ISSN 1990-7478, Biochemistry (Moscow) Supplement Series A: Membrane and Cell Biology, 2009, Vol. 3, No. 3, pp. 254–260. © Pleiades Publishing, Ltd., 2009. Original Russian Text © R.A. Romanov, Y.E. Yatzenko, N.V. Kabanova, M.F. Bystrova, S.S. Kolesnikov, 2009, published in Biologicheskie Membrany, 2009, Vol. 26, No. 4, pp. 258–264. 254 1 The population of taste bud cells is heterogeneous and includes morphologically distinct spindle-like taste cells of three types, type I to type III, which serve the chemosensory function [1–3]. Previously, we have demonstrated that taste cells of the particular type are different in their electrophysiological characteristics [4, 5]. For example, voltage-gated (VG) Na + channels are functional in taste cells of the type II and type III but not in type I cells [4, 6], while activity of VG Ca 2+ channels is solely specific for taste cells of the type III [4, 6, 7]. In taste cells of the type I and type III, VG outward cur- rents are predominantly mediated by K + channels [4, 6]. In contrast, poorly selective cation channels, which are most likely formed by channel proteins from the connexin family, are largely responsible for VG out- ward currents in type II cells [8, 9]. Thus, electrophy- siological profiling provides the reliable basis for unambiguous identification of individual taste cells in physiological experiments [4, 5], wherein morphologi- cal criteria are poorly applicable. Here we aimed to characterize certain properties of VG Ca 2+ channels operative in taste cells of the type III. EXPERIMENTAL Electrophysiology . All experiments were con- ducted using individual taste cells isolated from cir- cumvallate papilla of the mice (6–12-week old) described previously [10]. Electrical activity of taste cells was analyzed with the perforated patch clamp approach using an Axopatch 200B amplifier, a Digi- Data 1322A interface, and the pClamp8 software (all 1 The article was translated by the authors. from MDS Analytical Technologies). The recording chamber and the perfusion system have been described previously [11]. Recording pipettes were filled with a solution containing (in mM): 140 KCl/CsCl, 1 MgCl 2 , 0.5 EGTA, 10 HEPES-CsOH, pH 7.4, and 400 μg/ml amphotericin B. The basic bath solution contained (in mM): 140 NaCl, 2.5 KCl, 1 CaCl 2 , 1 MgCl 2 , 10 HEPES-NaOH (pH 7.4), and 5 glucose. All chemicals were from Sigma-Aldrich. Experiments were per- formed at 23–25°ë. Photometry . Calcium transients in taste cells were visualized with an ICCD camera (IC-200; Photon Tech- nology International, USA) using an Axioscope-2 microscope and a water-immersion objective (Achro- plan 40; NA = 0.8; Zeiss, Germany). For Ca 2+ imaging, taste cells were loaded with 2–4 μM Fluo-4AM for 35 min in the presence of pluronic (0.02%; both from Invitrogen, USA) at room temperature. Fluo-4 fluores- cence was excited with a light emitting diode (Luxion, USA) at 480 ± 10 nm [12]. Cell emission was filtered at 535 ± 20 nm (Chroma, USA), and sequential fluores- cence images were acquired every 0.5–2 s with Work- bench 5.2 software (INDEC Biosystems, USA). Molecular biology. Total RNA was isolated from mouse circumvallate papilla and retina using RNeasy Mini kit (Qiagen, USA), following the protocol of the manufacturer. First-strand cDNA was synthesized using SuperScript III reverse transcriptase (Invitrogen) and oligo(dT) primer. For PCR amplification of cDNA encoding Ca v 1.4, three sets of gene-specific primers were used (5’-TCTTTGCCATGCTAACTGT and 5’- TCTGAAAGAGTGAGGAGG; 5’-GCCCTCCT- CACTGTCTTT and 5’-GCTGAAAGCACGT- Voltage-Gated Ca 2+ Channels in Type III Taste Cells 1 R. A. Romanov, Y. E. Yatzenko, N. V. Kabanova, M. F. Bystrova, and S. S. Kolesnikov Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow oblast, 142290 Russia; e-mail: cheglok@rambler.ru Received February 19, 2009 Abstract—In the mammalian taste bud, the heterogeneous cell population includes three morphologically dis- tinct types of cells, type I to type III, which are also different in their electrophysiological features. Particularly, voltage-gated (VG) Ca 2+ channels are functional solely in taste cells of the type III. These channels were studied here with external Ba 2+ ions as current carriers. It was specifically shown that VG Ba 2+ currents were almost completely blockable with nifedipine as well as with ionic blockers, such as Cd 2+ , Ni 2+ , and Co 2+ . Kinetic prop- erties of VG Ba 2+ currents in type III cells and their sensitivity to the blockers indicated that these currents were largely mediated by VG Ca 2+ channels of the L-type. The expression of genes, which encode pore-forming α1-subunits of Ca 2+ channels, was analyzed using methods of molecular biology. Among four genes encoding L-type Ca 2+ channel α1-subunits (Ca v 1.1–Ca v 1.4), the expression of Ca v 1.2 was demonstrated in taste cells. Key words: taste cell, voltage-gated Ca 2+ channel, dihydropyridine DOI: 10.1134/S1990747809030039