Important contribution of alpha-neurexins to Ca2+-triggered exocytosis of secretory granules

Abstract

Alpha-neurexins constitute a family of neuronal cell surface molecules that are essential for efficient neurotransmission, because mice lacking two or all three alpha-neurexin genes show a severe reduction of synaptic release. Although analyses of alpha-neurexin knock-outs and transgenic rescue animals suggested an involvement of voltage-dependent Ca2+ channels, it remained unclear whether alpha-neurexins have a general role in Ca2+-dependent exocytosis and how they may affect Ca2+ channels. Here we show by membrane capacitance measurements from melanotrophs in acute pituitary gland slices that release from endocrine cells is diminished by >50% in adult alpha-neurexin double knock-out and newborn triple knock-out mice. There is a reduction of the cell volume in mutant melanotrophs; however, no ultrastructural changes in size or intracellular distribution of the secretory granules were observed. Recordings of Ca2+ currents from melanotrophs, transfected human embryonic kidney cells, and brainstem neurons reveal that alpha-neurexins do not affect the activation or inactivation properties of Ca2+ channels directly but may be responsible for coupling them to release-ready vesicles and metabotropic receptors. Our data support a general and essential role for alpha-neurexins in Ca2+-triggered exocytosis that is similarly important for secretion from neurons and endocrine cells.

Figure 1.

α-Neurexin-deficient mice exhibit a small pituitary gland. A, Double knock-out mice (DKO; right) show a hypomorphic phenotype as compared with single knock-out littermate controls (SKO; left). B, Body weight of 5- to 6-week-old female mice lacking Nrxn2α and Nrxn3α (D2/3), Nrxn1α and Nrxn2α (D1/2), or Nrxn2α alone (control). Error bars represent the mean ± SEM. C, D, Manganese-enhanced MRI of adult wild-type (C) and α-neurexin double knock-out mice (D). The sample images show horizontal T₁-weighted scans at the level of the base of the skull. Arrows point to the pituitary glands that highly enrich MnCl₂, indicating reduced uptake of MnCl₂ and/or size of the gland in mutant mice. E, RT-PCR demonstrates the presence of two α-neurexins and two β-neurexins in the RNA from adult pituitary gland. Syntag1, Synaptotagmin 1 (positive control); dH₂O, distilled water. F, α-Neurexin isoforms are expressed in the pituitary glands at levels comparable to or higher than their expression in the brain. The relative quantification was performed by SYBR Green-based real-time PCR with β-actin as a reference gene. Error bars represent the mean ± SEM. G, H, Hematoxylin and eosin-stained frontal sections of the pituitary glands from control (G) and double knock-out mice (H). aL, Anterior lobe; iL, intermediate lobe; pL, posterior lobe. I, J, High-magnification pictures of the intermediate lobe of control (I) and double knock-out animals (J). Cells appear to be packed more densely in the latter (arrows). Scale bars are as indicated; level of statistical significance is indicated above the bars.

Figure 2.

Melanotrophs show a normal expression of peptides in α-neurexin mutant mice. Control (A, C, E, G) and double knock-out (B, D, F, H) pituitaries were labeled with antibodies against intermediate lobe markers: POMC (A–D) and its cleavage products β-endorphin (E, F) and α-MSH (G, H), expressed by melanotrophs (arrows). aL, Anterior lobe; iL, intermediate lobe; pL, posterior lobe. Scale bars are as indicated.

Figure 3.

Release from pituitary gland melanotrophs is reduced in adult α-neurexin-deficient mice. A, B, Resting membrane capacitance was measured in melanotrophs of the intermediate lobes from adult wild-type (WT), single knock-out (SKO2), and double knock-out (DKO) pituitary glands (A), revealing that the average cell volume of DKO melanotrophs is reduced (B). C, Ca²⁺-dependent secretion was evoked by a train of 200 depolarizing pulses from −80 to +10 mV at 10 Hz. The secretory response was measured as ΔC _m in WT and SKO2 controls and in α-neurexin DKO cells and is shown as the mean ± SEM in the traces. D, Higher-resolution plot of the first 4 s of capacitance change (boxed area in C). E, Cumulative membrane capacitance change normalized to the resting C _m in WT and SKO2 controls compared with α-neurexin DKO cells. F, Membrane capacitance changes evoked by the single pulses of the depolarization train. Data are represented as the mean ± SEM (A–C, E, F) and have been normalized to the resting capacitance (C–E). Level of statistical significance is indicated above the bars.

Figure 4.

The functional defect, but not the structural phenotype, is present in the pituitary glands of newborn mutant mice. A, B, Representative sections of the pituitary gland from newborn control (A) and triple knock-out (B) mice stained with hematoxylin and eosin, showing intermediate lobes of similar thickness and cell density. C, Resting membrane capacitance of melanotrophs from newborn double knock-out (DKO; red) and triple α-neurexin knock-out (TKO; gray) mice is not different from littermate control animals (SKO2; green). D, Ca²⁺-dependent secretion was evoked from melanotrophs of newborn control animals (SKO2; green trace) and α-neurexin double (DKO; red) and triple (TKO; gray) knock-out mice; responses are represented as changes in membrane capacitance, using the same protocol as for adult mice (see Fig. 3 C). E, Higher-resolution plot of the first 4 s of capacitance change (boxed area in D). F, Cumulative membrane capacitance change normalized to the resting C _m. Data are represented as the mean ± SEM (C, D, F) and have been normalized to the resting capacitance (D–F). Level of statistical significance is indicated above the bars; n.s., not significant. Scale bar (in B) is as indicated.

Figure 5.

The impaired secretion from melanotrophs affects the release-ready pool of vesicles. A, Dual-pulse protocol applied to elicit depletion of the IRP of vesicles. Paired pulses of 40 ms duration to −10 and 0 mV were applied 60 ms apart, and the voltage of the pulses was adjusted to give an equal amount of Ca²⁺ influx during both pulses. B, Comparison of the IRP size among wild-type (WT) and α-neurexin single knock-out (SKO2) and double knock-out (DKO) mice by dual-pulse protocol as follows: S = ΔC _m1 + ΔC _m2. Level of statistical significance is indicated above the bars. C, Horrigan–Bookman plot identifies alterations in the IRP of vesicles. At 2 min after the beginning of whole-cell dialysis, single depolarization pulses of different duration (between 2 and 200 ms) from −80 to 0 mV were applied in random order. Then, 15 s was allowed for pool refilling after pulses shorter than 40 ms, and 30 s was allowed for longer pulses. Membrane capacitance changes were recorded from control (SKO2; green) and α-neurexin double knock-out (DKO; red) cells. Pulses <40 ms presumably evoke a release of vesicles from the IRP; this exponential phase reaches a plateau at ∼20 ms, indicating pool depletion. τ represents the time constant for the exponential fit of control data, although knock-out data could not be fit satisfactorily with an exponential equation. The slower component of release was fit with a straight line; k indicates the slope of the linear fit. Data are represented as the mean ± SEM.

Figure 6.

Whole-cell Ca²⁺currents are not reduced in α-neurexin-deficient melanotrophs but show a small kinetic difference. A, Representative Ca²⁺ current recordings evoked by 300 ms voltage ramps from −80 to +60 mV in melanotrophs of α-neurexin DKO (red), and littermate control (SKO2; green) mice. B, Comparison of high voltage-activated peak Ca²⁺ current densities in melanotrophs of adult and newborn control (WT and SKO2) and α-neurexin DKO and TKO mice. C, Rundown of normalized Ca²⁺ currents is similar among genotypes. D, Ca²⁺ channel subtype contributions to Ca²⁺ currents were measured in 10 mm [Ca²⁺]_e by sequentially adding 1 μm ω-conotoxin GVIA, 100 nm of ω-agatoxin TK plus 100 nm ω-conotoxin MVIIC, and 10 μm nifedipine. E, The densities of Ca²⁺-activated Cl⁻ currents (Turner et al., 2005) are comparable between the genotypes. F, Voltage dependence of the averaged Ca²⁺ current densities shows a shift in α-neurexin double knock-out cells (arrows; ΔV _peak). All data in bar histograms are represented as the mean ± SEM; n.s., not significant.

Figure 7.

Melanotrophs in α-neurexin-deficient mice receive reduced synaptic inputs from hypothalamic neurons. A, Representative current traces show GABA-mediated SPCs before and after the addition of 60 μm α-latrotoxin recorded under voltage clamp from adult melanotrophs of control and double knock-out mice (DKO). B, Quantification of the SPC frequency demonstrates that synaptic release from hypothalamic neurons is reduced in mutant mice and cannot be stimulated by the neurotoxin as in controls. C, Similar recording as in A before (0 mm sucrose) and after the addition of hypertonic solution (500 mm sucrose). D, Quantification of the SPC frequency shows that the hypothalamic terminals on both control and mutant melanotrophs respond to sucrose stimulation. Data are represented as the mean ± SEM. Level of statistical significance is indicated above the bars; n.s., not significant.

Figure 8.

Neurexin 1α has no direct effect on biophysical parameters of recombinant N-type (Ca_V2.2) calcium channels. A, Representative Ca²⁺ channel currents recorded from HEK293 cells stably expressing the α1B, β3, and α2δ subunits of high voltage-activated Ca²⁺ channels (HEK293_Ca_V2.2). Currents were induced by 150 ms pulses from −40 to +50 mV in 10 mV increments. B, Ca²⁺ channel currents recorded from the stably transfected HEK293 cells can be blocked completely by 1 μm ω-conotoxin GVIA (ω-Ctx), suggesting that they are mediated specifically by the recombinant N-type (Ca_V2.2) channels. Time course was obtained by applying 50 ms voltage pulses to 0 mV every 10 s. The inset shows sample traces before (black) and 5 min after (red) the addition of ω-Ctx. C, Cotransfection experiments of HEK293_Ca_V2.2 cells with Nrxn1α cDNA or Nrxn1α plus CASK and Mint1 cDNAs. The diagram depicts the expression vector for Nrxn1α that coexpresses a reporter gene, GFP. A representative cell shows neurexin at the plasma membrane (red), and cytosolic GFP (green). D, E, Whole-cell voltage-clamp recordings reveal no differences in averaged current density–voltage relationships (D) and in averaged steady-state inactivation curves (E) in HEK293_Ca_V2.2 cells coexpressing Nrxn 1α, Nrxn 1α together with CASK and Mint1, or untransfected HEK293_Ca_V2.2 cells. Curves were generated from voltage-step protocols as indicated in the diagrams. The numbers of cells from more than three independent transfections are as indicated. Data are represented as the mean ± SEM.

Figure 9.

GABA_B receptor modulation of Ca²⁺currents is impaired in α-neurexin mutant mice. A, B, Ca²⁺ currents were recorded before and after the addition of 30 μm baclofen from brainstem neurons of newborn control (A; WT) and α-neurexin triple knock-out mice (B; TKO). Representative current traces (A₁, B₁), current–voltage relationships (A₂, B₂), and quantitated peak current densities (A₃, B₃) show a decrease of Ca²⁺ currents in the presence of baclofen in control neurons, but not in mutant neurons. C, Peak amplitude of high voltage-activated Ca²⁺ currents in response to 30 μm baclofen plotted against time in control (WT) and α-neurexin knock-out mice (TKO), demonstrating similar onset within <30 s. D, Dose–response curve showing the concentration-dependent effect of baclofen in control (WT; IC₅₀ = 2.4 μm; n = 5 mice) and α-neurexin knock-outs (TKO; IC₅₀ = 4.2 μm; n = 5). Data are represented as the mean ± SEM. Level of statistical significance is indicated above the bars; n.s., not significant.