Three types of K(+) currents in murine osteocyte-like cells (MLO-Y4).
Osteocytes play an important role in signaling within bone. Communication of osteocytes with each other and with bone lining cells may have a function in mineral homeostasis and mechanotransduction. However, very little is known of the expression of ion channels in these cells. Using the whole-cell patch-clamp technique, we have detected three types of K(+) currents in the mouse osteocyte-like cell line MLO-Y4. The most commonly observed current (48% of cells) activated rapidly (20 msec) in response to depolarizing steps from -40 mV and exhibited voltage-dependent inactivation. The current was inhibited by 20 mmol/L tetraethyl ammonium (TEA) and abolished by intracellular 2 mmol/L 4-aminopyridine (4-AP). Biophysical and pharmacological characteristics of the current differed from those of inactivating K(+) currents in osteoblastic cells. In 22% of cells, a slowly activating, voltage-activated current was observed (threshold at 20-30 mV). This current was TEA insensitive, was abolished by intracellular application of 2 mmol/L 4-AP, and was strongly inhibited by apamin, a selective inhibitor of small conductance (SK) Ca(2+)-activated K(+) channels. A third current developed during whole-cell dialysis (37% of cells). This current showed little voltage sensitivity. It was abolished by intracellular application of 2 mmol/L 4-AP, high-extracellular Ba(2+) (108 mmol/L), or by inclusion of ATP in the intracellular solution, but was insensitive to TEA, apamin, Cs(+), and glibenclamide. None of these currents was affected by replacement of chloride with acetate in the bath or pipette salines. Reverse-transcription polymerase chain reaction confirmed the presence of mRNA for the types 1 and 2 SK channels, but message for the large conductance (BK) Ca(2+)-activated K(+) channel was not detected in these cells. Message for the sulphonylurea receptor SUR2, a subunit of glibenclamide-insensitive ATP-dependent K(+) channels (K(ATP)), was also detected, but the glibenclamide-sensitive SUR1 subunit was not. These data are the first descriptions of SK- and ATP-sensitive, glibenclamide-insensitive channels in cells of osteoblastic lineage. Our findings are consistent with a change in K(+) channel expression during differentiation from osteoblast to osteocyte. K(+) channels of osteocytes will contribute to maintenance of the cell membrane potential and thus may participate in mechanosensitivity and osteocyte intercellular communication. In addition, they may be involved in homeostatic maintenance of the extracellular fluid occupying the periosteocytic space.
Gu
Y
Y
School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
Preston
M R
MR
El Haj
A J
AJ
Howl
J D
JD
Publicover
S J
SJ
D015761:4-Aminopyridine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001030:Apamin / Q000494:pharmacology; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D019070:Cell Lineage / Q000502:physiology; D002586:Cesium / Q000493:pharmacokinetics; D002712:Chlorides / Q000494:pharmacology; D003094:Collagen; D017931:DNA Primers; D015870:Gene Expression / Q000502:physiology; D005905:Glyburide / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007004:Hypoglycemic Agents / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D010011:Osteocytes / Q000166:cytology* / Q000502:physiology*; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012965:Sodium Chloride / Q000494:pharmacology; D019789:Tetraethylammonium / Q000494:pharmacology
D002712:Chlorides; D017931:DNA Primers; D007004:Hypoglycemic Agents; D015221:Potassium Channels; D002586:Cesium; D001030:Apamin; D012965:Sodium Chloride; D019789:Tetraethylammonium; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D003094:Collagen; D015761:4-Aminopyridine; D005905:Glyburide