Precise control of ion channel and gap junction expression is required for patterning of the regenerating axolotl limb

Abstract

Axolotls and other salamanders have the capacity to regenerate lost tissue after an amputation or injury. Growth and morphogenesis are coordinated within cell groups in many contexts by the interplay of transcriptional networks and biophysical properties such as ion flows and voltage gradients. It is not, however, known whether regulators of a cell’s ionic state are involved in limb patterning at later stages of regeneration. Here we manipulated expression and activities of ion channels and gap junctions in vivo, in axolotl limb blastema cells. Limb amputations followed by retroviral infections were performed to drive expression of a human gap junction protein Connexin 26 (Cx26), potassium (Kir2.1-Y242F and Kv1.5) and sodium (NeoNav1.5) ion channel proteins along with EGFP control. Skeletal preparation revealed that overexpressing Cx26 caused syndactyly, while overexpression of ion channel proteins resulted in digit loss and structural abnormalities compared to EGFP expressing control limbs. Additionally, we showed that exposing limbs to the gap junction inhibitor lindane during the regeneration process caused digit loss. Our data reveal that manipulating native ion channel and gap junction function in blastema cells results in patterning defects involving the number and structure of the regenerated digits. Gap junctions and ion channels have been shown to mediate ion flows that control the endogenous voltage gradients which are tightly associated with the regulation of gene expression, cell cycle progression, migration, and other cellular behaviors. Therefore, we postulate that mis-expression of these channels may have disturbed this regulation causing uncoordinated cell behavior which results in morphological defects.

Potassium channel-driven bioelectric signaling regulates metastasis in triple-negative breast cancer

“…To study the effect of K + conductance and change in the RMP on breast cancer cell metastatic potential, we engineered MDA-MB-231 and MDA-MB-468 cell lines to stably express two different types of GFP-tagged K + channels: Kv1.5, a voltage-gated channel (231-Kv1.5 and 468-Kv1.5), and Kir2.1, a constitutively open, inwardly-rectifying channel (231-Kir2.1 and 468-Kir2.1) (Figure 2a). These channels were chosen as a tool to hyperpolarize the RMP, as has been previously demonstrated [28][29][30][31] and compared against their respective negative control expressing a fluorophore alone. The observed nuclear localization of GFP in the Kv1.5 expressing cells (Figure 2a) is due to the Thosea asigna virus 2A (T2A) sequence linking Kv1.5 and histone GFP in the Kv1.5 construct, which during translation initiates ribosomal skipping and as a result the histone GFP is cleaved and shuttled to the nucleus 32 .…”

“…Viruses were produced as described previously 31 . For infections, 1µg of constructs expressing human ion channel coding proteins RFP, Kv1.5-T2A-his-EGFP, or KCNJ2(Kir2.1)-Y242F-EGFP were used.…”

Cell Systems Bioelectricity: How Different Intercellular Gap Junctions Could Regionalize a Multicellular Aggregate

“…In addition, a complementary distributed control based on the cell membrane bioelectricity is also emerging [3][4][5][6][7]. The cell potential V, defined as the electric potential difference between the cell inside and the external microenvironment, regulates the distribution of signaling cations and biochemical messengers.…”

“…Patterns in biology result from the interplay between biochemical [ 1 ] and biomechanical [ 2 ] signals that establish spatio-temporal correlations in multicellular aggregates. In addition, a complementary distributed control based on the cell membrane bioelectricity is also emerging [ 3 , 4 , 5 , 6 , 7 ]. The cell potential V , defined as the electric potential difference between the cell inside and the external microenvironment, regulates the distribution of signaling cations and biochemical messengers.…”

“…At the multicellular scale, two general mechanisms for cells coordination are the extracellular signaling and the intercellular gap junctions. The connexin proteins forming the gap junction channels allow the latter communication by the exchange of biochemical and electrical signals between adjacent cells [ 5 , 7 , 9 , 10 , 11 , 12 , 13 , 14 ]. Different junction types can be identified on the basis of their constituent proteins and distinct voltage-gated conductances.…”