Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Abstract

Ca(2+) activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca(2+)] was used to investigate if Ca(2+)-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca(2+)-buffering conditions that produced brief, localized increases in [Ca(2+)] after focal laser photolysis of caged Ca(2+). Conditions were used to isolate K(+) and Cl(-) conductances. Photolysis at the apical PM resulted in a robust increase in K(+) and Cl(-) currents. A localized reduction in [Ca(2+)] at the apical PM after photolysis of Diazo-2, a caged Ca(2+) chelator, resulted in a decrease in both K(+) and Cl(-) currents. The K(+) currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance "maxi-K" (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34-sensitive K(+) currents were also observed in BK-null parotid acini. In contrast, when the [Ca(2+)] was increased at the basal or lateral PM, no increase in either K(+) or Cl(-) currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

Figure 1.

Polarized morphology of parotid acini. (A) A transmitted laser light image is shown of a small group of cells characteristic of those used throughout this study. The positions of secretory granules (SG), nucleus (NU), apical membrane (AM), lateral membrane (LM), and basal membrane (BM) are annotated. The position of apical (red dot), lateral (green dot), and basal (blue dot) photolysis sites is shown. (B) A maximum projection image, constructed from a z-series, is shown from a small group of cells stained with live cell markers of the nucleus (blue), granules (red), and PM (green). (C) A similar triplet was stained to indicate PM (green), nucleus (blue), and mitochondria (red). (D) Similar staining was performed in a parotid lobule not subjected to enzymatic digestion and indicates the similar localization of granules (red), PM (green), and nuclei (blue).

Figure 2.

Establishing conditions for local manipulation of [Ca²⁺]. (A) A bright field image of a parotid acinar cell is shown together with pseudocolored fluorescence image series of cells immediately before and after flash photolysis. The pipette solution contained 10 mM NP-EGTA and 5 mM Ca²⁺ (free Ca²⁺, ∼160 nM). The images were acquired every 45 ms, and the single image containing the flash artifact was removed. Release of Ca²⁺ after laser exposure (green arrow) resulted in a marked increase in fluorescence at the photolysis site in the basal pole (blue dot), which decayed rapidly but spread toward the apical pole (red dot). (B) The change in ΔF/F₀ ratio in either the basal (blue line) or apical (red line) region, demonstrating that the increase in the “untargeted” pole was >10% when compared with basal values. (C) A similar experiment is shown, except the pipette solution contained 10 mM NP-EGTA and 2 mM Ca²⁺ (free [Ca²⁺], ∼40 nM). The series of images demonstrates that the increase in [Ca²⁺] remains localized to the apical pole (red dot) under these conditions. (D) The change in ΔF/F₀ ratio values for this experiment, indicating that the marked increase in fluorescence after laser exposure in the apical domain (red line) results in little increase (<10% increase over basal) in fluorescence in the untargeted region (blue line). (E) A spatial analysis of the peak change in fluorescence after photolysis with buffering conditions as in C. The peak change in fluorescence shown in E (left) was compared with an average image generated from the previous five frames under basal conditions. The change in ΔF/F₀ ratio along the yellow line is shown in the right panel and demonstrates that the fluorescence decays exponentially from the photolysis site, with a length constant of 2.5 ± 0.2 µm. The fit for the decay is shown in the red line. (F) A similar analysis in which the individual experiments are normalized to the peak fluorescence in the individual cell. The red line shows the mean fit of the data.

Figure 3.

Apically localized increase in [Ca²⁺] activates K and Cl channels. Experiments were performed with the buffering conditions established in Fig. 2 (C–F). (A) A bright field and pseudocolored image series in which the [Ca²⁺] was increased close to the apical PM (red dot). (B) In these images and kinetic plot, a marked increase in signal was evoked in the apical domain (red line) and remained highly localized. A minimal increase is observed in the basal region (blue line) after laser exposure (green arrow). (C; top) The increase in whole cell K⁺ current evoked by the Ca²⁺ transient in B is shown. (Bottom) The increase in Cl⁻ current after a subsequent apical laser exposure after the bath solution had been exchanged for 140 mM TEA-Cl. A robust increase in whole cell Cl⁻ current is evoked. (D)Paired experiments before and after flash.

Figure 4.

Basolaterally localized increases in [Ca²⁺] fail to activate K and Cl channels. (A) A bright field and pseudocolor image series is shown for a cell in which [Ca²⁺] was increased under the basal PM (blue dot) using laser parameters identical to Fig 3. (B) The kinetic demonstrates that a substantial and localized increase in fluorescence was evoked. Increasing the [Ca²⁺] at the basal PM failed to significantly activate K channels (C, top) or Cl channels in TEA-Cl solution (C, bottom). (D) Paired experiments before and after flash. (E) Similar experiments were performed, but the laser power output was increased to generate a larger increase in [Ca²⁺], as shown in E (images) and F (kinetic). (G) Under these conditions, no increase in K⁺ conductance could be evoked. (H) Paired experiments demonstrating that no significant increase in either K⁺ (left) or Cl⁻ (right) conductance occurred under these conditions.

Figure 5.

An apical reduction in [Ca²⁺] reduces the activity of K and Cl channels. Local photolysis of Diazo-2 was used to reduce the [Ca²⁺] specifically at either the basal or apical pole in cells dialyzed with Ca²⁺ to preactivate Ca²⁺-sensitive currents. (A) A bright field and pseudocolored image series is shown from a cell dialyzed with ∼550 nM [Ca²⁺]. After laser exposure, the fluorescence decreased specifically in the apical pole. (B) The kinetic showing the decrease in fluorescence after laser exposure in the apical pole (red line) and basal pole (blue line). (C) The spatial analysis along the yellow line of the peak change in fluorescence compared with the mean of the five previous basal frames. The single-exponential fit is shown in the red line, and the mean of these analysis yields a length constant of 5.02 ± 0.76 µm. (D; top) The decrease in [Ca²⁺] evoked in A resulted in a significant reduction in K⁺ and Cl⁻ conductance (bottom). (E) Paired experiments are shown before and after flash for either 550 or 175 nM [Ca²⁺], “high” or “low” [Ca²⁺], respectively. In F (images) and G (kinetic), basal photolysis of Diazo-2 resulted in a similar decrease in [Ca²⁺] in the basal domain, which again remained localized as demonstrated by the spatial analysis shown in H. (I and J) Basal photolysis failed to reduce either K⁺ or Cl⁻ currents in cells dialyzed with the “high [Ca²⁺]” pipette solution.

Figure 6.

BK and IK are present in the apical PM. Experiments were performed using the techniques described in Fig. 3 to analyze the contribution of IK and BK to the currents evoked by apical photolysis. (A) K⁺ currents could be evoked in the presence of either TRAM-34 (left) or paxilline (middle) but were essentially abolished when cells were incubated with both agents. (B) Similar experiments were performed in BK-null animals (Slo^−/−). The baseline current was reduced, but apical photolysis resulted in an increase in the remaining IK current, which was markedly reduced in the presence of TRAM-34. However, Cl⁻ currents could be evoked by apical photolysis in the same cells (right).

Figure 7.

Localization of IK in parotid clumps by immunofluorescence. Immunofluorescence experiments were performed as described in Materials and methods. (A and E) Transmitted laser light images of the parotid clusters. (B and F) The localization of IK is shown and illustrates a prominent PM distribution with enriched localization of channels to the apical domain of the cells. (C) The distribution of the tight junction marker ZO-1 in the clump of cells stained with IK antibodies in B. (G) The localization of InsP₃R-3, known to localize to the ER juxtaposed to the apical PM, is shown in the same cells. (D) The overlap of IK and ZO-1 is shown. (H) The co-distribution of IK and InsP₃R-3. Bar, 10 µm. Each image is a single z slice acquired at an approximately equatorial plane.