly by bath K+ at -100 mV) (mean decreased insignificantly by 9.8%; from I-100 = -476 ± 112 pA to -429 ± 130 pA, n = 8). A representative experiment is shown in Figure and a summary of similar experiments is shown in Figure and Table .
Li+/Na+ permeability ratio of benzamil-sensitive current
The high Na+ selectivity of the benzamil-sensitive current over K+ suggested that ENaC might mediate part or all of that current. One salient characteristic of ENaC is a higher permeability to Li+ than to Na+. We compared (Series 3, 4) whole-cell currents in the presence of Li+ and Na+ in the bath and the effects of benzamil. The comparisons were conducted in two series of paired measurements from the same experimental data set in order to minimize the effects of rundown. In the first series of measurements (Series 3), inward Li currents were tested for sensitivity to benzamil. A representative experiment is shown in Figure and its I-V releationship in Figure . The I-V relationships were very similar for both Na+ (B2) and Li+ (B4) and benzamil markedly decreased the inward currents for both ions. Specifically, benzamil significantly inhibited the inward Li+ whole-cell current (at -100 mV) by 58.8%; from I-100 = -1656 ± 126 pA to -682 ± 86 pA (n = 5) (Figure and Table ). Similarly, benzamil decreased (Series 3) the inward Na+ current at -100 mV by 66.8% (from I-100 = -1922 ± 96 pA to -639 ± 151 pA), a decrease that was not significantly different from the decrease by benzamil seen in the previous series (Series 2) of experiments with Na+ in the bath (Figure and Table ).
Figure 3 Whole-cell patch clamp currents with only Na+ or Li+ as the major permeant ions. The compositions of the bath perfusates are indicated by the horizontal bars and the colors of the bars are identical to the colors in the corresponding graphs. A) Representative (more ...)
It was to be expected that there would be no change in the observed Vr between the presence and absence of benzamil since the reversal potential for both Na+-selectective and NSC channels are both = 0. By contrast, if Li+ is more permeable than Na+, Vr would be expected to become more positive (see second series, below).
In the second series of measurements (Series 4), Na+ and Li+ I-V relationships were compared immediately before and after the solution change (points 3 and 4' in Figure ). Rapid change of the bath solution from a Na+-rich solution (B2) to a Li+-rich solution (B4) led to small but consistent changes in the current. The average inward current and conductance at large negative voltage (-100 mV) were significantly larger for Li+ than for Na+, by 19.9% (I-100 = -1699 ±149 pA vs -1416 ±181 pA, n = 5) and 14.9% (g-100 = 16.2 ± 1.4 nS vs 14.1 ± 1.6 nS, n = 5). Further, the reversal voltage was significantly more positive (7.1 ± 1.2 mV vs 0.1 ± 0.4 mV) (Figure ).
Figure 4 Summary whole-cell patch clamp currents and reversal voltage with only Na+ or Li+ as the major permeant ions. A) Bar graph of the reversal voltage (left panel), the currents at -100 mV (middle panel) and the conductance at -100 mV (right panel) in Na (more ...)
The shift in Vr
was analyzed with the Goldman-Hodgkin-Katz (GHK) equation as applied to bionic substitutions to estimate the relative membrane permeability to Li+
is the change in reversal potential, R the universal gas constant, T the absolute temperature, z the valence, F the Faraday constant, [Li]o
the extracellular Li+
the extracellular Na+
concentration and PLi
the permeabilities of the membrane for Li+
. The shift of Vr
by 7.0 mV in the current study corresponds to a PLi
ratio of 1.3. This result is within the range of reports by others for the ratio in αβγ-ENaC (1.3 [13
], 1.6 [11
], 1.8 [15
Benzamil-insensitive cation channels
We hypothesized that the residual current after inhibition by benzamil could be transported through nonselective cation channels, although a substantial contribution of NSC channels was unlikely since K+ conductance was relatively small. Alternatively, the residual current could be a patch leakage current.
It was first observed that transcripts of TRPV4, an amiloride-insensitive NSC channel, were present by both gene array and RT-PCR (Table ). The amiloride-insensitive channels HCN1-4 are permeable to K+
]) and were found to be absent by RT-PCR, although some false positives in the gene array were found (Table ).
We tested for the presence of benzamil-insensitive currents by comparing the inward current in the presence and absence of benzamil with a substitution of the impermeable cation NMDG+ for Na+ in the bath.
In this series of experiments (Series 5), the initial phase before benzamil did not quickly reach a stable condition so that parameters in benzamil were compared to those after washout. Nonetheless, it is clear that the inward current and conductance in benzamil were significantly less than the inward Na+ current and conductance after washout (Figure , Table ). Importantly, there was no significant difference between the inward current in benzamil (I-100 = -741 ± 65 pA, n = 5) and in NMDG+ (I-100 = -802 ± 118 pA, n = 5, B5). These results demonstrate that there were no cation currents other than the benzamil-sensitive channels under these conditions.
Figure 5 Whole-cell patch clamp currents with only Na+ or NMDG+ as the major permeant ions. The compositions of the bath perfusates are indicated by the horizontal bars and the colors of the bars are identical to the colors in the corresponding graphs. A) Representative (more ...)
Na+ in the pipette was reduced to 15 mM (P4) in order to confirm the Na+-selectivity of the current in this experimental series. The large decrease in Vr between Na+ (B2) and NMDG+ (B5) (from 29.8 ± 1.0 mV to -27.8 ± 3.6 mV, n = 5; Figure and Table ) is consistent with a large Na+ conductance. The small, but significant, decrease in Vr between ± benzamil could conceivably be due to the presence of an H+ conductance and the small pH gradient between intra- and extracellular solutions. The equilibrium potential for Na+ was +60 mV and for H+ was +6 mV. The absence of any significant difference (Table ) in I+100 (14834 ± 342 pA vs 1666 ± 243 pA, n = 5) in NMDG+ and Na+ baths (B5 and B2) is consistent with the current at +100 mV being carried predominantly by the efflux of intracellular Na+, since the pipette concentration (P4) remained the same for both bath solutions.