Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14917–14921, December 1996 Neurobiology

Single-channel properties of the nonselective cation conductance induced by neurotensin in dopaminergic neurons (cell culture͞G protein͞ventral tegmental area͞outside–out patch͞neurotensin antagonist)

PEI-YU CHIEN*, RONALD H. FARKAS*, SHIGEHIRO NAKAJIMA†, AND YASUKO NAKAJIMA*‡

Departments of *Anatomy and Cell Biology and †Pharmacology, University of Illinois, College of Medicine, Chicago, IL 60612

Communicated by Susan E. Leeman, Boston University School of Medicine, Boston, MA, October 3, 1996 (received for review June 25, 1996)

ABSTRACT Slow nonselective cation conductances play a described (14) except that 2% (instead of 5%) rat serum was central role in determining the excitability of many neurons, used, and neurons were dissociated with 12 (instead of 20) but heretofore this channel type has not been analyzed at the units͞ml of papain. We recorded from large neurons cultured single-channel level. Neurotensin (NT) excites cultured dopa- for Ϸ2 weeks (soma diameter Ϸ 21 ␮m), Ϸ75% of which are minergic neurons from the ventral tegmental area primarily dopaminergic (9). by increasing such a cation conductance. Using the outside– Recordings were made using the outside–out or cell- out configuration of the patch clamp, we elicited single- attached configuration of the patch clamp (15). Single-channel channel activity of this NT-induced cation channel. Channel currents were recorded on videotape from a List EPC-7 activity was blocked by the nonpeptide NT antagonist amplifier (List Electronic, Darmstadt, Germany) and analyzed SR48692, indicating that the response was mediated by NT by using PCLAMP software (Axon Instruments, Foster City, receptors. The channel opened in both solitary form and in CA) as described (16). NT (10 nM) was applied by pressure bursts. The reversal potential was ؊4.2 ؎ 1.7 mV, and the ejection (0.5 psi) from glass pipettes with a tip diameter of 3–4 ؉ -␮m, placed Ϸ45 ␮m from the patch. Experiments were con ؍ elementary conductance was 31 pS at ؊67 mV with [Na ]o ؉ ؉ ؉ .duced at Ϸ23ЊC. Values are given as mean Ϯ SEM 74 ؍ mM, and [Cs ]i 88 ؍ mM, [Na ]i 5 ؍ mM, [Cs ]o 140 –mM. Thus, the channel was permeable to both Na؉ and Cs؉. The standard pipette solution for whole-cell and outside From these characteristics, it is likely that this channel is out recording contained 144 mM K-D-gluconate, 8.5 mM responsible for the whole-cell current we studied previously. In Na-D-gluconate, 1.5 mM NaCl, 5 mM Hepes⅐KOH, 0.5 mM ,guanosine 5؅-[␥-thio]triphosphate-loaded cells, NT irrevers- EGTA⅐KOH, 0.25 mM CaCl2, 3 mM MgCl2,2mMNa2ATP ibly activated about half of the channel activity, suggesting and 100 ␮MNa3GTP (pH 7.2). When guanosine 5Ј-[␥- that at least part of the response was mediated byaGprotein. thio]triphosphate (GTP[␥S]; 100 ␮M) was used, GTP was Similar channel activity could be induced occasionally in the omitted. A Csϩ-containing internal solution used for deter- cell-attached configuration by applying NT outside the patch mining the reversal potential contained 74 mM CsCl, 80 mM region. NaCl, 5 mM Hepes⅐NaOH, 0.5 mM EGTA⅐NaOH, 0.25 mM CaCl2, 3 mM MgCl2,2mMNa2ATP, and 100 ␮MNa3GTP Neurotensin (NT), a peptide neurotransmitter originally iso- (pH 7.2). lated from bovine hypothalamus (1), excites many central In early experiments, Ca2ϩ was omitted from the standard nervous system neurons (2–6) including dopaminergic neurons external solution because our previous whole-cell data sug- in the ventral tegmental area (VTA) (7–9). The mechanism by gested that the NT-induced conductance is blocked by external which NT excites VTA neurons has been shown to be an Ca2ϩ. This nominally Ca2ϩ-free external solution contained overlapping increase of nonselective cation conductance and 155 mM NaCl, 5 mM KCl, 1.3 mM MgCl2, 5 mM Hepes⅐NaOH decrease of inward rectifier Kϩ conductance (8, 9). (pH 7.4) and had a Ca2ϩ concentration of Ϸ1 ␮M, measured We previously used whole-cell recording to study the nonse- by Ca2ϩ electrode. In later experiments, external Ca2ϩ was lective conductance increased by NT in cultured VTA neurons buffered to 1 ␮M with EGTA. This Ca2ϩ-buffered external ϩ ϩ (9). The conductance was equally permeable to Na ,K , and solution contained 140 mM NaCl, 5 mM KCl, 4.7 mM CaCl2, ϩ Ϫ Cs , but was impermeable to Cl , indicating that it was indeed a 5 mM EGTA⅐NaOH, 1.3 mM MgCl2, and 5 mM Hepes⅐NaOH nonselective cation conductance. Activation of the NT-induced (pH 7.4). To determine the reversal potential, we used a nonselective current did not involve cAMP, cGMP, or internal Csϩ-containing external solution, in which KCl was replaced Ca2ϩ, while the latency of activation was long, indicating the by CsCl. Tetrodotoxin (0.5 ␮M) was added for all recordings. involvement of second messenger(s). Therefore, this NT-induced The membrane potential was corrected for the liquid junction nonselective cation conductance might be a member of a unique potential as measured in reference to a saturated KCl micro- class of slow nonselective cation conductance, different from electrode. ligand-gated (10, 11), cyclic nucleotide-gated (12), and calcium- When analyzing burst time, we used a 3-kHz filter (Ϫ3dbby activated nonselective (13) cation conductances. 8-pole Bessel filter) with 25-kHz digitization, and the level The purpose of this paper was to characterize the single- changes registered if longer than 40 ␮sec. As will be described, channel properties of the nonselective cation conductance the close time histogram was fit by two exponentials with time induced by NT. We also examined the involvement of G constants ␶g,f (short gaps) and ␶g,s (long closings), indicating proteins in the NT response. that the channel opened in bursts, with the long openings interrupted by brief closures. After measuring close times, MATERIALS AND METHODS burst times were determining by analyzing the same data file, but this time a level change from the open state was registered VTA neurons were cultured from 2- to 4-day-old postnatal only when the transition from the open state lasted longer than Long-Evans rats (Charles River Breeding Laboratories) as a critical time (␶c) (17). For the rest of the experiments, the

The publication costs of this article were defrayed in part by page charge Abbreviations: NT, neurotensin; VTA, ventral tegmental area; payment. This article must therefore be hereby marked ‘‘advertisement’’ in GTP[␥S], guanosine 5Ј-[␥ -thio]triphosphate. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed.

14917 Downloaded by guest on September 24, 2021 14918 Neurobiology: Chien et al. Proc. Natl. Acad. Sci. USA 93 (1996)

frequency response was 2 kHz with 20-kHz digitization, and Block by NT Antagonists. The nonpeptide NT antagonist level changes registered if longer than 100 ␮sec. SR48692 was used to characterize the receptor mediating the Npo was calculated by (18): NT response. This antagonist competitively inhibits NT bind- ing to the high-affinity binding site present in the rat mesen- N cephalon (19). NT was first applied to the patch to establish a baseline response. Two hundred seconds later, SR48692 (10 Npo ϭ ͸ ͕nP͑n͖͒ nϭ1 nM) was applied for 5 sec, followed in 15–23 sec by a second application of NT. After another 200 sec, NT was applied for in which N is the number of the channels in the patch, po is the a third time (Fig. 2). Using different patches as controls, the open probability of an individual channel, and P(n)isthe solvent alone (0.001% dimethyl sulfoxide) instead of SR48692 probability of the record staying at a level at which n channels was applied before the second NT application. The results open simultaneously. were summarized in Fig. 2. For control patches (open bars), The chemicals used were NT (Peninsula Laboratories) and the average peak Npo for the first NT response was 0.11 Ϯ 0.03 GTP[␥S] (Sigma). The nonpeptide NT antagonist SR48692 (n ϭ 5). The second NT response decreased to 0.06 Ϯ 0.02 was obtained from Sanofi Recherche (Tolouse, France). because of desensitization. The third response decreased fur- ther to 0.03 Ϯ 0.01. For the NT antagonist experiment (solid bars), the average peak Npo of the first NT response was 0.14 Ϯ RESULTS 0.04 (n ϭ 7), fairly close to the response in control patches. NT-Induced Channel Activity. As previously described (9), After SR48692 was applied, the second NT response was NT induced an inward current in whole-cell recording (Fig. dramatically decreased to 0.007 Ϯ 0.002. Compared with the 1A). The average whole-cell response induced by 10 nM NT control, the difference was significant (P ϭ 0.0045). After washout of the NT antagonist, the third response partially was 240 Ϯ 154 pA (n ϭ 5). NT applied to outside–out recovered, to 0.05 Ϯ 0.02. The single-channel current ampli- membrane patches induced long-lasting channel activity (Fig. tude was not affected by the NT antagonist (data not shown). 1B1). On an expanded time base, Fig. 1B2 shows that there was These results showed that the channel activity was blocked by no channel activity before NT application, while channel the NT antagonist, indicating that NT was acting through NT activity occurred after NT was applied (Fig. 1B3). Fig. 1B4 receptors. shows the change of Npo induced by NT. Npo reached a peak Kinetic Analysis of the Burst Behavior. Kinetic properties of (Ϸ0.1) 25 sec after NT application, then decreased to zero the NT-induced channel were studied in standard external and after Ϸ60 sec. More than half of the outside–out patches internal solutions (Fig. 3). The NT-induced channel opened in responded to NT (28͞52). We also investigated whether the solitary form as well as in bursts, with the long openings nonselective channel could be induced using the cell-attached interrupted by brief closures (Fig. 3A). The close time histo- configuration, with NT (1 ␮M) applied outside the pipette. No gram is shown in Fig. 3B. This histogram was fit by the sum of channel activity was detected in our original experiments (n ϭ two exponentials. The faster component (␶g,f) of close time 32). However, in later experiments a few cell-attached patches represents the brief closures within a burst. The slower com- (4͞22) responded to NT (Fig. 1C). The rest of the experiments ponent (␶g,s) reflects intervals between individual bursts. The were done in the outside–out configuration. average of ␶g,f was 0.34 Ϯ 0.02 ms, and the average of ␶g,s was

FIG. 1. NT (10 nM)-induced channel activity. (A) In whole-cell recording, NT elicited an inward current. Holding potential was Ϫ79 mV. Depolarizing (20 mV) and hyperpolarizing (Ϫ30 mV) command voltages were applied to monitor the conductance. (B1) NT-induced single-channel activity from an outside–out patch held at Ϫ80 mV. On an expanded time base, there was no channel activity before NT application (B2), while single-channel openings appeared after NT was applied (B3). (B4) Npo of the NT response in B1 was calculated at 4-sec intervals. The Npo reached a peak (ϭ0.097) then decreased to zero. (C1) Similar channel activity occasionally could be induced in the cell-attached configuration. Potential was held at 30 mV intracellular side more negative than resting potential. NT was applied outside the patch region, and the patch pipette solution contained standard external solution. On an expanded time base, there was no channel activity before NT application (C2), while single-channel openings appeared after NT was applied (C3). The frequency response was 2 kHz (B2, B3, C2, and C3) and 250 Hz (B1 and C1) for display purposes. Downloaded by guest on September 24, 2021 Neurobiology: Chien et al. Proc. Natl. Acad. Sci. USA 93 (1996) 14919

(n ϭ 8). Based on this value and the average peak inward current (Ϸ2500 pA) induced by NT in whole-cell recordings (9), we have estimated that 1 ␮M NT opened Ϸ900 channels per neuron during the peak response (or Ϸ0.6 channels͞␮m2, assuming the neuron is sphere with an average diameter of 21 ␮m). This channel density is on the same order as that of an inward rectifier K channel (1.3 channels͞␮m2) in frog skeletal muscle (21). Current–Voltage Relation of the Channel. The current– voltage relation of the NT-induced channel activity was first examined in standard internal and external solutions. How- ever, channel activity of unknown origin occurred at depolar- ized potentials, obscuring the NT response. This compelled us to determine the reversal potential of the NT-induced channels using Csϩ in place of Kϩ to suppress Kϩ conductance, and increasing internal Naϩ to shift the reversal potential toward negative potential. An example of single-channel records from a patch held at various membrane potentials is shown in Fig. 4A. Before NT application (Fig. 4A1) there were no channel openings, while the NT-induced channel activity is shown in FIG. 2. Effect of NT antagonist. NT (10 nM) was applied three Fig. 4A2, as well as in Fig. 4A3 on an expanded time base. The times to elicit first, second, and third responses. Either the NT receptor antagonist SR48692 (10 nM in 0.001% dimethyl sulfoxide) or dimethyl relation between amplitude and voltage was plotted in Fig. 4B. sulfoxide alone (0.001%) as control was applied before the 2nd NT On average, the reversal potential was Ϫ4.2 Ϯ 1.7 mV (n ϭ 6), application. In control experiments, NT responses decreased steadily which was between ECs (Ϫ90 mV) and ENa (ϩ13 mV). The because of desensitization. In contrast, in NT antagonist experiments unitary chord conductance (␥) was 31 pS at Ϫ67 mV using the the second NT response was much decreased by SR48692, and the Csϩ-containing solutions. P ϭ We also estimated what the elementary conductance of the ,ء) difference between control and antagonist was very significant 0.0045). After the antagonist was washed away, the third NT response nonselective channel would be in the standard external and showed partial recovery. internal solutions, containing normal Kϩ and Naϩ concentra- tions. From the single-channel amplitude (Ϫ2.85 pA) and the 8.45 Ϯ 1.30 ms (n ϭ 8). Fig. 3C shows the burst time histogram. permeability ratio data from whole-cell experiments (9), the Again, the histogram was fit by two exponentials. The faster elementary chord conductance at Ϫ80 mV would be 36 pS, not time constant (␶b,f) represents the short openings (short bursts) much different from the value measured in the Csϩ-containing while the longer time constant (␶b,s) represents the long bursts. solutions. This value is close to the conductance of cyclic The average of ␶b,f was 0.102 Ϯ 0.013 ms with a weight factor nucleotide-gated channels (20–25 pS in photoreceptor, see ref. (ab,f) of 0.528 Ϯ 0.015, and the average of ␶b,s was 1.12 Ϯ 0.13 12; 40 pS in olfactory epithelium, see ref. 22) and calcium- ms. The average burst time (weighted average of fast and slow activated nonselective channels (20–30 pS, see ref. 13). components) was 0.528 Ϯ 0.046 ms (n ϭ 8). NT-Induced Irreversible Activation in GTP[␥S]. The signal Fig. 3D shows a histogram of burst amplitude that was fit by transduction mechanism of the NT-induced nonselective cat- a single Gaussian distribution. The average single-channel ion channel was characterized by using GTP[␥S], a nonhydro- current at a holding potential of Ϫ80 mV was Ϫ2.85 Ϯ 0.38 pA lyzable GTP analogue. The pipette solution contained either

FIG. 3. Kinetic analysis with standard external and internal solutions. (A) The NT (10 nM)-induced channel opened in solitary form as well as in bursts. (B–D) Analysis of the data shown in record A.(B) Close time histogram. (C) Burst time histogram. (D) Histogram of burst amplitudes; only openings lasting longer than one-half of the average burst time were included (following the procedure of ref. 20). The frequency response was 3 kHz (Ϫ3 db). The potential was held at Ϫ80 mV. Downloaded by guest on September 24, 2021 14920 Neurobiology: Chien et al. Proc. Natl. Acad. Sci. USA 93 (1996)

FIG. 4. Current–voltage relation. Single-channel records from a patch held at various membrane potentials are shown (A1–A3). (A1) No channel opened before NT application. (A2) Channel activity was induced by NT (10 nM). (A3) Segments of A2 on an expanded time base. The current through the NT-induced channels reversed from inward at Ϫ67 mV to outward at 33 mV. The current-voltage relation for the patch in A was plotted in B.Csϩ-containing external and internal solutions were used.

GTP[␥S] (100 ␮M), or, for control, GTP (100 ␮M), and the through a membrane-delimited pathway, similar to muscarinic ϩ time course of Npo after NT application was calculated (Fig. 5). K channels (25–27). Alternatively, other unknown messen- In control, the Npo reached maximum and decreased to almost gers might be involved, but any such messengers are not 0% at 80 sec. In contrast, the Npo of the patch with GTP[␥S] dialyzed out by the pipette solution. reached the peak and then decreased to about 40% of the peak, We occasionally (4͞54) could elicit the nonselective channel where it remained steady. This irreversible component of in the cell-attached configuration when NT was applied out- channel activity indicated that at least some of the NT-induced side the patch region. This might indicated that a ‘‘diffusible’’ nonselective cation channels were linked to a signal transduc- messenger activated these channels. This does not necessarily tion pathway involving G proteins. contradict the apparently local effect of NT in outside–out recordings; the same pathway activated in outside–out patches DISCUSSION might have the mobility to cross the barrier of the recording electrode. On the other hand, the cell attached activity might Comparison of Single-Channel with Whole-Cell Current. represent a distinct, diffusible pathway, independent from the The characteristics of the channel induced by NT were very activity observed in outside–out patches. similar to those of the whole-cell current previously examined. Burst Analysis. The NT-induced channel opened in bursts, Both are mediated by NT receptors, as indicated by inhibition as do ligand-gated (17, 28) and cyclic nucleotide-gated non- by the NT antagonist SR48692. The reversal potential for the selective cation channels (29) (Bursts also occur in inward single-channel current (Ϫ4.2 Ϯ 1.7 mV) was close to the rectifier Kϩ channels; see refs. 16 and 30). The mechanism of reversal potential in the whole cell (Ϫ7 Ϯ 2 mV) using about bursts for the nicotinic acetylcholine receptor channel was the same internal and external solutions. Furthermore, the proposed by Colquhoun and Sakmann (17) to be a long single-channel activity and the whole-cell current had a very opening interrupted by gaps from the channel briefly closing similar time course (Fig. 1). These findings suggest that we several times during a single acetylcholine receptor occupancy. recorded the same channels that produced the NT-induced The bursts we observed could represent the nonselective whole-cell current. cation channel opening and closing several times during a Signal Transduction. About half of the channel activity single messenger binding. On the other hand, bursts can also induced by NT was irreversibly activated by GTP[␥S], suggest- result from divalent ion block as in the case of the N-methyl- ing that at least part of the signal transduction mechanism D-aspartate receptor (31) and cyclic nucleotide-gated nonse- involves G proteins. This agrees with previous work showing lective channels (32). that the cloned NT receptor belongs to the family of G Significance of the Slow Nonselective Cation Channel. Brain protein-coupled receptors (23), and that NT excites VTA neurons constantly receive slow excitatory and slow inhibitory neurons by activating a pertussis toxin-insensitive G protein signals from neighboring neurons, and the interplay of these (8), probably G␣q/11 (24). The portion of the NT-induced signals sets neuronal excitability. Many transmitters, including channel activity that was not irreversibly activated by GTP[␥S] muscarine, substance P, luteinizing hormone-releasing hor- might have been inactivated by an unknown mechanism, or mone, and NT, induce slow excitation in various neurons by possibly this activity might not be G protein-related. dual ionic mechanisms: inhibition of a resting Kϩ conductance The NT-induced channel activity was recorded in more than and activation of a slow nonselective cation conductance (6, 8, half (28͞52) of the outside–out patches. When outside–out 9, 33–36). The transmitter-modulated Kϩ channels (inward patches are formed, soluble second messengers, such as Ca2ϩ, rectifier Kϩ channels) have been studied at the single-channel cAMP, and cGMP are believed to be dialyzed out of the patch. level (16, 30, 37), and several have recently been cloned Thus, the outside–out results suggest that NT induced the (38–40). In contrast, the nonselective cation channels acti- channels locally. NT receptors and the nonselective cation vated by slow transmitters have not, until now, been examined channels we studied might be coupled by a G protein acting at the single-channel level, and the first member of this family Downloaded by guest on September 24, 2021 Neurobiology: Chien et al. Proc. Natl. Acad. Sci. USA 93 (1996) 14921

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