title: Two-pore domain K⁺ channels regulate membrane potential of isolated human articular chondrocytes.
abstract: Potassium channels that regulate resting membrane potential (RMP) of human articular chondrocytes (HACs) of the tibial joint maintained in short-term (0-3 days) non-confluent cell culture were studied using patch-clamp techniques. Quantitative PCR showed that transcripts of genes for two-pore domain K(+) channels (KCNK1, KCNK5 and KCNK6), and 'BK' Ca(2+)-activated K(+) channels (KCNMA1) were abundantly expressed. Immunocytological methods detected α-subunits for BK and K(2p)5.1 (TASK-2) K(+) channels. Electrophysiological recordings identified three distinct K(+) currents in isolated HACs: (i) a voltage- and time-dependent 'delayed rectifier', blocked by 100 nM α-dendrotoxin, (ii) a large 'noisy' voltage-dependent current that was blocked by low concentrations of tetraethylammonium (TEA; 50% blocking dose = 0.15 mM) and iberiotoxin (52% block, 100 nM) and (iii) a voltage-independent 'background' K(+) current that was blocked by acidic pH (5.5-6), was increased by alkaline pH (8.5), and was not blocked by TEA, but was blocked by the local anaesthetic bupivacaine (0.25 mM). The RMP of isolated HACs was very slightly affected by 5 mM TEA, which was sufficient to block both voltage-dependent K(+) currents, suggesting that these currents probably contributed little to maintaining RMP under 'resting' conditions (i.e. low internal [Ca(2+)]). Increases in external K(+) concentration depolarized HACs by 30 mV in response to a 10-fold increase in [K(+)], indicating a significant but not exclusive role for K(+) current in determining RMP. Increases in external [K(+)] in voltage-clamped HACs revealed a voltage-independent K(+) current whose inward current magnitude increased with external [K(+)]. Block of this current by bupivacaine (0.25-1 mM) in 5 and 25 mM external [K(+)] resulted in a large (8-25 mV) depolarization of RMP. The biophysical and pharmacological properties of the background K(+) current, together with expression of mRNA and α-subunit protein for TASK-2, strongly suggest that these two-pore domain K(+) channels contribute significantly to stabilizing the RMP of HACs.
authors.affiliation: Roger Jackson Centre for Health and Wellness Research, Faculty of Kinesiology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Robert B
authors.initials: RB
authors.lastname: Clark
authors.forename: Colleen
authors.initials: C
authors.lastname: Kondo
authors.forename: Darrell D
authors.initials: DD
authors.lastname: Belke
authors.forename: Wayne R
authors.initials: WR
authors.lastname: Giles
mesh_terms: D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D004546:Elapid Venoms / Q000494:pharmacology; D006801:Humans; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000235:genetics / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D011188:Potassium / Q000502:physiology; D024683:Potassium Channels, Tandem Pore Domain / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D019789:Tetraethylammonium / Q000494:pharmacology; D013977:Tibia
chemical_list: D002121:Calcium Channel Blockers; D004546:Elapid Venoms; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D010455:Peptides; D024683:Potassium Channels, Tandem Pore Domain; D012333:RNA, Messenger; D019789:Tetraethylammonium; C020273:dendrotoxin; C064719:iberiotoxin; D011188:Potassium