Regulation of membrane potential and fluid secretion by Ca2+-activated K+ channels in mouse submandibular glands

Abstract

We have recently shown that the IK1 and maxi-K channels in parotid salivary gland acinar cells are encoded by the K(Ca)3.1 and K(Ca)1.1 genes, respectively, and in vivo stimulated parotid secretion is severely reduced in double-null mice. The current study tested whether submandibular acinar cell function also relies on these channels. We found that the K(+) currents in submandibular acinar cells have the biophysical and pharmacological footprints of IK1 and maxi-K channels and their molecular identities were confirmed by the loss of these currents in K(Ca)3.1- and K(Ca)1.1-null mice. Unexpectedly, the pilocarpine-stimulated in vivo fluid secretion from submandibular glands was essentially normal in double-null mice. This result and the possibility of side-effects of pilocarpine on the nervous system, led us to develop an ex vivo fluid secretion assay. Fluid secretion from the ex vivo assay was substantially (about 75%) reduced in animals with both K(+) channel genes ablated - strongly suggesting systemic complications with the in vivo assay. Additional experiments focusing on the membrane potential in isolated submandibular acinar cells revealed mechanistic details underlying fluid secretion in K(+) channel-deficient mice. The membrane potential of submandibular acinar cells from wild-type mice remained strongly hyperpolarized (-55 +/- 2 mV) relative to the Cl(-) equilibrium potential (-24 mV) during muscarinic stimulation. Similar hyperpolarizations were observed in K(Ca)3.1- and K(Ca)1.1-null mice (-51 +/- 3 and -48 +/- 3 mV, respectively), consistent with the normal fluid secretion produced ex vivo. In contrast, acinar cells from double K(Ca)3.1/K(Ca)1.1-null mice were only slightly hyperpolarized (-35 +/- 2 mV) also consistent with the ex vivo (but not in vivo) results. Finally, we found that the modest hyperpolarization of cells from the double-null mice was maintained by the electrogenic Na(+),K(+)-ATPase.

Figure 5. Membrane potential of submandibular acinar cells from wild-type and K_Ca3.1-, K_Ca1.1- and double-null mice

Cell were stimulated with 0.3 μm CCh with or without K⁺ channel blockers (with 1 μm paxillin, with 3 μm clotrimazole, or with 1 μm paxilline and 3 μm clotrimazole). A, membrane potential of a submandibular acinar cell from wild-type mice recorded in perforated-patch configuration. B–D, membrane potentials recorded in cells from K_Ca3.1-, K_Ca1.1- and double-null mice. E, time-averaged mean membrane potentials in cells from wild-type or mutant mice as indicated. *P < 0.05.

Figure 1. Analysis of whole-cell and single channel K⁺ currents in wild-type submandibular acinar cells

A, expression of mRNA of IK1 and maxi-K channels in submandibular glands from wild-type mice was determined using RT-PCR analysis. B, whole-cell currents recorded with 250 nm Ca²⁺ in the pipette. Currents were recorded immediately (10–20 s) after achieving whole-cell mode (Early) and after a steady level of IK1 current developed (Later, 5–10 min). Top panel, typical raw currents in response to 40 ms voltage pulses to −110, −30, +10 and +50 mV from −70 mV holding potential. Calibration bars are 1 nA and 10 ms. Bottom panel, mean current densities determined at the end of the 40 ms pulses to the indicated potentials at Early (^) and Later (•) times (n = 12). Dashed line represents the voltage-independent (IK1-like) component. C, sensitivity of whole-cell currents to the maxi-K channel inhibitor paxilline and the IK1 channel inhibitor clotrimazole. Raw traces and I–V plot are presented in the same format as in B; •– no inhibitors, ▵– with 1 μm paxillin, ▴– with 1 μm paxilline and 3 μm clotrimazole (n = 7). D, top panel, single channel recordings from 3 inside-out patches exposed to 250 nm Ca²⁺ with either IK1 (top patch) or maxi-K (middle patch), or both (bottom patch) channels present. Bottom panel, I–V plot of single channel IK1 (^) and maxi-K (•) currents at the indicated potentials (n = 7 cells).

Figure 2. Whole-cell submandibular acinar K currents in K_Ca3.1- and K_Ca1.1- and double-null mice

A, whole-cell currents in a cell from K_Ca3.1-null mouse recorded with 250 nm Ca²⁺ in the pipette. Currents were recorded immediately after achieving whole-cell mode (Early and ^) and after a steady level of IK1 current developed (Later and •) and after addition of 1 μm paxillin, a maxi-K channel inhibitor (▴). Typical raw traces and average I–V plot are presented in the same format as in Fig. 1B (n = 7). B and C, same as described in A but currents recorded in cells from K_Ca1.1-null mice (n = 5) and from double-null mice (n = 7). B, the identity of the developed current was tested by addition of 3 μm clotrimazole, the specific inhibitor of IK1 channel (▴).

Figure 3. K⁺ currents in resting and stimulated cells from wild-type mice and K_Ca3.1- and K_Ca1.1-null mice

IK1 and maxi-K currents were simultaneously recorded using perforated-patch mode in cells from wild-type mice (A and B), K_Ca3.1-null (C and D) and K_Ca1.1-null (E and F) mice. For A, C and E, every 0.4 s IK1 currents (upper panels) were measured at −100 mV (10 ms voltage step) and maxi-K currents (lower panels) were determined as a time-dependent component of the current elicited by 40 ms steps to +30 mV from holding potential of −60 mV. At time points indicated by the arrows in A, C and E full I–V plots were obtained. The average I–V plots in B (n = 7), D (n = 6), and F (n = 4) show K⁺ currents before (^) and during stimulation with 0.3 μm CCh (•) as well as after blocking K⁺ channels without (▵) and with (▴) stimulation. Insets of B, D and F, raw currents in response to 40 ms voltage pulses to −110, −30, +10 and +50 mV from −70 mV holding potential. Calibration bars: 1, 2 and 0.5 nA for B, D and F, respectively, versus 10 ms.

Figure 4. Membrane potential, membrane conductance and K⁺ and Cl⁻ currents in submandibular acinar cells from wild-type mice

Membrane voltage and currents were recorded in perforated-patch configuration. Stimulation with 0.3 μm CCh induced either membrane oscillations (A) or non-oscillatory responses (B, upper panel) with concurrent increase in membrane conductance (B, lower panel). Typical current traces of ‘oscillatory’ (C) and ‘non-oscillatory’ (D) cells were recorded during 10 ms steps to either −25 mV (upper traces) or to −85 mV (lower traces) with 10 ms interpulse interval applied every 0.4 s from holding potential of −60 mV.

Figure 6. In vivo stimulated fluid secretion from submandibular glands

A, amount of saliva secreted over 30 min from K_Ca3.1-null (^) and wild-type (•) mice in response to 10 mg (kg body weight)⁻¹ of pilocarpine (n = 11 and 10 mice, respectively). B, saliva secreted from K_Ca1.1-null (^) and wild-type (•) mice (n = 7 and 6 mice, respectively). C, volume of saliva secreted from K_Ca3.1/K_Ca1.1-double-null (^) and wild-type (•) mice (n = 7 and 11 mice, respectively). Inset, respective average saliva flow rates calculated for 5 min time intervals. *P < 0.05.

Figure 7. Muscarinic-stimulated saliva secretion from isolated submandibular glands

A, saliva secretion from ex vivo submandibular glands from either wild-type or K_Ca3.1-null mice, the artery and main excretory duct cannulated. Secretion was stimulated by vascular perfusion with 0.5 μm CCh: •– wild-type, ^–K_Ca3.1-null glands (n = 8 and 9 mice, respectively). B and C, same as described for A but fluid flow rates were compared in K_Ca1.1-null (n = 5 mice) and double-null (n = 5 mice) glands with their wild-type controls (n = 4 and n = 4 mice, respectively).

Figure 8. Membrane depolarization upon inhibition of Na⁺,K⁺-ATPase

A and B, membrane potential traces recorded in wild-type cells stimulated with 0.3 μm CCh prior to or along with superfusion of ouabain alone (A) or with paxilline and clotrimazole (B). C, the mean values of V_m from cells during stimulation with no inhibitors (open bars), with K⁺ channel inhibitors (grey bars), or with both K⁺ channel inhibitors and Na⁺,K⁺-ATPase inhibitor (black bars). *P < 0.05.