Immunolocalization of the mNav2.3 Na+ channel in mouse heart: upregulation in myometrium during pregnancy.

mNav2.3 is a putative voltage-dependent sodium channel (NaC) gene expressed in both mouse heart and uterus that shares only 45% amino acid identity with NaCs from gene subfamily 1. Immunofluorescence studies using polyclonal antibodies against two distinct epitopes revealed that mNav2.3 protein in heart colocalized with nerve-specific antibody binding. Similar mNav2.3-specific antibody staining was observed in virgin uterus. However, mNav2.3 expression in uterine nerve disappeared during late pregnancy, concurrent with an appearance in both the longitudinal and circular uterine smooth muscle, which reached a maximum at term and quickly declined within 2 days postpartum. mNav2.3 expression in term uterus often colocalized on the myocyte surface with connexin 43. The immunofluorescence results are supported by Western analysis in which the 217-kDa NaC increased during late pregnancy and declined 2 days postpartum. These data provide perhaps the most dramatic example of NaC regulation. The acute and transient upregulation in myometrium during gestation suggests the Nav2.3 channel plays a role in uterine function at term.

Primary structure and differential expression during development and pregnancy of a novel voltage-gated sodium channel in the mouse.

Until recently, all cloned vertebrate voltage-dependent sodium channels exhibited high sequence homology to one another and appeared to comprise a single multigene subfamily. An exception is the human Nav2.1 channel proposed to represent a second Na+ channel (NaCh) gene subfamily since comparison with previously cloned voltage-gated NaChs revealed only 40-45% identity. We have now cloned a mouse NaCh (mNav2.3) from an atrial tumor cell line that shows high amino acid sequence identity to hNav2.1 in functionally relevant regions such as the pore-forming segments, S4 segments, and inactivation gate sequence. Overall sequence identity is 68%. mNav2.3 mRNA was most abundant in heart and uterus, and the transcript levels in heart, brain, and skeletal muscle were differentially regulated during development. Transcript levels in heart were greatest immediately after birth. mNav2.3 transcript levels in pregnant uterus increased 3-fold between day 15 of pregnancy and birth and then declined 15-fold during the 2 days following delivery. The mNav2.3 amino acid sequence indicates that the Nav2 NaCh gene subfamily is well conserved across species, and the tissue-specific and developmental regulation of mRNA expression suggests these channels play important physiological roles in cardiac and uterine muscle.

Differential expression of Isk mRNAs in mouse tissue during development and pregnancy.

The molecular isoform of the cDNA clone Isk present in the AT-1 atrial tumor cell line was characterized by molecular cloning of Isk cDNA. Since Isk mRNA was found in mouse heart, kidney, and uterus, a complete study of its expression during development in the heart and kidney was performed, in addition to its expression in the uterus during pregnancy. In the heart, Isk showed a 4-fold upregulation during the perinatal period followed by a 20-fold decrease between birth and the adult state. Furthermore, the two 0.9- and 3.4-kb transcripts were differentially regulated after birth. In the kidney, Isk progressively increased 10-fold, reaching steady-state adult values at 21 days. Isk mRNA levels in the uterus increased threefold at late pregnancy and decreased sixfold rapidly after birth. The Isk gene is differentially expressed during development in kidney and cardiac tissue, and both Isk transcripts appeared to be differentially regulated. Furthermore, the drastic changes in transcript levels before delivery and after birth suggest that Isk plays a significant role in myometrium during late pregnancy and delivery.

Molecular cloning of an atypical voltage-gated sodium channel expressed in human heart and uterus: evidence for a distinct gene family.

Previously cloned voltage-dependent sodium channels exhibit a high degree of homology to one another and appear to comprise a single multigene family. We have now isolated and characterized cDNAs from both human adult heart and fetal skeletal muscle that encode a sodium channel alpha subunit that exhibits only moderate primary structure identity with other sodium channels and is prominently expressed in both heart and uterus. The approximately 7.2-kilobase cDNA sequence, designated hNav2.1, predicts a 1682-amino acid protein that bears 52%, 49%, and 46% overall identity with sodium channels cloned from rat brain, skeletal muscle, and heart, respectively. Positively charged S4 segments are present in hNav2.1, but there are fewer basic residues in repeat domains 1, 3, and 4 than in other cloned sodium channels. The cloning of hNav2.1 provides evidence for greater evolutionary divergence among voltage-dependent sodium channels and suggests that other sodium channel gene subfamilies may exist. The unique amino acid sequences in regions known to be involved in voltage-dependent activation and inactivation suggest that hNav2.1 will have novel gating properties.