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(Ih Channels) Contributes to Activity-Evoked Neuronal Secretion

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(Ih Channels) Contributes to Activity-Evoked Neuronal Secretion Calcium influx through hyperpolarization-activated cation channels (Ih channels) contributes to activity-evoked neuronal secretion Xiao Yu*†, Kai-Lai Duan*†, Chun-Feng Shang*, Han-Gang Yu‡, and Zhuan Zhou*§ *Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China; and ‡Department of Physiology and Biophysics, New York College of Osteopathic Medicine, New York Institute of Technology, Old Westbury, NY 11568 Edited by Bert Sakmann, Max Planck Institute for Medical Research, Heidelberg, Germany, and approved December 1, 2003 (received for review August 12, 2003) The hyperpolarization-activated cation channels (Ih) play a distinct pore region, indicating that their ion selectivity should be similar. role in rhythmic activities in a variety of tissues, including neurons HCN messenger RNA is widely and nonuniformly expressed in and cardiac cells. In the present study, we investigated whether central neurons (18), photoreceptors (19), dorsal root ganglion .(Ca2؉ can permeate through the hyperpolarization-activated pace- (DRG) neurons and cardiac myocytes (19, 20 maker channels (HCN) expressed in HEK293 cells and Ih channels in Recently, Ih channels in neurons have received increasing dorsal root ganglion (DRG) neurons. Using combined measure- attention because the activation of Ih affects a variety of neural -ments of whole-cell currents and fura-2 Ca2؉ imaging, we found functions including synaptic plasticity. At the crayfish neuro 2؉ that there is a Ca influx in proportion to Ih induced by hyper- muscular junction, activation of Ih channels by cAMP or by ؉ polarization in HEK293 cells. The Ih channel blockers Cs and hyperpolarization induces synaptic facilitation (21). In hip- -ZD7288 inhibit both HCN current and Ca2؉ influx. Measurements of pocampal neurons, the presynaptic mossy fiber long-term po ؉ the fractional Ca2 current showed that it constitutes 0.60 ؎ 0.02% tentiation (LTP) is also dependent on Ih activation (ref. 22, but of the net inward current through HCN4 at ؊120 mV. This fractional see refs. 23 and 24). Biophysical studies have shown that the Ih current is similar to that of the low Ca2؉-permeable AMPA-R channel is permeable only to monovalent cations in physiological (␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) solutions (25), and Ih-mediated membrane depolarization is not channels in Purkinje neurons. In DRG neurons, activation of Ih for responsible for Ih modulation of synaptic plasticity. Thus, it is not s also resulted in a Ca2؉ influx and an elevated action potential- surprising that possible mechanisms for any Ih-mediated events 30 2ϩ induced secretion, as assayed by the increase in membrane capac- are assumed to be downstream from Ca (21, 22). However, this itance. These results suggest a functional significance for I chan- interpretation may need to be revised, considering the possibility h 2ϩ .nels in modulating neuronal secretion by permitting Ca2؉ influx at of Ca influx through the Ih channel itself negative membrane potentials. Using a combined whole-cell patch clamp recording and fluorescence Ca2ϩ imaging method, we show that Ca2ϩ perme- 2ϩ ates through Ih channels. Furthermore, activation of Ih channels s the most important second messenger, Ca controls many causes a marked facilitation of action potential-induced secre- Aphysiological events, such as neurotransmitter release and tion from DRG neurons, as revealed by membrane capacitance muscle contraction (1, 2). Whereas the classic voltage-dependent 2ϩ measurements. Thus, Ca entry through Ih channels may pro- 2ϩ calcium channels provide an important pathway for Ca entry into vide a cellular basis for Ih-mediated events, such as presynaptic neurons (3, 4), many ligand-gated cation channels are also perme- facilitation and LTP. abletoCa2ϩ, providing another pathway for Ca2ϩ influx (5–8). Fractional Ca2ϩ current, the percentage of current carried by Ca2ϩ Materials and Methods in the total current through cation channels, has been determined Heterologous Expression of HCN Channels. Human HCN4 sub- NEUROSCIENCE for nicotinic acetylcholine receptors (nAChRs) (6), for glutamate cloned into HindIII͞XbaI sites in pcDNA1.1͞Amp vector was receptors [N-methyl-D-aspartate receptors (NMDA-Rs) and generously provided by U. B. Kaupp (Forshungszentrum Julich, ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors Germany). HEK293 cells were grown in DMEM, supplemented (AMPA-Rs)] (7, 9), for cyclic nucleotide-gated (CNG) channels with 10% FBS, 100 units͞ml penicillin, and 100 ␮g͞ml strepto- ϩ (8), and for voltage-dependent Ca2 channels (VDCC) (3). The mycin. When cells approached confluence, they were seeded ϩ Ca2 influx through these channels may result in transmitter release into 35-mm dishes and subsequently transfected with the HCN or muscle contraction (e.g., CNG and AMPA-Rs, see refs. 8–10; plasmid by using a calcium phosphate method. HCN4 was VDCC and NMDA-Rs, see refs. 3 and 11; and nAChRs and P2X-R, cotransfected with the GFP to guide selection of cells expressing see refs. 12 and 13). HCN channels. After 48–96 h, transfected cells with green The voltage-dependent hyperpolarization-activated cyclic nu- fluorescence were selected for patch clamp experiments. cleotide-gated (HCN) channels generate a hyperpolarization- activated cation inward current, named Ih (for hyperpolariza- Cell Dissociation. Adrenal chromaffin cells from Wistar rats tion-activated current) in neurons and If (for funny current) in (SLACCAS Inc., Shanghai) were isolated and cultured as de- cardiac cells. Opened by hyperpolarization, these channels are thought to be permeable only to Naϩ and Kϩ ions (14–17). Near ϩ This paper was submitted directly (Track II) to the PNAS office. the membrane resting potential, Ih channels conduct more Na ϩ into and less K out of the cell, generating a net inward current. Abbreviations: NMDA-R, N-methyl-D-aspartate receptor; AMPA-R, ␣-amino-3-hydroxy-5- methyl-4-isoxazolepropionic acid receptor; VDCC, voltage-dependent Ca2ϩ channel; HCN, In rhythmically pacing cells, this inward current contributes to hyperpolarization-activated cyclic nucleotide-gated; DRG, dorsal root ganglion; Cm, mem- the slow depolarization toward the threshold for firing (4). brane capacitance; LTP, long-term potentiation; HEK, human embryonic kidney; AP, action Four pore-forming subunits (HCN1 to -4) of Ih channels have potential. been identified in brain (16, 17). They resemble the voltage-gated †X.Y. and K.-L.D. contributed equally to this work. potassium channel superfamily. HCN channel subunits contain six §To whom correspondence should be addressed at: Institute of Neuroscience, 320 Yue- transmembrane domains (S1–S6), with a pore-forming P region Yang Road, Shanghai 200031, China. E-mail: [email protected]. between S5 and S6 (16). All four HCN subunits have an identical © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0305167101 PNAS ͉ January 27, 2004 ͉ vol. 101 ͉ no. 4 ͉ 1051–1056 Downloaded by guest on September 26, 2021 Table 1. Composition of internal solutions* (in mM) Fura-2 (0.1–1.0 mM) was loaded into the cell via a patch-pipette HEK cells DRG neurons Chromaffin cells in the whole-cell configuration. The fluorescence was sampled at a frequency of 1 Hz (28). NaCl 10 ——Fractional Ca2ϩ current, Pf, is defined as the percentage of KCl 145 150† — Ca2ϩ current in the total current passing through a cation CsCl —— 153 channel (say, IHCN4 in this case). According to the original MgCl2 11 1definition (6), Hepes 5 10 5 ATP-Mg — 4 — pH 7.2 7.2 7.2 ϭ ͵ ͞͵ ϭ ⌬ ͞ ⅐͵ Pf IHCN,Ca dt IHCN dt Fd (fmax IHCN dt), [1] *For fluorescence calibration experiments, 1 mM fura-2 potassium salt was added to internal solutions. For calcium imaging experiments, 0.1 mM fura-2 where IHCN is the HCN4 current, and IHCN,Ca is the proposed salt was added to internal solutions. 2ϩ ⌬ † fractional IHCN4 current carried by Ca . Fd is the change of Fd, For capacitance measurement of DRG neurons, 150 mM KCl was replaced 2ϩ ϩ which is the ‘‘modified Ca -sensitive fura-2 signal’’ immediately with 153 mM CsCl to block K channels. before (FdЈ) and after (FdЉ) the voltage-pulse induced Ca2ϩ ϭ Ϫ ⌬ ϭ ЉϪ Ј influx (3), Fd F340 F380, Fd Fd Fd , and fmax is a 2ϩ scribed (26, 27). Cells were used in the experiments after 2–6 constant, which is determined by measuring Ca influx through days in culture. voltage-gated calcium channels in chromaffin cells under the Ͼ DRG neurons were isolated as described with slight modifi- condition that intracellular fura-2 is sufficiently high ( 0.4 mM) (6). Under physiological conditions, all ions contributing to the cation and used 4–16 h after preparation (28). We used small- ϩ ϩ ␮ current through Ca2 channels are Ca2 (3), or Pf ϭ 100%. From to middle-sized (25–40 m) neurons. ϭ⌬ ͞ ͐ Eq.1, we have fmax Fd ( ICa dt), where ICa is the current The use and care of animals used in this study complied with 2ϩ the guidelines of the Animal Research Advisory Committee at through voltage-gated Ca channels. the Shanghai Institutes of Biological Sciences. To record the time course of fura-2 dialysis, following ref. 6, we used the Ca2ϩ-independent fluorescence signal F360, which ϭ ϩ ␣ Electrophysiology. Ionic currents were studied in the whole-cell can be calculated from F340 and F380. F360 F340 F380, where ␣ is the ‘‘isocoefficient’’ and can be determined by any configuration under voltage-clamp by using an EPC-9 amplifier 2ϩ (HEKA Electronics, Lambrecht͞Pfalz, Germany). For switching experimental recording that shows rapid changes in Ca con- centration. In our setup, ␣ ϭ 0.35. Because F360 is Ca2ϩ external solutions, we used an RCP-2B perfusion system, which independent, it can be used as an indicator of the intracellular has a fast exchange time (100 ms) controlled electronically fura-2 concentration [fura] .
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