Novel isoforms of the beta and gamma subunits of the Xenopus epithelial Na channel provide information about the amiloride binding site and extracellular sodium sensing

Abstract

We have previously identified three homologous subunits alpha, beta, and gamma of the highly selective amiloride-sensitive Na channel from the Xenopus laevis kidney A6 cell line, which forms a tight epithelium in culture. We report here two novel genes, termed beta2 and gamma2, which share 90 and 92% sequence identity with the previously characterized beta and gamma XENaC, respectively. beta2 and gamma2 transcripts were detected in lung, kidney, and A6 cells grown on porous substrate. The physiological and pharmacological profile of the Na channel expressed after alphabeta2gamma XENaC cRNA injection in Xenopus oocyte did not differ from alphabetagamma XENaC. By contrast, the channel expressed after alphabetagamma2 injection showed: (i) a lower maximal amiloride-sensitive sodium current, (ii) a higher apparent affinity for external sodium and inactivation of the sodium current by high sodium concentrations, and (iii) a lower apparent affinity for amiloride (KI alphabetagamma2; 1.34 microM versus alphabetagamma 0.35 microM). These data indicate that the gamma (and/or gamma2) subunit participates in amiloride binding and the sensing of the extracellular sodium concentration. The close homology between gamma and gamma2 will help to define the domains involved in sensing external sodium and in the structure of this important drug receptor.

Figure 7

Amiloride affinity in γ/γ2 chimeric constructs. The inhibition constant (K_I) for amiloride was measured in oocytes injected with α and β subunit cRNA and either γ, γ2, or one of the four chimeric constructs cRNA. The scheme shows the structure of the chimera with the γ part hatched and the γ2 part shaded. The position of the two hydrophobic segments is indicated by solid boxes. The statistical significance of the differences is indicated as follows: ∗ and ∗∗ indicate P < 0.01 and P < 0.001, respectively, when compared with the γ group. • and •• indicate P < 0.01 and P < 0.001, respectively, when compared  ith the γ2 group.

Figure 1

Sequence of the novel β2 and γ2 subunit of XENaC. The deducted amino acid sequences of the β2 and γ2 isoforms are aligned with the corresponding β and γ XENaC subunits [output of the pileup program of the Genetics Computer Group sequence analysis software package (18)]. Dots indicate that the residue in the β isoform is identical to the same position in the β2 isoform, and similarly for the γ and γ2 subunits. A dash indicates that a gap was introduced for obtaining a best-fit alignment. Lines above sequences indicate the positions of the putative transmembrane segments.

Figure 2

Tissue distribution by Northern blot analysis. Northern blot analysis of α, β, β2, γ, and γ2 transcript in lung, kidney, and A6 cells grown either on plastic or on permeable support. poly(A)⁺ RNA (3 μg) were loaded in lanes a (A6 cells grown on plastic) and b (A6 cells grown on filters), whereas 5 μg of poly(A)⁺ RNA was loaded in lanes c (kidney) and d (lung). The probes used to recognize α (A), β (B), β2 (C), and γ (D) have been described. The probe for β XENaC (B) also recognizes β2 XENaC and four other transcripts, but their presence was not consistent from one RNA extraction to another. (C) The same blot was probed with a fragment, which specifically recognized β2XENaC.

Figure 3

Tissue distribution by RT-PCR. RT-PCR analysis in the same tissues. The transcripts of all five subunits were present in kidney, lung, and A6 cells grown on a porous substrate. Transcripts of the expected size for β2 and γ2 XENaC could never be detected when single-stranded cDNA derived from RNA from A6 cells grown on plastic was used as a template. Considering the high sensitivity of the RT-PCR methods, this makes unlikely the presence of a significant number of these transcripts in cells grown on plastic.

Figure 4

Amiloride-sensitive current in isoforms of XENaC. Oocytes injected with the indicated combination of cRNA α, β, and γ subunits were studied under voltage-clamp conditions at −100 mV in a 100 mM sodium amphibian Ringer. (a) The current inhibited by a high concentration of amiloride (20 or 50 μM). The number of observations were 41, 25, 23, and 18 for the αβγ, αβ2γ, αβγ2, and αβ2γ2 groups, respectively. (b) The inhibitory constant of amiloride in each group. The number of measurements were 10, 4, 7, and 8 in αβγ, αβ2γ, αβγ2, and αβ2γ2, respectively.

Figure 5

Kinetics of the activation by sodium of the amiloride-sensitive current. The current sensitive to 50 μM amiloride at −100 mV, I_(am), was measured in solutions containing 0, 1, 3, 10, 20, and 100 mM Na⁺. All current values were normalized to the value of the amiloride-sensitive current in 100 mM Na, which was 3,630 ± 1,070 nA (n = 12) in the αβγ group and 167 ± 73 nA (n = 8) in the αβγ2 group. In the αβγ group the sodium concentration/I_(am) relationship was well described by simple Michaelis–Menten kinetics (upper equation) with a K_d of 11.3 mM. In the αβγ2 group the sodium concentration/I_(am) relationship was obviously different, with a significantly lower value in 100 mM Na⁺ than in 20 mM Na⁺. The curve fitted to the αβγ2 data points (lower equation) represents a double effect of Na⁺ concentration on the amiloride-sensitive current; first, an activation effect with a K_d of 4.8 mM and second, an inhibitory effect with a K_I of 154 mM.

Figure 6

Na-/amiloride interaction. The graph reports the apparent affinity for amiloride as a function of the extracellular Na⁺ concentration. Each point represent a group of 8–14 oocytes in which the response to 5 concentrations of amiloride was measured in a concentration of extracellular Na⁺ current. Experiments were performed in parallel on a similar number of oocytes expressing the αβγ and αβγ2 isoforms of XENaC. The parameters of the regression lines for the two isoforms are indicated above the graph.