Cao-sensing receptor (CaR)-mediated activation of K+ channels is blunted in CaR gene-deficient mouse neurons.

The extracellular Ca2+ (Cao)-sensing receptor (CaR) is expressed in hippocampus and other brain regions, suggesting that it could mediate some of the well recognized but poorly understood direct actions of Cao on neuronal function. This study presents evidence that the CaR is functionally coupled to Ca(2+)-activated K+ channels. The effects of CaR agonists on these channels in neurons from wild type (WT) and CaR-deficient (CaR -/-) mice were compared. Neomycin (100 mM) and elevation of Cao from 0.5 to 3 mM significantly increased the probability of channel opening (Po) in neurons from WT but not in those from CaR -/- mice. Thus the CaR activates neuronal K+ channels and could potentially inhibit neuronal excitability and neurotransmission via membrane repolarization.

A flickery block of a K+ channel mediated by extracellular Ca2+ and other agonists of the Ca2+-sensing receptors in dispersed bovine parathyroid cells.

Single K+ channel activities in parathyroid cells were studied using the patch-clamp technique. A K+ channel modulated by external Ca2+ (Ca2+o) was identified. This channel had a unitary conductance of 109pS at 150 mM K+o in the pipette solution. An increase in Ca2+o from 0.5-0.75 to 2-3 mM induced a flickery partial block of the channel over a wide voltage range. The mechanism of channel blockade included a significant increase in the number of closings per burst and a reduction of the mean open times. Addition of other divalent and polyvalent agonists of the Ca2+-sensing receptor (CaR) induced a similar channel blockade. With its typical characteristics and flickery block by CaR agonists, this channel differs from previously described types of K+ channels. It is probably strongly coupled to the CaR and may contribute to the depolarization of parathyroid cells which is known to occur at elevated levels of Ca2+o.

Intracellular Ca(2+)-activated K+ channels modulated by variations in extracellular Ca2+ in dispersed bovine parathyroid cells.

The modulation of K+ channels by Ca2+ may have important functional implications in parathyroid cells, since in most endocrine cells they control membrane voltage regulating Ca2+ influx and hormone secretion. To characterize specific channel mechanisms regulating membrane voltage in parathyroid cells, the patch-clamp technique was used to determine the activities of K+ channels at different levels of intracellular Ca2+ concentration (Ca2+i) associated with changes in extracellular Ca2+ concentration (Ca2+o). This study shows that the membranes of dispersed bovine parathyroid cells contain a K+ channel that is activated by elevated Ca2+o through an indirect mechanism (i.e. exposure of the entire cell to high Ca2+o activates the channel despite a low Ca2+ concentration within the pipette solution on the external side of the channel under study). This K+ channel has a unitary conductance of 191 pS and is highly selective for K+, similar to the so-called maxi type of Ca(2+)-activated K+ channel previously defined in a number of other cell types. Like the latter channel, the activity of this channel in excised patches from parathyroid cells is markedly increased when an EGTA-containing buffer on the cytoplasmic face of the membrane is replaced with one containing 0.5 microM Ca2+. Changes in Ca2+ on the intracellular side of the membrane also shift the level of voltage necessary for half-maximal activation of the channel from 103 mV at 0.1 microM Ca2+ to 79 mV and 54 mV at 0.25 and 0.5 microM Ca2+, respectively. When similar studies were carried out using cell-attached patches on parathyroid cells exposed to 0.5, 1.5, or 2.0 mM Ca2+o, the values for half-maximal activation were approximately 105, 56, and 29 mV, respectively. The latter result suggests that in intact parathyroid cells, the channel is exposed to Ca2+i concentrations of about 0.15-0.2, 0.4 and 0.6-0.7 microM at these three extracellular Ca2+ concentrations, values that are in excellent agreement with those previously measured using Ca(2+)-sensitive fluorescent dyes. Thus, parathyroid cells express a maxi type of Ca(2+)-activated K+ channel that is indirectly regulated by Ca2+o, presumably through concomitant changes in Ca2+i. The latter may limit the extent of the cellular depolarization produced in response to elevated Ca2+o in this cell type.

Ca2+ channels from brain microsomal membranes reconstituted in patch-clamped bilayers.

Single Ca2+ channels from brain microsomal membranes were reconstituted in bilayers made at the tips of patch-clamp micropipettes. The single-channel conductance was defined to be 107 pS in 50 mM Ca2+. The channel activity was stimulated by nucleotides and inositol 1,4,5-trisphosphate (Ins-P3), and was inhibited by ruthenium red. Na+ added asymmetrically to the membrane bilayer induced an increase in the Ca2+-channel activity. The described characteristics of these Ca2+ channels suggest that they may be responsible for the Ca2+ transport across the membranes of the endoplasmic reticulum system triggering and modulating various neurosecretory and excitatory processes in nerve cells.

Changes in calcium channel activity in membranes from cis-diammine-dichloroplatinum(II)-resistant and -sensitive L1210 cells.

Ca2+ channels from lipid and proteolipid fractions of cisplatin-sensitive and cisplatin-resistant cells were reconstituted and characterized in bilayer lipid membranes formed at the tips of patch-clamp micropipets. The characteristics of the Ca2+ channels were typical for the endoplasmic reticulum membrane channel activity. They had a relatively large unit conductance and were modified by typical activators (nucleotides) and inhibitors (ruthenium red, verapamil). Different doses of nifedipine did not inhibit Ca2+ channel activity. A substantial difference between the single-channel properties of the two types of investigated membranes was observed. The mean open time and the open state probability of channels reconstituted in bilayer lipid membranes from the membrane components of cisplatin-resistant cells were larger than those in bilayer lipid membranes made from components of cisplatin-sensitive cells. Ruthenium red (7 X 10(-7) M) inhibited the channel activity in both types of membranes to the same level. The observed effects could be related to an increased Ca2+ release from the intracellular Ca2+ stores (endoplasmic reticulum system) accompanied by an enhanced intracytoplasmic Ca2+ concentration in cisplatin-resistant cells. These changes in the Ca2+ concentration level may be responsible for the higher antitumor drug efflux rate and the development of the drug resistance. The suggestion is made that specific inhibitors of the Ca2+ transport across the membranes of the subcellular Ca2+-storing organelles may be tested as agents for overcoming the antitumor drug resistance.

Cell-mediated tumor-killing effect studied by using bilayer lipid membranes.

The bilayer lipid membrane (BLM) system was used to investigate the tumor killing effect of natural killer (NK) cells under various experimental conditions. It was found that NK cells interact specifically with BLMs made from lipids and proteolipids isolated from target K562 cells inducing an increase of the membrane conductance. This effect was more pronounced when the NK cells were pretreated with interferon. A similar effect was observed when NK cells were pretreated with sodium selenite. The results suggest that changes in membrane conductance and permeability are involved in the mechanism of the tumor-killing effect mediated by NK cells.