Swelling-induced and depolarization-induced C1-channels in normal and cystic fibrosis epithelial cells.

Cl- currents induced by cell swelling were characterized at the whole cell and single-channel levels in primary cultures of normal and cystic fibrosis (CF) epithelial cells and in the T84 cell line. Currents recorded in normal and CF cells were indistinguishable. At 22-24 degrees C with isotonic CsCl in the pipette, initial whole cell outward current density at 100 mV in unswollen cells was 2-4 pA/pF. The current density increased with time during whole cell recording up to 100 pA/pF in isotonic solutions and up to 200 pA/pF in a hypotonic bath, though values typically ranged between 10 and 70 pA/pF. Currents were outwardly rectifying, active at negative voltages, started to inactivate above approximately 40 mV, and were blocked by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS). Single Cl- channels (approximately 50 pS near 0 mV) with an outwardly rectifying current-voltage relation were recorded in cell-attached and outside-out patches from swollen cells. The channels were mostly open at negative voltages and inactivated at positive voltages with a voltage dependence similar to the whole cell currents. Channel activity decreased rapidly (channel rundown) after seal formation. After swelling-induced channel activity had ceased, outwardly rectifying, depolarization-induced Cl- channels (ORDIC channels) were activated in some patches. The swelling-induced and ORDIC single-channel currents were similar, but some consistent differences were observed. ORDIC channels were often closed at resting voltages (-70 to -50 mV), while swelling-induced channels were always open in this voltage range. In addition, ORDIC channels started to inactivate at more positive voltages (approximately 90 vs. approximately 50 mV), rectified more, and had smaller conductances (approximately 25 pS near 0 mV), shorter mean open durations (approximately 70 vs. approximately 350 ms), and more open-channel noise than swelling-induced channels. The two types of currents might arise from separate channel proteins or from a single channel molecule in different states.

Cystic fibrosis, the CFTR, and rectifying Cl- channels.

The human genetic disease cystic fibrosis is caused by a single defective gene on chromosome 7 that codes for a 1480 amino acid protein called the cystic fibrosis transmembrane conductance regulator (CFTR). The defect causes a profound reduction of Cl- permeability in several tissues, which in turn impairs salt absorption and fluid secretion. A 25-80 pS, rectifying Cl- channel has been targeted as the exclusive or primary channel affected in CF. However, we have found no evidence for significant activation or spontaneous activity of this channel in cell-attached patches of normal lymphoblasts or dog tracheal cells. However, in dog tracheal cells, we find lower conductance, linear Cl- channels that are spontaneously active in unstimulated cells and may show increased activity in stimulated cells. Attempts to correlate the expression of mRNA for the CFTR protein in various types of cells with the presence of the rectifying Cl- channel show a lack of correlation: i.e., depolarization-activated rectifying Cl- channesl have been found in excised, inside-out patches from all cell types that we have examined to date, but the CFTR mRNA has so far only been detected in a subset of epithelial cells.

Gating of single non-Shaker A-type potassium channels in larval Drosophila neurons.

The voltage-dependent gating of transient A2-type potassium channels from primary cultures of larval Drosophila central nervous system neurons was studied using whole-cell and single-channel voltage clamp. A2 channels are genetically distinct from the Shaker A1 channels observed in Drosophila muscle, and differ in single-channel conductance, voltage dependence, and gating kinetics. Single A2 channels were recorded and analyzed at -30, -10, +10, and +30 mV. The channels opened in bursts in response to depolarizing steps, with three to four openings per burst and two to three bursts per 480-ms pulse (2.8-ms burst criterion). Mean open durations were in a range of 2-4 ms and mean burst durations in a range of 9-17 ms. With the exception of the first latency distributions, none of the means of the distributions measured showed a consistent trend with voltage. Macroscopic inactivation of both whole-cell A currents and ensemble average currents of single A2 channels was well fitted by a sum of two exponentials. The fast time constants in different cells were in a range of 9-25 ms, and the slow time constants in a range of 60-140 ms. A six-state kinetic model (three closed, one open, two inactivated states) was tested at four command voltages by fitting frequency histograms of open durations, burst durations, burst closed durations, number of openings per burst, and number of bursts per trace. The model provided good fits to these data, as well as to the ensemble averages. With the exception of the rates leading to initial opening, the transitions in the model were largely independent of voltage.

Voltage-gated potassium channels in larval CNS neurons of Drosophila.

The availability of genetic, molecular, and biophysical techniques makes Drosophila an ideal system for the study of ion channel function. We have used the patch-clamp technique to characterize voltage-gated K+ channels in cultured larval Drosophila CNS neurons. Whole-cell currents from different cells vary in current kinetics and magnitude. Most of the cells contain a transient A-type 4-AP-sensitive current. In addition, many cells also have a more slowly inactivating TEA-sensitive component and/or a sustained component. No clear correlation between cell morphology and whole-cell current kinetics was observed. Single-channel analysis in cell-free patches revealed that 3 types of channels, named A2, KD, and K1 can account for the whole-cell currents. None of these channels requires elevated intracellular calcium concentration for activation. The A2 channels have a conductance of 6-8 pS and underlie the whole-cell A current. They turn on rapidly, inactivate in response to depolarizing voltage steps, and are completely inactivated by prepulses to -50 mV. The KD (delayed) channels have a conductance of 10-16 pS and can account, in part, for the more slowly inactivating component of whole-cell current. They have longer open times and activate and inactivate more slowly than the A2 channels. The K1 channels have a slope conductance, measured between 0 and +40 mV, of 20-40 pS. These channels do not inactivate during 500 msec voltage steps and thus can contribute to the sustained component of current. They exhibit complex gating behavior with increased probability of being open at higher voltages. Although the K1 channels are sufficient to account for the noninactivating component of whole-cell current, we have observed several other channel types that have a similar voltage dependence and average kinetics.

Single-channel and genetic analyses reveal two distinct A-type potassium channels in Drosophila.

Whole-cell and single-channel voltage-clamp techniques were used to identify and characterize the channels underlying the fast transient potassium current (A current) in cultured myotubes and neurons of Drosophila. The myotube (A1) and neuronal (A2) channels are distinct, differing in conductance, voltage dependence, and gating kinetics. The myotube currents have a faster and more voltage-dependent macroscopic inactivation rate, a larger steady-state component, and a less negative steady-state inactivation curve than the neuronal currents. The myotube channels have a conductance of 12 to 16 picosiemens, whereas the neuronal channels have a conductance of 5 to 8 picosiemens. In addition, the myotube channel is affected by Shaker mutations, whereas the neuronal channel is not. Together, these data suggest that the two channels are separate molecular structures, the expression of which is controlled, at least in part, by different genes.