DEVELOPMENT OF BK CHANNELS IN NEOCORTICAL PYRAMIDAL NEURONS

1. Postnatal development of a large conductance Ca2+-activated K+ chan nel(BK channel) was investigated in neocortical infragranular pyramida l neurons with inside-out and outside-out patch-clamp configurations. Neurons were acutely isolated from slices of 1- to 28-day-old rats (P1 -P28) by using a vibrating glass probe after preincubation with low co ncentrations of enzymes. Patch membrane area was estimated by measurin g membrane capacitance. The density, distribution, voltage dependence, Ca2+ sensitivity, kinetics, and pharmacological properties of BK chan nels were examined in neurons from animals of different ages. 2. In so mata, the density of BK channels was 0.056 +/- 0.011/mu m(2) in P1 neu rons and 0.312 + 0.008/mu m(2) in P28 neurons. There was an abrupt inc rease between P5 and P7 at a rate of similar to 0.032/mu m(2)/day. Bef ore P5 and after P7, the density of BK channels also increased but at slower rates. 3. The density of BK channels in proximal apical dendrit es underwent a similar developmental sequence. There was a relatively large increase between P5 and P7 with a rate of similar to 0.021/mu m( 2)/day, and after P7, channel density increased more slowly (similar t o 0.002/mu m(2)/day). In P1 neurons, channel density in apical dendrit es was 0.039 +/- 0.008/mu m(2), which was close to that in somata, whe reas in P28 neurons, channel density (0.134 +/- 0.008/mu m(2)) was les s than one-half of that in somata. 4. The distribution of BK channels was different in immature and mature neurons. In somata of P1 neurons, BK channels were distributed singly without evidence of clustering, w hereas in P28 neurons BK channels were clustered in groups of similar to 4. 5. BK channels in both P1 and P14 neurons showed a steep increas e in the probability of opening (P-o) as intracellular Ca2+ concentrat ion was raised from 50 to 100 nM, especially at positive membrane pote ntials. The Ca2+ dependence, as measured by the [Ca2+](i) that provide d half-maximal P-o at a variety of membrane potentials, was not differ ent in patches from P1 and P14 neurons. On the other hand, the voltage dependence of BK channels shifted during ontogeny such that P-o was l arger at negative potentials in P14 than in P1 neurons. 6. The voltage dependence of P1 BK channels was bimodally distributed with 57% of ch annels exhibiting an ''immature'' pattern consisting of a more positiv e V-1/2, and a smaller change in voltage required to produce an e-fold increase in P-o. Immature type P1 BK channels showed a longer mean cl osed time at negative membrane potentials than either P14 or ''mature' ' P1 BK channels. 7. No postnatal developmental changes in pharmacolog ical properties of BK channels were observed. In both mature and immat ure neurons, BK channels were partially inhibited by 30 or 100 nM char ybdotoxin(ChTX) and fully blocked by 1 mu M ChTX. The IC50 for ChTX wa s 100 nM, indicating that BK channels in neocortical pyramidal neurons are much less sensitive to ChTX than those in muscle cells and sympat hetic ganglion neurons. BK channels were also inhibited by 0.5 mM tetr aethylammonium chloride (TEA) and 50 mu M trifluoperazine. 8. These da ta indicate that functional somatic and dendritic BK channels are inse rted into neuronal membranes during neocortical development, with an e specially rapid increment in density occurring around P5-P7. These cha nges, which occur at a time when other voltage-gated ion channels are known to be increasing in density, contribute to the development of ne ocortical excitability.