Nav1.3 and FGF14 are primary determinants of the TTX-sensitive sodium current in mouse adrenal chromaffin cells

Adrenal chromaffin cells (CCs) in rodents express rapidly inactivating, tetrodotoxin (TTX)-sensitive sodium channels. The resulting current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate via two independent pathways which differ in their time course of recovery. One subpopulation recovers with time constants similar to traditional fast inactivation and the other ∼10-fold slower, but both pathways can act within a single homogenous population of channels. Here, we use Nav1.3 KO mice to probe the properties and molecular components of Nav current in CCs. We find that the absence of Nav1.3 abolishes all Nav current in about half of CCs examined, while a small, fast inactivating Nav current is still observed in the rest. To probe possible molecular components underlying slow recovery from inactivation, we used mice null for fibroblast growth factor homology factor 14 (FGF14). In these cells, the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. The use dependence of Nav current reduction during trains of stimuli in WT cells is completely abolished in FGF14 KO mice, directly demonstrating a role for slow recovery from inactivation in determining Nav current availability. Our results indicate that FGF14-mediated inactivation is the major determinant defining use-dependent changes in Nav availability in CCs. These results establish that Nav1.3, like other Nav isoforms, can also partner with FGF subunits, strongly regulating Nav channel function.

Download Full-text

Nav1.3 and fibroblast growth factor homologous factor 14 are primary determinants of the TTX-sensitive sodium current in mouse adrenal chromaffin cells

10.1101/2020.10.31.363416 ◽

2020 ◽

Author(s):

P.L. Martinez-Espinosa ◽

C. Yang ◽

X.M. Xia ◽

C.J. Lingle

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Chromaffin Cells ◽

Slow Component ◽

Sodium Current ◽

Fibroblast Growth ◽

Adrenal Chromaffin Cells ◽

Fast Inactivation ◽

Recovery From Inactivation ◽

Adrenal Chromaffin

AbstractAdrenal chromaffin cells (CCs) in rodents express a rapidly inactivating, TTX-sensitive sodium current. The current has generally been attributed to Nav1.7, although a possible role for Nav1.3 has also been suggested. Nav channels in rat CCs rapidly inactivate into two separable pathways, which differ in their time course of recovery from inactivation. One population recovers with time constants similar to traditional fast inactivation and the other about 10-fold slower. Inactivation properties suggest that the two pathways result from a single homogenous population of channels. Here we probe the properties and molecular components of the Nav current present in mouse CCs. We first confirm that functional properties of Nav current in rat and mouse cells are generally similar in terms of activation range, steady-state inactivation, and dual pathway fast inactivation. The results then show that all inward Nav current is absent in CCs from Nav1.3 KO mice. Subsequently, in a mouse with KO of fibroblast growth factor homology factor 14 (FGF14), we find that the slow component of recovery from fast inactivation is completely absent in most CCs, with no change in the time constant of fast recovery. Experiments probing the use-dependence of Nav current diminution between WT and FGF14 KO mice directly demonstrate a role of slow recovery from inactivation in determination of Nav current availability. Overall, the results indicate that the FGF14-mediated inactivation is the major determinant in defining use-dependent changes in Nav availability in CCs. We also consider the potential impact that inactivating FGF’s with different recovery kinetics can exert on differential use-dependent changes in Nav availability.

Download Full-text

Fast inactivation of Na+ current in rat adrenal chromaffin cells involves two independent inactivation pathways

10.1101/2020.10.31.363341 ◽

2020 ◽

Cited By ~ 1

Author(s):

Pedro L. Martinez-Espinosa ◽

Alan Neely ◽

Jiuping Ding ◽

Christopher J. Lingle

Keyword(s):

Chromaffin Cells ◽

Time Course ◽

Adrenal Chromaffin Cells ◽

Fast Inactivation ◽

Fast Recovery ◽

Slow Recovery ◽

Recovery Processes ◽

Voltage Dependent ◽

Adrenal Chromaffin ◽

Recovery Pathways

AbstractVoltage-dependent sodium (Nav) current in adrenal chromaffin cells (CCs) is rapidly inactivating and TTX-sensitive. The fractional availability of CC Nav current has been implicated in regulation of action potential (AP) frequency and the occurrence of slow-wave burst firing. To ascertain whether features of CC Nav inactivation might influence AP firing, we recorded Nav current in rat CCs, primarily from adrenal medullary slices. A key feature of CC Nav current is that recovery from inactivation, even following brief (5 ms) inactivation steps, exhibits two exponential components of generally similar amplitude. Variations of standard paired pulse protocols support the view that entry into the fast and slower recovery processes result from largely independent, competing inactivation pathways, both of which occur with similar onset times at depolarizing potentials. Over voltages from −120 to −80 mV, faster recovery varies from ~3 to 30 ms, while slower recovery from about 50-400 ms. At strong activation voltages (+0 mV and more positive), the relative entry into slow or fast recovery pathways is similar and independent of voltage. Trains of brief inactivating steps result in cumulative increases in the slower recovery fraction. This supports idea that brief recovery intervals preferentially allow recovery of channels from fast recovery pathways, thereby increasing the fraction of channels in the slow recovery pathway with each subsequent inactivation step. This provides a mechanism whereby differential rates of recovery produce use-dependent accumulation in slower recovery pathways. Consistent with use-dependent accumulation of channels in slow recovery pathways, repetitive AP clamp waveforms at 1-10 Hz frequencies reduce Nav availability to 10-20% of initial amplitude dependent on holding potential. The results indicate that there are two distinct pathways of fast inactivation, one that leads to normal fast recovery and the other with a slower time course, which together are well-suited to mediate use-dependent changes in Nav availability.

Download Full-text

Fast inactivation of Nav current in rat adrenal chromaffin cells involves two independent inactivation pathways

The Journal of General Physiology ◽

10.1085/jgp.202012784 ◽

2021 ◽

Vol 153 (4) ◽

Author(s):

Pedro L. Martinez-Espinosa ◽

Alan Neely ◽

Jiuping Ding ◽

Christopher J. Lingle

Keyword(s):

Chromaffin Cells ◽

Adrenal Chromaffin Cells ◽

Fast Inactivation ◽

Fast Recovery ◽

Slow Recovery ◽

Recovery Processes ◽

Voltage Dependent ◽

Dual Pathway ◽

Adrenal Chromaffin ◽

Recovery Pathways

Voltage-dependent sodium (Nav) current in adrenal chromaffin cells (CCs) is rapidly inactivating and tetrodotoxin (TTX)–sensitive. The fractional availability of CC Nav current has been implicated in regulation of action potential (AP) frequency and the occurrence of slow-wave burst firing. Here, through recordings of Nav current in rat CCs, primarily in adrenal medullary slices, we describe unique inactivation properties of CC Nav inactivation that help define AP firing rates in CCs. The key feature of CC Nav current is that recovery from inactivation, even following brief (5 ms) inactivation steps, exhibits two exponential components of similar amplitude. Various paired pulse protocols show that entry into the fast and slower recovery processes result from largely independent competing inactivation pathways, each of which occurs with similar onset times at depolarizing potentials. Over voltages from −120 to −80 mV, faster recovery varies from ∼3 to 30 ms, while slower recovery varies from ∼50 to 400 ms. With strong depolarization (above −10 mV), the relative entry into slow or fast recovery pathways is similar and independent of voltage. Trains of short depolarizations favor recovery from fast recovery pathways and result in cumulative increases in the slow recovery fraction. Dual-pathway fast inactivation, by promoting use-dependent accumulation in slow recovery pathways, dynamically regulates Nav availability. Consistent with this finding, repetitive AP clamp waveforms at 1–10 Hz frequencies reduce Nav availability 80–90%, depending on holding potential. These results indicate that there are two distinct pathways of fast inactivation, one leading to conventional fast recovery and the other to slower recovery, which together are well-suited to mediate use-dependent changes in Nav availability.

Download Full-text

Distinct effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis.

The Journal of Cell Biology ◽

10.1083/jcb.130.5.1063 ◽

1995 ◽

Vol 130 (5) ◽

pp. 1063-1070 ◽

Cited By ~ 132

Author(s):

L H Chamberlain ◽

D Roth ◽

A Morgan ◽

R D Burgoyne

Keyword(s):

Membrane Fusion ◽

Chromaffin Cells ◽

Time Course ◽

Mechanisms Of Action ◽

Catecholamine Secretion ◽

Adrenal Chromaffin Cells ◽

Regulated Exocytosis ◽

Distinct Stage ◽

Adrenal Chromaffin

We have used stage-specific assays for MgATP-dependent priming and for Ca(2+)-activated triggering in the absence of free MgATP to examine the effects of alpha-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca(2+)-dependent increase in catecholamine secretion. Both alpha-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of alpha-SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca(2+)-dependency, time course and extent of stimulation and their effects were additive. alpha-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that alpha-SNAP acts in an early MgATP-requiring stage and not in the late Ca(2+)-triggered steps immediately prior to membrane fusion as previously suggested.

Download Full-text

Time course of release of catecholamine and other granular contents from perifused adrenal chromaffin cells of guinea-pig.

The Journal of Physiology ◽

10.1113/jphysiol.1983.sp014798 ◽

1983 ◽

Vol 341 (1) ◽

pp. 153-167 ◽

Cited By ~ 18

Author(s):

S Ito

Keyword(s):

Guinea Pig ◽

Chromaffin Cells ◽

Time Course ◽

Adrenal Chromaffin Cells ◽

Adrenal Chromaffin

Download Full-text

Fast inactivation of Nav1.3 channels by FGF14 proteins: An unconventional way to regulate the slow firing of adrenal chromaffin cells

The Journal of General Physiology ◽

10.1085/jgp.202112879 ◽

2021 ◽

Vol 153 (5) ◽

Author(s):

Emilio Carbone

Keyword(s):

Chromaffin Cells ◽

Adrenal Chromaffin Cells ◽

Fast Inactivation ◽

New Findings ◽

Inactivation Gating ◽

Adrenal Chromaffin ◽

Channel Availability

Using Nav1.3 and FGF14 KO mice, Martinez-Espinosa et al. provide new findings on how intracellular FGF14 proteins interfere with the endogenous fast inactivation gating and regulate the “long-term inactivation” of Nav1.3 channels that sets Nav channel availability and spike adaptation during sustained stimulation in adrenal chromaffin cells.

Download Full-text

Morphological Observations on the Granule Types in Rabbit Extra-Adrenal Chromaffin Cells.

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100115213 ◽

1975 ◽

Vol 33 ◽

pp. 318-319

Author(s):

Joe A. Mascorro ◽

Robert D. Yates

Keyword(s):

Chromaffin Cells ◽

Sodium Phosphate ◽

Ganglion Cells ◽

Ultrastructural Level ◽

Osmic Acid ◽

Adrenal Chromaffin Cells ◽

Potassium Iodate ◽

Perfusion Fluid ◽

New Zealand White Rabbits ◽

Adrenal Chromaffin

Extra-adrenal chromaffin organs (abdominal paraganglia) constitute rich sources of catecholamines. It is believed that these bodies contain norepinephrine exclusively. However, the present workers recently observed epinephrine type granules in para- ganglion cells. This report investigates catecholamine containing granules in rabbit paraganglia at the ultrastructural level.New Zealand white rabbits (150-170 grams) were anesthetized with 50 mg/kg Nembutal (IP) and perfused with 3% glutaraldehyde buffered with 0.2M sodium phosphate, pH 7.3. The retroperitoneal tissue blocks were removed and placed in perfusion fluid for 4 hours. The abdominal paraganglia were dissected from the blocks, diced, washed in phosphate buffer and fixed in 1% osmic acid buffered with phosphate. In other animals, the glutaraldehyde perfused tissue blocks were immersed for 1 hour in 3% glutaraldehyde/2.5% potassium iodate buffered as before. The paraganglia were then diced, separated into two vials and washed in the buffer. A portion of this tissue received osmic acid fixation.

Download Full-text