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Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh Medical School, Edinburgh, Scotland EH8 9AG, United Kingdom
Address all correspondence and requests for reprints to: Michael J. Shipston, Ph.D., Membrane Biology Group, Department of Biomedical Sciences, University of Edinburgh Medical School, Teviot Place, Edinburgh, Scotland EH8 9AG, United Kingdom. E-mail: mike.shipston{at}ed.ac.uk
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In many neurones and other excitable cells, hyperpolarization-activated cation currents (previously termed Ih, If, or Iq) act to depolarize membrane potential to threshold required for action potential generation (for reviews see Refs. 7, 8). Importantly, these currents are targets for regulation by the signal transduction pathways activated by distinct G protein-linked receptors that enhance cellular excitability (8, 9, 10). Furthermore, the ion channel subunits that underlie these currents have recently been cloned [termed HCN 1 to 4: (7, 11, 12, 13)].
Although preliminary studies have suggested that corticotropes express a hyperpolarization-activated cation current whose activation kinetics are accelerated by stimulation of the CRH pathway (14), the properties and regulation of hyperpolarization-activated cation currents have not been defined in anterior pituitary corticotropes.
To examine whether hyperpolarization-activated cation currents play an important role in corticotrope function we have examined the properties, subunit expression, and regulation of the hyperpolarization-activated cation current in mouse anterior pituitary corticotrope, AtT20 D16:16, cells. AtT20 cells have been widely exploited as a model system to explore the regulation of corticotrope function by CRH and cAMP-mediated signal transduction pathways (1, 2, 3, 4). Our data suggest that mouse AtT20 D16:16 corticotropes express HCN 1 channel subunits that give rise to hyperpolarization-activated cation (Ih) currents that are tonically modulated by the cAMP pathway. However, the activation kinetics of the endogenous Ih current, and lack of effect of Ih blockers on resting membrane potential, suggest that Ih does not play a significant pacemaker role in corticotropes, rather may act as a brake of membrane hyperpolarization.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Electrophysiology
Whole cell currents were recorded in the conventional whole cell
mode of the patch clamp technique at room temperature (21–23 C). The
bath solution (extracellular) unless otherwise stated in the figure
legend contained in mM: 140 NaCl, 5 KCl, 5
MgCl2, 0.1 CaCl2, 10 HEPES,
10 TEA, 1 BaCl2, 20 glucose at pH 7.4 and
containing 0.002 tetrodotoxin. The patch pipette (intracellular)
contained in mM: 140 KCl, 2 MgCl2, 10
HEPES, 30 Glucose, 5 BAPTA
(1,2-bis-o-aminophenoxy)ethane-N,N,N',N'-tetracetic
acid, tetrasodium salt), 1 mM ATP pH 7.3 with
intracellular free calcium
[Ca2+]i buffered to 100
nM. These solutions minimized outward
voltage-dependent potassium currents, including A-type currents, and
inward calcium and sodium currents as well as inwardly rectifying
potassium currents predominant in AtT20 cells (16, 17). Cells were
voltage clamped at -40 mV and voltage protocols applied as described
in respective figure legends protocol with series resistance
compensation of >50%. In some experiments, online leak subtraction
was applied using a p/8 protocol. All traces shown in figures are
representative nonleak subtracted records. Steady-state
hyperpolarization-activated inward currents were stable for >30 min
under these recording conditions. The current density (measured as
pA/pF) of Ih current showed up to a 3-fold
variation between cells. Data presented is from cells that displayed at
least 100 pA (measured at -140 mV) of Ih current
and normalized to the maximal Ih current for each
cell. No significant differences in channel properties or regulation
were observed between cells with different Ih
current densities (not shown). For current clamp experiments, the
extracellular bath solution in mM was: 140 NaCl,
5 KCl, 2 MgCl2, 1 CaCl2, 10
HEPES, 20 glucose at pH 7.4 and containing 0.002 tetrodotoxin.
All data acquisition and voltage protocols were controlled by an
Axopatch 200B amplifier and pCLAMP 6 software (Axon Instruments Inc.,
Foster City, CA). All data were sampled at 5 or 10 kHz and filtered at
2 kHz. Pipettes were manufactured from Garner no. 7052 glass, coated
with Sylgard, with resistance’s of 1–3 M
in physiological saline
after fire polishing. Series resistance was < 8 M before
series resistance compensation.
Data analyses were done using pCLAMP v6.0 (Axon Instruments) and Igor
Pro 3.1 (Wavemetrics Inc., Lake Oswego, NY). The steady-state
activation curve was determined from the amplitude of tail currents
measured immediately after stepping to -140 mV after the respective 1
sec pre-pulse voltage step (-20 to -140 mV) as in Fig. 1A
. Current amplitude (I, expressed
relative to peak value at -140 mV, Imax) was
plotted as a function of the pre-pulse hyperpolarizing potential and
fitted with a Boltzmann function: I =
Imax/(1+ exp(V50 - V)/k),
where V50 is the voltage for half maximal
activation, V is the activation potential, and k is the slope factor
reflecting the voltage dependence of activation. Activation time
constants were determined by fitting a single exponential to the rising
phase of the inward current.
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was determined from tail current
analysis of the I/V relationship of the fully activated current using a
subtraction protocol. Currents were either fully activated by stepping
to -140 mV (protocol I), or depolarized to -20 mV (protocol II) and
then stepped back to different test potentials (-100 to + 40 mV, 20 mV
steps). The peak amplitude of tail currents at the respective test
potentials using protocol II were subtracted from those obtained using
protocol I and I/V relationships constructed. Permeability ratios
(pNa/pK) were calculated from the reversal potential in each ionic
condition using the Goldmann-Hodgkin-Katz equation.
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were as
follows. HCN2 [primers span base pairs 1691–2064 of HCN2 (7, 12, 13)]: forward primer (5'-gAA gAT gTA CTT CAT CCA gCA); reverse primer
(5'-CTg gCC AAg CTC TgC CTg). HCN1: [primers span base pairs 587-1067
of HCN1 (7, 12, 13)]: forward primer (5'-gAT TAT CCA TCC gTA CAg T);
reverse primer (5'-gAg gTC ATA ggT CAT gTg g). HCN3 [primers span base
pairs 1484–1874 of HCN3 (12, 13)]: forward primer (5'-gAA gAT gTA CTT
CAT CCA gCA); reverse primer (5'-CTT TCC ACT gAg TCg AgC T). RT-PCR
products of the correct size (not shown) were also obtained using the
HCN1 primer set spanning residues 1731–2116 of HCN1 (7, 12, 13):
forward (5'-gAA AAT gTA TTT CAT CCA gCA C); reverse (5'-gTT gAg ggC TgT
CAT TTg Ag).
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Statistics
Data are expressed as means ± SEM. Statistical
significance was determined by ANOVA as appropriate. A P
value of less than 0.05 was considered to be significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The amplitude
and rate of activation of the inward current increased with
hyperpolarization being maximal at -140 mV. The activation of
Ih was steeply voltage dependent and was well
fitted by a Boltzmann function with a half-maximal voltage of
activation (V0.5) of -96.2 ± 3.1 mV
(n = 9, Fig. 1B). The rate of current activation was well fitted
by a single exponential with a time constant of 168 ± 13 ms
(n = 9, Fig. 1C) at -140 mV.
The ion selectivity of the current was determined from the
current/voltage relationship (I/V) of tail currents from the fully
activated channel using a subtraction pulse protocol (Fig. 2A). The
reversal potentials of the subtracted tail currents were -35.5 ±
1.0 mV (n = 4) and -23.3 ± 1.4 mV (n = 5) using 5
mM and 25 mM extracellular
K+ respectively (Fig. 2B). The corresponding
relative permeability ratios for Na+ and
K+ (pNa/pK), determined from the
Goldmann-Hodgkin-Katz equation, were 0.24 and 0.25 at 5 mM
and 25 mM KCl, respectively. The reversal potentials
determined from the I/V relationship of the fully activated slowly
activating current are significantly more depolarized than would be
predicted for a pure K+ conductance. The
calculated reversal potential if the slowly activating inward current
was entirely carried by K+ would be -78 and -40
mV at 5 and 25 mM extracellular KCl, respectively,
suggesting that this current is a mixed cation current.
The slowly activating inward current was reversibly blocked (79.1
± 6.9%, n = 5) by 2 mM extracellular
Cs+ and partially, but irreversibly, blocked
(46.6 ± 8.0%, n = 5) by 100 µM ZD 7288 (Fig. 3, A and B) an inhibitor of
Ih in some, but not all, systems (19, 20).
Furthermore, 1 mM extracellular Ba2+
only inhibited the total inward current (including
IKIR) by 17 ± 6.3% (n = 4), and the
inward current was not blocked by millimolar TEA (not shown). Taken
together, the kinetic and pharmacological characteristics of the slowly
activating hyperpolarization-activated inward cation current has
properties similar to that of the hyperpolarization-activated cation
current (Ih) present in many neurones and cardiac
cells (8, 12, 13).
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]. However, RT-PCR products with two
independent primer sets failed to amplify HCN2 or HCN3 subunits in this
cell line (Fig. 4). RT-PCR analysis demonstrated that HCN1 was also
expressed in the adult mouse anterior pituitary gland (not shown).
Regulation of Ih by cAMP in AtT20 cells
In many systems, Ih currents are modulated
by activation of the cAMP pathway by direct binding to the cyclic
nucleotide domain in the HCN subunit itself or via PKA dependent
phosphorylation (8, 10, 12, 13, 21). Furthermore, modulation of the
cAMP cascade in corticotropes represents one of the major signaling
pathways by which neuropeptides regulate corticotrope excitability and
hormone secretion (1, 2, 3, 4).
In preliminary experiments, bath application of the cell permeable cAMP analog 8-CPT-cAMP, at concentrations up to 1 mM, had no significant effect on Ih current properties over a time course of 15 min (mean activation at -100 mV was 0.3 ± 4.2%, n = 8). The lack of effect of 8-CPT-cAMP was not a result of low membrane permeability of this analog as 8-CPT-cAMP is effective in other whole-cell assays of ion channel function in this system (17). Higher concentrations of 8-CPT-cAMP were not possible, as 1 mM was the limit of solubility under the recording conditions used for Ih analysis.
However, in four out of five experiments in which 1 mM cAMP was included in the patch pipette, in individual experiments 10 min diffusion of cAMP into the cell resulted in a small, but reproducible, right shift in V0.5 by 2–4 mV compared with the V0.5 determined immediately after establishment of the whole cell configuration. This small current activation was maximal at -100 mV (mean activation 20.3 ± 3.6%, n = 4; at -130 mV activation was 14.2 ± 4.2%, n = 4) but was not associated with a statistically significant increase in current activation kinetics when comparing the control and cAMP-treated groups. To verify that the effect of cAMP was specific for the cyclic nucleotide analog of AMP parallel experiments were performed with 1 mM AMP in the patch pipette. Over the same time course of cAMP action (10 min) AMP had no significant effect on Ih (mean activation at -100 mV was 0.3 ± 5.4%, n = 4).
In contrast, bath application of the cell permeable cAMP analog
Rp-8-CPT-cAMPS (1 mM), that blocks cAMP binding to cyclic
nucleotide binding domains as well as inhibiting PKA (22), reduced the
amplitude of the inward Ih current (Fig. 5A). The effect of Rp-8-CPT-cAMP was
maintained during continued application of the cAMP analog (not shown).
The inhibitory effect of Rp-8-CPT-cAMP was detectable within 1 min of
bath application and maximal between 3–5 min. The mean inhibition of
the inward current determined at -130 mV was 29.2 ± 4.5% after
10 min (n = 5). This inhibition resulted in a left shift (to more
negative potentials) in the V0.5 of current
activation by 11 mV (Fig. 5B, n = 5) and a significant
(P < 0.01, ANOVA, n = 5) slowing of current
activation at all potentials examined (Fig. 5C). This suggests that at
resting free calcium levels, the endogenous Ih
current is tonically activated by cAMP, and this can be relieved by the
cAMP antagonist Rp-8-CPT-cAMPS. Taken together, these data suggest that
the cAMP intracellular signaling pathway tonically regulates the
endogenous Ih current in AtT20 cells.
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, A–C). The time constant
of activation, determined at -140 mV, was 184.3 ± 11.1 msec
(n = 7) in cells treated with PMA for 10 min compared with
189.6 ± 22.3 ms in the corresponding pretreatment controls (Fig. 6C, n = 7). The V0.5 was -98.8 ± 3.1
mV (n = 7) and -95.1 ± 2.1 mV (n = 7) in PMA-treated
and control cells, respectively (Fig. 6B). Furthermore, treatment of
cells for > 10 min with the protein kinase C inhibitor
bisindolylmaleimide I (100 nM), that blocks PKC activity in
this system (15) had no significant effect on the endogenous
Ih current (not shown).
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To examine whether Ih acts as a brake to
excessive hyperpolarization, we examined the membrane potential
response to hyperpolarizing current injection before and after complete
block of Ih with 2 mM extracellular
CsCl (Fig. 7). Hyperpolarizing current
injection (-40 pA, 1 sec duration) resulted in a transient membrane
hyperpolarization that relaxed to a steady-state level in control cells
(Fig. 7). Bath application of 2 mM CsCl reversibly enhanced
the membrane hyperpolarization to the current injection (Fig. 7, A and
B). The mean steady-state membrane potential determined at 950 ms into
the pulse was -70.0 ± 6.5 mV, n = 5 in control and
-111.4 ± 10.9 mV, n = 5 after bath application of 2
mM CsCl. This data suggests that Ih
acts as a brake on membrane hyperpolarization in AtT20 cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Properties of Ih in AtT20 cells.
The mixed cation hyperpolarization-activated
(Ih) inward current in AtT20 D16:16 cells
displayed characteristics similar to those described in central
neurones, cardiac cells and other endocrine cells termed
Ih, If and
Iq (for reviews see Refs. 7, 8).
First, the slowly activating voltage-dependent inward currents had half maximal voltage of activation and activation time constant values within the ranges typically reported in other systems and for the cloned mouse HCN2 and HCN1 subunits expressed in HEK 293 cells and Xenopus oocytes, respectively (7, 8, 12, 13). Importantly, the inward currents were robust in ‘physiological’ K+ and Na+ gradients, suggesting they contribute a significant inward current during periods of hyperpolarization.
Second, the weak ionic selectivity of the current for K+ relative to Na+ (relative permeability pNa/pK = 0.24) and sensitivity to millimolar external Cs+, but not Ba2+, support a mixed cation-selective current with pharmacology similar to that observed in native tissues as well as with cloned subunits (8, 12, 13). In contrast to the robust inhibition of Ih by low micromolar concentrations of ZD7288, the Ih current in AtT20 cells was only partially inhibited by ZD7288 concentrations up to 100 µM. ZD7288 is a relatively specific inhibitor of Ih in some, but not all, central neurones (19, 20). However, as the potency of ZD7288 on cloned HCN channels has not been reported, and the subunit composition of Ih channels are largely unknown in native tissue the lack of effect of ZD7288 may reflect the HCN subunits expressed in AtT20 cells. Although ZD7288 did not fully inhibit Ih and CsCl, that fully blocks the current, is nonselective in AtT20 cells (23), neither Ih blocker had any significant effect on the resting membrane potential of AtT20 cells. This suggests that Ih does not play an important role in maintenance of the resting membrane potential, but rather may provide a negative feedback mechanism to factors that induce significant hyperpolarization as suggested for other anterior pituitary cells (24). Indeed, complete blockade of Ih with CsCl resulted in an exaggerated membrane hyperpolarization in response to hyperpolarizing current injection. Such a function is further supported by the observation that the half-maximal voltage required for Ih activation in AtT20 cells is approximately 50 mV more negative than the normal resting potential of these cells.
Third, Ih in AtT20 cells was regulated by modulation of cAMP levels in line with the activation of Ih currents by cAMP either by direct binding to the cyclic nucleotide binding domain in the HCN channel pore-forming subunits or via protein kinase A (11, 12, 13, 21). This suggests that Ih may be an important target for regulators of corticotrope function that modulate intracellular cAMP levels; however, as discussed above, this most likely plays a significant role only during conditions of significant hyperpolarization.
Finally, RT-PCR analysis revealed robust expression of HCN1, but not HCN2 or HCN3, channel subunits suggesting that the endogenous Ih current in AtT20 D16:16 corticotropes is composed of HCN1 [also referred to as HAC2 or mBCNG-1: (7, 11, 12, 13)] subunits.
Regulation of Ih in AtT20 D16:16 corticotropes
The two major stimulatory intracellular signal transduction
pathways in anterior pituitary corticotropes are the cAMP and
IP3/protein kinase C cascades. As
Ih currents in other systems are regulated by
activation of these cascades, we addressed whether manipulation of
either pathway resulted in modulation of Ih in
corticotropes. Intracellular perfusion of cAMP resulted in a small but
reproducible enhancement of Ih current with a
shift in half activation potential between 2 and 4 mV, a value similar
to that observed in hippocampal neurones and for HCN1 subunits
expressed in Xenopus oocytes (10, 13). Bath application of the cell
permeable cAMP antagonist, Rp-8-CPT-cAMP resulted in a rapid (< 1 min)
inhibition of Ih with a left shift of
V0.5 by > 10 mV with a corresponding
significant slowing of activation. Rp-8-CPT-cAMP antagonizes cAMP
binding to cyclic nucleotide binding domains in other cyclic nucleotide
regulated ion channels (22) as well as blocking protein kinase A
activation. The rapidity of onset of Rp-8-CPT-cAMP action would suggest
that a protein phosphorylation dependent mechanism is not involved in
the action of cAMP. In further support of a phosphorylation-independent
action of cAMP, blockade of serine threonine phosphatase activity by
100 nM okadaic acid had no significant effect on
Ih (not shown).
As Rp-8-CPT-cAMP significantly attenuated Ih under basal recording conditions, these data would suggest that Ih is tonically activated by cAMP at near basal intracellular calcium concentrations in corticotropes. Thus agents that elevate (e.g. CRF) or inhibit (e.g. somatostatin) cAMP levels in corticotropes may regulate Ih directly via cAMP. Indeed, preliminary data in rat corticotropes suggests that CRF modulates Ih (14).
Previous studies in anterior pituitary lactotropes and somatotropes suggested that Ih was not activated by cAMP (24). However, no inhibitors of cAMP were used, and intracellular free calcium was buffered to below 10 nM in these latter studies. As anterior pituitary cells express calcium-regulated adenylate cyclases and Ih can be regulated via calcium dependent regulation of adenylate cyclase (9), and hence cAMP levels in central neurones, the regulation of Ih by cAMP in other pituitary cell types cannot be excluded.
Although activation of the protein kinase C pathway has been shown to regulate Ih currents in some systems (8), activation (using maximally effective concentrations of PMA: (15) or inhibition (using maximally effective concentrations of BIS: (15) of protein kinase C activity in AtT20 D16:16 corticotropes had no significant effect on Ih activation. Thus, as for most systems investigated Ih does not appear to be a major target for regulation by the protein kinase C pathway.
Role for Ih in corticotropes
Ih has been proposed to underlie the
pacemaker depolarization in pacemaker cells, determine the resting
membrane potential in neurones and act as a mechanism to limit
hyperpolarization in other systems (8, 24). Ih is
unlikely to play a significant role in pacemaker activity or contribute
significantly to resting membrane potential in AtT20 corticotropes as
the resting membrane potential rarely hyperpolarizes to within the
activation range (<-65 mV) required for Ih
activation. Thus Ih is unlikely to play a
significant role in the depolarizing potentials from rest leading to
calcium-dependent action potential generation or play a major role in
setting the resting membrane potential. Rather, as proposed for other
anterior pituitary cell types, Ih may serve as a
brake of excessive hyperpolarization in response to hyperpolarizing
stimuli (24). Rat and human corticotropes in primary culture have
similar resting membrane potentials to AtT20 corticotropes (4).
However, as different HCN subunits can confer distinct activation and
regulatory properties to endogenous Ih currents,
analysis of HCN subunit expression and Ih
properties in rat and human corticotropes is required to determine
whether Ih plays a similar role in these
systems.
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Acknowledgments |
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Footnotes |
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Received January 19, 2000.
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References |
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2-adrenoceptor that blocks coupling to
potassium but not calcium currents. Science 257:977–980This article has been cited by other articles:
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