Cang C et al. (2014), A non-inactivating high-voltage-activated two-p...

XB-ART-51487

Nat Commun 2014 Sep 26;5:5015. doi: 10.1038/ncomms6015.

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A non-inactivating high-voltage-activated two-pore Na⁺ channel that supports ultra-long action potentials and membrane bistability.

Cang C , Aranda K , Ren D .

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Action potentials (APs) are fundamental cellular electrical signals. The genesis of short APs lasting milliseconds is well understood. Ultra-long APs (ulAPs) lasting seconds to minutes also occur in eukaryotic organisms, but their biological functions and mechanisms of generation are largely unknown. Here, we identify TPC3, a previously uncharacterized member of the two-pore channel protein family, as a new voltage-gated Na(+) channel (NaV) that generates ulAPs, and that establishes membrane potential bistability. Unlike the rapidly inactivating NaVs that generate short APs in neurons, TPC3 has a high activation threshold, activates slowly and does not inactivate-three properties that help generate long-lasting APs and guard the membrane against unintended perturbation. In amphibian oocytes, TPC3 forms a channel similar to channels induced by depolarization and sperm entry into eggs. TPC3 homologues are present in plants and animals, and they may be important for cellular processes and behaviours associated with prolonged membrane depolarization.

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Species referenced: Xenopus laevis
Genes referenced: sh2b2

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	Figure 2. TPC3 forms a high voltage-activated plasma membrane Na+ channel(a–d) Whole-cell currents recorded with symmetrical [Na+] (150 mM) in the bath and pipette from nontransfected HEK293T cells (a), or cells transfected with GFP-tagged drTPC2 (GFP-drTPC2) (b), GFP-drTPC3 (c) or non-tagged drTPC3 (d). (e) Normalized conductance (G/Gmax) – voltage (V) relationships reconstructed from d. (f) Similar to panel c but recorded under asymmetrical [Na+] conditions (150 mM in the bath and 8 mM in the pipette). Left panels show representative traces obtained with a step voltage protocol (shown in a for a–d, and in f). Current (I)-voltage (V) relationships constructed using the current sizes at the end of the voltage steps are in right panels. Data are represented as mean ± s.e.m.
	Figure 3. TPC3 channels do not inactivate(a) Representative whole-cell currents activated by 20-s-long step pulses to +50 and +100 mV (Vh = −70 mV). (b) Average amplitudes of the current peak and those at the end of pulses (20 s). (c) Current amplitudes obtained at the end of pulses normalized to those at the beginning (2 s). (d) Steady-state inactivation of TPC3. Currents were recorded with a test pulse to +100 mV following a pre-pulse of 2 s in duration, at voltages ranging from −100 to +150 mV (10-mV step). (e) Current amplitudes at the end of the test pulses (arrow in d) were plotted against pre-pulse voltages. Whole-cell currents were recorded from HEK293T cells transfected with drTPC3 (non-tagged). GFP-tagged drTPC3 generated similar non-inactivating currents (not shown; see also Fig. 2). Numbers of cells are indicated in parentheses. Data are represented as mean ± s.e.m.
	Figure 4. TPC3 channels are Na+- selectiveWhole-cell currents were recorded from GFP-drTPC3-transfected HEK293T cells. Representative traces (a) and average amplitudes (b) of currents recorded with a pipette solution containing 150 mM NMDG+ and bath containing 150 mM Na+, 150 mM K+, 100 mM Ca2+, or 150 mM NMDG+ as indicated. (c) Representative currents elicited by voltage pulses (illustrated) under bi-ionic condition (150 mM K+ in pipette and 50 mM Na+ in bath). (d) Current sizes obtained from c plotted against test pulse voltages to obtain reversal potential (Erev) for the calculation of relative permeability. Data are represented as mean ± s.e.m.
	Figure 5. TPC3 is insensitive to the NaV blocker TTX but is inhibited by CaV blockersWhole-cell currents were elicited with a test pulse to +100 mV (Vh = −70 mV) and recorded under the symmetric [Na+] (150 mM in the bath and pipette) condition from GFP-drTPC3-transfected HEK293T cells. Inhibitors were added as indicated. Representative currents were recorded in the presence and absence of each drug. (a) Representative currents and (b) average current amplitudes in the presence or absence of TTX. (c) Representative currents and (d) dose response curve for Cd2+. (e) Representative currents and (f) dose response curve for verapamil. (g) Representative currents and (h) dose response curve for nifedipine. Current amplitudes normalized to that obtained without drug were fitted with the Hill equations. Data are represented as mean ± s.e.m.
	Figure 6. TPC3 is insensitive to PI(3,5)P2 and PI(4,5)P2Endolysosomal currents were recorded from GFP-drTPC3-transfected HEK293T cells. Concentrations of PI(3,5)P2 and PI(4,5)P2 are indicated in the figure. (a) Representative current traces and (b) average current amplitudes in the absence (basal) or presence of PI(3,5)P2. (c) Representative current traces and (d) average current amplitudes in the absence or presence of PI(4,5)P2. Data are represented as mean ± s.e.m.
	Figure 7. TPC3 supports ultra-long action potentials and membrane bistabilityRepresentative whole-cell current clamp recordings were performed in HEK293T cells without (a) or with (b–d) GFP-drTPC3 transfection. Depolarizing current injection stimuli (illustrated below each recording with the sizes indicated) were applied. ulAPs were elicited from resting membrane potentials of −50 mV in b and c, or −20 mV in d.
	Figure 8. TPC3-like current in Xenopus oocytesTwo-electrode voltage-clamp recordings were performed with Xenopus oocytes without (a–c) or with (d–f) xlTPC3 RNA injection. (a, b, d, e) Representative currents recorded with a voltage step protocol (−50 to +80 mV with a 10-mV step, Vh = −60 mV, shown in a) before (a, d) and after (b, e) the oocyte was “induced” with a 60-s depolarization of +50 mV. Capacitive transient currents are masked for clarity. (c, f) Average current-voltage relationships. Data are represented as mean ± s.e.m.

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