Expression of the Voltage-Gated Sodium Channel NaV1.5 in the Macrophage Late Endosome Regulates Endosomal Acidification

Abstract

Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.

Regulation of Podosome Formation in Macrophages by a Splice Variant of the Sodium Channel SCN8A

“…Because TTX does not readily cross membranes, it is likely that it was taken up by pinocytosis or by endocytosis/ phagocytosis. Rapid distribution of TTX throughout the endosomal compartment is suggested by its ability to block NaV1.5 function when applied for 1 h in primed human cells during endocytosis or phagocytosis (6). In contrast, the present results suggest TTX does not appear to enter the membrane compartment where NaV1.6 resides unless there is more prolonged exposure, possibly because access to these compartments requires a combination of pinocytotic, endocytic, and phagocytic mechanisms.…”

“…This concentration of TTX functionally blocks TTX-sensitive channels such as NaV1.6 but not NaV1.5 (1,6). In addition, NaV1.6, but not NaV1.5, is expressed in differentiated, but unprimed THP-1 cells (see Table 1 for real time PCR data on sodium channel expression), and a prior study did not demonstrate the expression of any other voltage-gated sodium channels in this cell line (6).…”

“…Small Hairpin RNA-THP-1 cells were transduced with shRNA lentiviral clones as previously described (6). The following five clones, specific for human SCN8A (encodes NaV1.6), were obtained from Sigma: SHVRSC-TRCN0000044488-92.…”

“…This concentration of TTX functionally blocks TTX-sensitive channels such as NaV1.6 but not NaV1.5 (1,6). In addition, NaV1.6, but not NaV1.5, is expressed in differentiated, but unprimed THP-1 cells (see Table 1 for real time PCR data on sodium channel expression), and a prior study did not demonstrate the expression of any other voltage-gated sodium channels in this cell line (6). Although exposure to TTX for 1 h did not have a significant effect, treatment with TTX for 4 h significantly reduced the number of podosomes in unprimed, differ- A, unprimed THP-1 cells were loaded with the ratiometric sodium dye, SBFI, which like Sodium Green predominantly labels the cytoplasm and positively charged organelles.…”

“…In human macrophages, variants of the neuronal channel, NaV1.6, and the cardiac channel, NaV1.5, are expressed intracellularly but not at the plasma membrane (6). NaV1.5 localizes to the macrophage late endosome in interferon-␥ or lipolysaccharide-activated cells where it mediates endosomal acidification.…”