Proestrus Differentially Regulates Expression of Ion Channel and Calcium Homeostasis Genes in GnRH Neurons of Mice

Abstract

In proestrus, the changing gonadal hormone milieu alters the physiological properties of GnRH neurons and contributes to the development of the GnRH surge. We hypothesized that proestrus also influences the expression of different ion channel genes in mouse GnRH neurons. Therefore, we performed gene expression profiling of GnRH neurons collected from intact, proestrous and metestrous GnRH-GFP transgenic mice, respectively. Proestrus changed the expression of 37 ion channel and 8 calcium homeostasis-regulating genes. Voltage-gated sodium channels responded with upregulation of three alpha subunits (Scn2a1, Scn3a, and Scn9a). Within the voltage-gated potassium channel class, Kcna1, Kcnd3, Kcnh3, and Kcnq2 were upregulated, while others (Kcna4, Kcnc3, Kcnd2, and Kcng1) underwent downregulation. Proestrus also had impact on inwardly rectifying potassium channel subunits manifested in enhanced expression of Kcnj9 and Kcnj10 genes, whereas Kcnj1, Kcnj11, and Kcnj12 subunit genes were downregulated. The two-pore domain potassium channels also showed differential expression with upregulation of Kcnk1 and reduced expression of three subunit genes (Kcnk7, Kcnk12, and Kcnk16). Changes in expression of chloride channels involved both the voltage-gated (Clcn3 and Clcn6) and the intracellular (Clic1) subtypes. Regarding the pore-forming alpha-1 subunits of voltage-gated calcium channels, two (Cacna1b and Cacna1h) were upregulated, while Cacna1g showed downregulation. The ancillary subunits were also differentially regulated (Cacna2d1, Cacna2d2, Cacnb1, Cacnb3, Cacnb4, Cacng5, Cacng6, and Cacng8). In addition, ryanodine receptor 1 (Ryr1) gene was downregulated, while a transient receptor potential cation channel (Trpm3) gene showed enhanced expression. Genes encoding proteins regulating the intracellular calcium homeostasis were also influenced (Calb1, Hpca, Hpcal1, Hpcal4, Cabp7, Cab 39l, and Cib2). The differential expression of genes coding for ion channel proteins in GnRH neurons at late proestrus indicates that the altering hormone milieu contributes to remodeling of different kinds of ion channels of GnRH neurons, which might be a prerequisite of enhanced cellular activity of GnRH neurons and the subsequent surge release of the neurohormone.

Keywords: GnRH; gene expression; ion channels; mouse; neurons; proestrus; transcriptome.

FIGURE 1

Heat map of genes regulated differentially in GnRH neurons of proestrous versus metestrous mice. Expression levels of genes coding ion channels and regulators of calcium homeostasis. The rows represent differentially expressed probe sets with corresponding gene symbols on the right. The expression level of each probe is color coded. For decoding, see the color key. The individual samples are shown as columns. The six metestrous and six proestrous samples are coded in yellow and blue, respectively.

FIGURE 2

Predicted interactions among proteins encoded by upregulated ion channel/transport genes in GnRH neurons of proestrous mice. The gene network was constructed by using the STRING 10.5 Known and Predicted Protein–Protein Interactions program (http://string-db.org/). Evidence view, medium confidence. The majority of the proteins (19) form functional clusters belonging to voltage-gated sodium (yellow), potassium (red), and calcium (green) channels. Six proteins represent the non-networking group (Clcn3, Clcn6, Trpm3, Hpcal1, Kcnj9, and Itpkb). Color code of highlighted proteins: red: voltage-gated potassium channel, green: voltage-gated calcium channel, yellow: voltage gated sodium channel, blue: voltage-gated chloride channel. Color code for lines in evidence view: red line – presence of fusion evidence, green line – neighborhood evidence, blue line – co-occurrence evidence, purple line – experimental evidence, light blue line – database evidence, black line co-expression.

FIGURE 3

Predicted interactions among proteins encoded by downregulated ion channel/transport genes in GnRH neurons of proestrous mice. The gene network was constructed by using the STRING 10.5 Known and Predicted Protein–Protein Interactions program (http://string-db.org/). Evidence view, medium confidence. It contains 13 networking proteins representing predominantly calcium channel (blue), voltage-gated channel (green), and voltage-gated potassium channel activities (red). The non-networking proteins numbered 6 (Cib2, Cab39l, Kcnk12, Cabp7, Cacng8, and Kcnj11). Color code of highlighted genes: green: voltage-gated channel activity, red: voltage-gated potassium channel activity, blue: calcium channel activity. Color code for lines in evidence view: red line –presence of fusion evidence, green line – neighborhood evidence, blue line – co-occurrence evidence, light blue line – database evidence, black line co-expression.

FIGURE 4

Validation of microarray data with quantitative PCR. (A) The qPCR confirmed the differential expression of 7 selected genes. Six showed upregulation (Kcnj10, Trpm3, Calb1, Clcn6, Cacna1b, and Kcnq2), while one gene (Kcnc3) represented the downregulated category. The fold change (FC) values of these genes – gained from the microarray dataset – are also listed. (B) The plot illustrates the correlation between microarray and quantitative PCR data. The coefficient of determination (r²) is 0.7349. Pearson’s product-moment correlation: (r = 0.8685, df = 5; p = 0.0137).