Aa, instantaneous current–voltage (I–V) relationships of whole-cell currents obtained from a single bovine parotid acinar (BPA) cell. The pipette was filled with a NMDG-glutamate rich pipette solution (pipette solution A), and the bath contained either a Na-glutamate-rich solution (glutamate; bath solution C), a solution containing 25 mm NaCl (Cl⁻; bath solution D) or a solution containing 25 mm NaHCO₃ (HCO₃⁻; bath solution B). The currents were evoked by voltage ramps, each of 800 ms duration, from a holding potential of −78 mV (or −79 mV) between −98 mV (or −99 mV) and +52 mV (or +51 mV). Ab, summary of I–V relationships. Values are means ±s.e.m. of 13 independent experiments (•, HCO₃⁻; ▴, Cl⁻; ▪, glutamate). Error bars representing s.e.m. were omitted when so small as to lie within symbols. Ba, instantaneous I–V relationships obtained from a BPA cell bathed in solutions having 25 mm HCO₃⁻ in the presence (Na⁺; bath solution B) and absence of external Na⁺ (Na⁺_o) (NMDG; bath solution E). Currents were evoked by voltage ramps from a holding potential of −78 mV (or −80 mV) between −98 mV (or −100 mV) and +52 mV (or +50 mV). Bb, summary of I–V relationships. Values are means ±s.e.m. of nine independent experiments. (•, Na⁺; ♦, NMDG). Error bars representing s.e.m. were omitted when so small as to lie within symbols. Ca and b, traces of whole-cell currents associated with voltage steps for 400 ms from a holding potential of −78 mV between −98 mV and +52 mV in 10 mV intervals. Data were obtained from a BPA cell in the absence and presence of 0.5 mm DIDS in the bath solution (solution B). Cc, traces of the DIDS-sensitive currents obtained from traces of Fig. 1Ca and b. Cd, current traces obtained from a cell before (Control) and after (+DIDS) addition of 0.5 mm DIDS to the bath solution (bath solution B). The inhibitory effect of DIDS was completely reversible (not shown). Data from the same cell shown in Fig. 1Ca and b. Ce, comparison of the DIDS-sensitive I–V relationships obtained from ramp pulse (line; obtained from Fig. 1Cd) and voltage step pulse protocols (○, obtained from Fig. 1Cc).

A, tracings of instantaneous I–V relationships recorded from a BPA cell in the presence of different extracellular Na⁺ concentrations ([Na⁺]_o; 145, 100, 25, 9, 3, 0 mm). The solutions containing 145, 25 and 0 mm[Na⁺]_o were the bath solution B, J and I (Table 1), respectively, from which the solutions having 100, 9 and 3 mm[Na⁺]_o were prepared. The pipette was filled with a solution containing 25 mm HCO₃⁻ and 100 mm Hepes (pipette solution C). B, summary of I–V relations. Values are means ±s.e.m. of nine experiments (•, 145 mm; ○, 100 mm; ▴, 25 mm; ▵, 9 mm, □, 3 mm; ▪, 0 mm[Na⁺]_o). Error bars representing s.e.m. were omitted when so small as to lie within symbols. C, plot of the outward current amplitude at −40 mV (○), 0 mV (□), or +50 mV (▵) (n = 9) as a function of [Na⁺]_o. Data were normalized to the value obtained from those with 145 mm[Na⁺]_o and 0 mm[Na⁺]_o(I_xmM−I_0mM/I_145mM−I_0mM). Each continuous line represents the result of least-squares curve fit of the Michaelis–Menten equation (eqn (2)) to the data. Inset; voltage dependence of K_m values for [Na⁺]_o. The continuous line shows linear regression fit to the data. Values are means ±s.e.m. of nine experiments. Error bars representing s.e.m. were omitted when so small as to lie within symbols.

A, effect of external DIDS on I_{Na⁺/HCO3⁻}. Typical I–V relationships obtained in the absence (control; bath solution B) or presence of 0.01, 0.1, 0.5, 1.0 mm DIDS are shown. The pipette was filled with a NMDG-glutamate-rich solution (pipette solution B). B, summary of I–V relationships. Values are means ±s.e.m. of nine independent experiments. Error bars representing s.e.m. were omitted when so small as to lie within symbols. (•, control; ○, 0.01 mm; ▴, 0.1 mm; ▵, 0.5 mm; ▪, 1 mm) C, dose–response relationship for DIDS block of I_{Na⁺/HCO3⁻} at +50 mV. Data are from Fig. 5B (n = 9). *P < 0.001. D, voltage dependence of the DIDS block. Fractional currents in the presence of 0.1 mm DIDS at −40, −10, +20, and +50 mV are shown. Data are from Fig. 5B (n = 9). E, plot of fractional current as a function of DIDS concentration at a membrane potential of −40 (•), −10 (▵), +20 (○), or +50 mV (▴). Each point represents the mean ±s.e.m. of nine experiments. The lines are fits to the Hill equation (eqn (1)). Error bars representing s.e.m. were omitted when so small as to lie within symbols. Inset, voltage dependence of K_i values for DIDS block. K_i values are plotted on a semilogarithmic scale. The continuous line shows the linear regression fit to the data. Values are means ±s.e.m. of nine experiments. F, effects of anion channel blockers (0.1 mm of DIDS (n = 9), phloretin (n = 5), niflumic acid (NFA) (n = 5), NPPB (n = 5), glybenclamide (n = 3), and DPC (n = 5)) on I_{Na⁺/HCO3⁻} at 0 mV. Both the pipette and bath solutions were the same as shown in Fig. 5A*P < 0.05. **P < 0.001.

A, traces of DIDS-sensitive Na⁺–HCO₃⁻-dependent whole-cell currents evoked by voltage steps for 400 ms from a holding potential of −78 mV between −98 mV and +52 mV in 10 mV intervals, which were obtained from a HEK293 cell transfected with bNBCe1-B. Experimental conditions were identical to those described in Fig. 1Ca–c. B, RT-PCR analysis of bNBCe1-B. HEK293 cells transfected with bNBCe1-B (Construct: bNBCe1-B), but not mock transfected cells (Construct: mock) express the mRNA for bNBCe1-B. See Methods for details of the primers and experimental conditions. C, monovalent cation dependence of bNBCe1-B current. Test cations were Cs⁺, Li⁺, NMDG, choline (a) and K⁺ (b). Solutions and pulse protocol were the same as described in Fig 2A and C. D, [Na⁺]_o dependence of bNBCe1-B. Tracings of instantaneous I–V relationships obtained from a cell in the presence of different [Na⁺]_o (145, 100, 25, 9, 3, 0 mm) are shown. The pipette was filled with the pipette solution D. Bath solutions and pulse protocol were the same as described in Fig. 3A. Inset, voltage dependence of K_m for [Na⁺]_o. The continuous line shows linear regression fit to the data. Values are means ±s.e.m. of five experiments. E, dose–response relationship for DIDS block of bNBCe1-B currents at +50 mV. The pipette was filled with the pipette solution A, and bath solutions were the same as described in Fig. 5A. Values are means ±s.e.m. of seven experiments. *P < 0.05; **P < 0.001. Inset, voltage dependence of K_i for DIDS block. K_i values were plotted on a semilogarithmic scale. The continuous line shows the linear regression fit to the data. Values are means ±s.e.m. of seven experiments. F, effects of anion channel blockers (0.1 mm of DIDS (n = 7), Niflumic acid (NFA) (n = 5), NPPB (n = 4), Glybendclamide (n = 3) and DPC (n = 5)) on bNBCe1-B currents at 0 mV. The pipette was filled with the pipette solution A, and the bath solution was the same as shown in Fig. 5F. *P < 0.05, **P < 0.01. Phloretin was not tested, because it was found to increase a background current, probably attributable to a cation conductance (data not shown).

A, traces of whole-cell currents obtained from a BPA cell bathed in a solution containing 25 mm HCO₃⁻ rich in either Na⁺ (bath solution F), Li⁺ (bath solution G), Cs⁺ (bath solution I), NMDG (bath solution E), or choline (bath solution H). The pipette was filled with a NMDG-glutamate-rich solution (pipette solution A). B, summary of I–V relationships. Values are means ±s.e.m. of five or six independent experiments. Error bars representing s.e.m. were omitted when so small as to lie within symbols. (•, Na⁺; ▵, Li⁺; □, Cs⁺; ▴, NMDG; ○, choline) C, traces of whole-cell currents obtained from a cell bathed in a Cs⁺ (120 mm)-rich solution having either 25 mm Na⁺ (bath solution J), K⁺ (bath solution K), or choline (bath solution I). The pipette solution and ramp pulse protocols were identical, and similar to those described in Fig. 2A, respectively. D, summary of I–V relationships. Values are means ±s.e.m. of six independent experiments. Error bars representing s.e.m. were omitted when so small as to lie within symbols. (•, Na⁺; ▴, K⁺; □, choline)

A, representative traces of DIDS (0.5 mm)-sensitive currents obtained from a same BPA cell bathed in a HCO₃⁻ (25 mm)-containing solution having 3 or 9 mm[Na⁺]_o. The pipette and bath solutions were the same as described in Fig. 3A. B, the shift of the reversal potential of DIDS-sensitive current induced by increasing [Na⁺]_o from 3 mm to 9 mm, and the corresponding value of stoichiometry (Na⁺: HCO₃⁻= 1: q) calculated from the following equation: ΔE_rev=RT/F(q− 1) ln ([Na⁺]_o/[Na⁺]_o). Values are means ±s.e.m. of four experiments.

A, RT-PCR analysis of NBCe1-A, NBCe1-B, and β-actin. mRNA transcripts detected by gel electrophoresis. See Methods for details of the primers and experimental conditions. Total RNA isolated from rat kidney was also used as a template as a positive control for NBCe1-A. Lane M: size markers (1 kbp DNA ladder, Life Technologies, Inc.). RT, reverse transcription. B, schematic of nucleotide and amino acid sequences of the bovine parotid NBCe1-B coding region. Portions of the sequences that are identical to those of the bovine corneal NBCe1-B (AF308160) are not shown. The regions where these NBCe1-B differ are indicated in bold.

Aa, typical traces of single maxi-K⁺ channel current in a cell-attached patch. The patch pipette was filled with the KCl-rich (150 mm K⁺) solution (pipette solution E) and the bath solution was the NaCl-rich solution containing 25 mm HCO₃⁻ (bath solution L: Control and After control) or NMDG-Cl-rich solution containing 25 mm HCO₃⁻ (bath solution M: Na⁺free). Voltage (−V_p) was +43 (control and after control) or +45 mV (Na⁺ free). Atropine (1 µm) was included in the bath solutions. Ab, I–V relations of maxi-K⁺ channel obtained from the patch shown in (a) (•, control; □, Na⁺ free; ▵, after control). B, the reversal potential shifts induced by replacement of Na⁺ with NMDG and choline in the absence (left; bath solution A and N) or the presence of 25 mm HCO₃⁻ (middle; bath solution L and M) or the presence of 25 mm HCO₃⁻ and 1 mm DIDS (right). Values are means ±s.e.m. of 5 or 11 experiments. **P < 0.001. C, the reversal potential shifts induced by addition of 25 mm HCO₃⁻ to the bath solution in the continued presence of Na⁺_o (bath solution A and L) without (left) or with 1 mm DIDS (right). Values are means ±s.e.m. of six experiments. *P < 0.05.