Local Ca²⁺Rise Near Store Operated Ca²⁺Channels Inhibits Cell Coupling During Capacitative Ca²⁺Influx

Abstract

Using a new fluorescence imaging technique, LAMP, we recently reported that Ca(2+) influx through store operated Ca(2+) channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH(i)) and Ca(2+)([Ca(2+)](i)) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca(2+) influx, there was a modest decline of pH(i) measured by BCECF. Decreasing pH(i) below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH(i) drop with thioacetate and bulk [Ca(2+)(i) rise with ionomycin was much less effective in inhibiting cell coupling than Ca(2+) influx. Moreover, clamping pH(i) with a weak acid and a weak base during Ca(2+) influx largely suppressed bulk pH(i) drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca(2+)(i) with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca(2+) influx. We concluded that local Ca(2+) elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca(2+) influx. To assess how Ca(2+) influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca(2+) influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca(2+) influx may be a more general phenomenon.

Photoactivatable fluorophores and techniques for biological imaging applications

“…In order to detect changes in molecular permeability of gap junction channels and to determine whether a cellular change can alter the gating property of connexin proteins, it is essential to assay dye transfer rates in the same coupled cell pairs before and after a biochemical stimulation in order to address if and how such an event affects cell coupling. Exploiting this important and unique property of LAMP assay, we demonstrated that Ca 2+ influx through store operated calcium channels potently inhibited GJ coupling 52 , likely mediated through a high subplasmalemmal Ca 2+ activity and colocalization of store operated Ca 2+ channel and connexin channel 58 .…”

“…Since HCCC2 emits blue fluorescence, it spectrally complements a variety of fluorescent sensors emitting from green to red. The combinatory use of these probes allow multi-color imaging to follow cellular biochemistry and cell coupling simultaneously 52, 58 .…”

“…Exploiting this important and unique property of LAMP assay, we demonstrated that Ca 2+ influx through store operated calcium channels potently inhibited gap junction coupling, 52 likely mediated through a high subplasmalemmal Ca 2+ activity and colocalization of store operated Ca 2+ channel and connexin channel. 58 In short, NPE-caged coumarins, including NPE-HCCC2/AM, enables us to develop a non-invasive imaging assay, LAMP, that provides quantitative and dynamic information on cell coupling. Since HCCC2 emits blue fluorescence, it spectrally complements a variety of fluorescent sensors emitting from green to red.…”

Gap Junctions in the Nervous System: Probing Functional Connections Using New Imaging Approaches

“…In this respect, LAMP has the combined advantages of both methods in that it is non-invasive, provides quantitative data, and has relatively high temporal resolution. In addition, LAMP allows for multicolor imaging, as the uncaging of NPE-HCCC2 requires a small pulse of UV light and is therefore compatible with fluorescent indicators in the visible spectrum ( Dakin et al, 2005 ; Dakin and Li, 2006 ; Abbaci et al, 2008 ). This method can be improved further by incorporating caged fluorescent molecules with higher uncaging efficiency and a more penetrable red-shifted emission spectrum.…”

Advantages and Limitations of Commonly used Methods to Assay the Molecular Permeability of Gap Junctional Intercellular Communication

“…This technique is mainly applied to study the dynamic regulation of gap junction coupling by intracellular Ca 2+ in individual cell pairs of human primary fibroblasts, for instance, and also to assess how Ca 2+ influx affects junctional coupling on HeLa cells expressing Cx26 (77,78). LAMP is limited to examination of changes in coupling in individual cell pairs, although it is likely to be useful in multiwell plate assays with confluent cell monolayers as well.…”