Bernd Nilius

Modal gating behavior of cardiac sodium channels in cell-free membrane patches.

Single voltage-activated Na+ channel currents were obtained from membrane patches of isolated ventricular cells of guinea pig hearts. The currents were compared when measured from cell-attached patches and from the same patch but at least 20 minutes after manual excision. The averaged currents showed a distinctly delayed decay in the excised patches due to the appearance of long lasting openings or bursts of openings. In contrast to control patches, the open time distribution in excised patches requires at least two exponentials. A short mean open time was voltage independent for cell-attached patches (0.38 ms +/- 0.07 ms between -60 and -20 mV, 6 cell-attached patches; and 0.41 +/- 0.1 ms, 7 excised patches). The long mean open time found in excised patches was clearly voltage dependent and increased from 0.48 +/- 0.14 ms (-80 mV) to 2.87 +/- 0.35 ms (-20 mV, regression coefficient +0.88, 7 patches). Sweeps with long openings appeared in clusters. The clustering of records with long openings, short openings, or without openings (nulls) was quantified by a runs analysis which showed a highly significant nonrandom ordering. The results show that in excised patches inactivation is temporally hibernating.

Efficient K+ buffering by mammalian retinal glial cells is due to cooperation of specialized ion channels

Radial glial (Müller) cells were isolated from rabbit retinae by papaine and mechanical dissociation. Regional membrane properties of these cells were studied by using the patch-clamp technique. In the course of our experiments, we found three distinct types of large K+ conducting channels. The vitread process membrane was dominated by high conductance inwardly rectifying (HCR) channels which carried, in the open state, inward currents along a conductance of about 105 pS (symmetrical solutions with 140 mM K+) but almost no outward currents. In the membrane of the soma and the proximal distal process, we found low conductance inwardly rectifying (LCR) channels which had an open state-conductance of about 60 pS and showed rather weak rectification. The endfoot membrane, on the other hand, was found to contain non-rectifying very high conductance (VHC) channels with an open state-conductance of about 360 pS (same solutions). These results suggest that mammalian Müller cells express regional membrane specializations which are optimized to carry spatial buffering currents of excess K+ ions.

Sodium current in single myocardial mouse cells

Morphologically intact single myocardial cells of the adult mouse show a length of 132±20 μm, a width of 21±5 μ, and a height of 10±4 μm (all mean ± SD) and are brick-like in shape. A one suction pipette method is used for voltage clamp of those single cells. The determined time constant of capacitive current τ=35±14 μs is very short. Series resistancers, membrane resistancerm, and membrane capacitycm are calculated to be 192±48 kΩ, 6.1±1.1 MΩ, and 186±92 pF (all mean ± SD), respectively. Assuming the specific unit membrane capacitance of 1 μF/cm2, a total membrane area of 1.86×10−4 cm2 is determined yielding a specific membrane resistanceRm of 1,134 Ωcm2. Settling time of voltage clamp is 30 μs. TTX-block of sodium current is described by 1:1 binding with aKD value of 1.4×10−6M. Using a reduced extracellular sodium concentration the maximum Na current is between 25 and 40 nA at voltages between −40 and −30 mV. Currents of between +20 and +30 mV reverse in an outward direction. Inward currents are approximated by a m3h model. The time constant of activation decreases from 0.7 ms at −60 mV to 0.12 ms at +20 mV. The time constant of inactivation falls from 9.1 ms at −60 mV to 0.6 ms at +20 mV.Steady state inactivationh∞ is characterized by the half maximum valueVH=−76.1±4.3 mV and the slope parameters=−6.3±1.1 mV (mean ± SD). A prepulse duration of 500 ms is essential for real steady state inactivation. Steady state activationm∞ and inactivationh∞ overlap each other defining a maximum window current at −65 mV.

Modulation of calcium channel currents in guinea-pig single ventricular heart cells by the dihydropyridine Bay K 8644.

1. A single glass micropipette voltage clamp technique with intracellular dialysis was used to study Ba2+ currents in isolated ventricular cells from guinea‐pig hearts. Effects of the 1,4‐dihydropyridine Bay K 8644 on whole‐cell currents were evaluated at 37 degrees C. 2. Bay K 8644 increased the Ba2+ peak currents at test potentials between ‐50 and +20 mV and shifted the current‐voltage relationships towards hyperpolarizing potentials (leftward shift for Ca2+ channel activation, 13.8 +/‐ 4.1 mV; n = 9; Bay K 8644, 5 mumol/l). 3. The peak times of the Ba2+ currents were diminished over the voltage range tested between ‐40 and +20 mV after Bay K 8644 in parallel with a shortening of the time constant of activation that was estimated from fits of the recorded currents with a d2f model. 4. The decay of the Ba2+ currents was fitted with two exponentials including a pedestal. The compound Bay K 8644 accelerated the fast decay over the whole voltage range. The amplitude of the rapidly inactivated component of the Ba2+ currents was strikingly increased after application of Bay K 8644. 5. The steady‐state inactivation using a 0.5 or 5 s pre‐pulse was shifted towards hyperpolarizing potentials (leftward shift 10.3 +/‐ 5.2 mV; n = 4; Bay K 8644, 5 mumol/l). 6. The change in the time course of Bay K 8644‐modified Ba2+ currents cannot be described solely by a decrease of the backward rate coefficient from an open to a closed state of the Ca2+ channel (Sanguinetti, Krafte & Kass, 1986). The described effects of Bay K 8644 on the inactivation can be both qualitatively and quantitatively described by a model of current‐dependent inactivation (Standen & Stanfield, 1982), assuming a lower affinity of an internal binding site for Ba2+ than for Ca2+.

Permeation properties of a non-selective cation channel in human vascular endothelial cells.

SummaryEndothelial cells obtained from human umbilical chord have been studied by the patch clamp method. An ion channel is described that is activated by μM concentrations of histamine and shows a slow run-down in cell-attached patches. After excision, channel activity quickly runs down to zero open probability. In symmetrical potassium concentrations (140 mM K in the bath and the pipette), the single channel conductance is 28±2 pS and the reversal potential is 0.3±0.8 mV (mean ± SEM, n=4). With 140 mM Na in the pipette, the conductance is 26±2 pS. A reversal potential of -1.5±0.9 mV (n=7) was measured. With 60 mM Ca and 70 mM Na in the pipette, 140 mM K in the bath, the reversal potential was -11±3 mV, the single channel conductance 16±3 pS (n=5). The single channel conductance in 110 mM Ca (pipette) and 140 mM K (bath) is 8±2 pS and the reversal potential is −18±6 mV (n=3). From analysis of the reversal potentials, a permeation ratio of K∶Na∶Ca=1∶0.9∶0.2 was calculated. This ligand-gated non-selective cation channel in human endothelial cells is Ca permeable and could induce a sustained agonist mediated Ca influx.

Calcium block of guinea-pig heart sodium channels with and without modification by the piperazinylindole DPI 201-106.

1. External Ca2+ block of Na+ channels was studied by a gigaohm‐seal patch clamp technique in single cardiac ventricular cells from guinea‐pig. Single‐channel currents were recorded from cell‐attached patches. 2. Increasing external Ca2+ concentrations in the patch pipette from 0.1 to 20 mM reduced the single‐channel conductance of normal Na+ channels from 27 to 14 pS without causing flickering (obtained from linear regression, eight patches). 3. Exposed to external Ca2+ concentrations of 20 mM, the single‐channel currents decreased at potentials negative to ‐60 mV in spite of an increased driving force for inward Na+ currents. 4. An external concentration of 35 mM‐Mg2+, which is supposed to exert a screening of surface charges nearly equal to that of 20 mM‐Ca2+ (Hille, Woodhull & Shapiro, 1975), reduced the single‐Na+‐channel conductance only from 26 (1 mM‐Mg2+) to 20 pS (linear regression, eight patches). A weaker voltage‐dependent block at potentials negative to ‐50 mV was observed in 35 mM‐Mg2+ than in 20 mM‐Ca2+. Therefore, surface charge effects cannot explain the obvious reduction of the conductance of single Na+ channels found when the external Ca2+ concentration was increased. 5. Single Na+‐channel currents increased with an increase in the external Na+ concentration [( Na+]o) but showed saturation. The Na+o‐single‐channel current relationship could be described by i = imax/(1 + kd/[Na+]o) with imax = 5.4 pA and kd = 359 mM (seventeen patches). 6. The mean open time of Na+ channels varied between 0.18 and 0.59 ms (potentials between ‐80 and ‐20 mV). No significant changes in the mean open time could be obtained when Ca2+ was varied between 0.1 and 20 mM. 7. The piperazinylindole compound DPI 201‐106 was used as a tool to prolong the open time of single Na+ channels. If the external Ca2+ concentration was increased from 0.1 to 20 mM the currents through the modified channels were reduced. The reduction of single‐channel currents was accentuated at potentials negative to ‐60 mV (20 mM‐Ca2+) similar to the control channels. 8. In contrast to non‐modified Na+ channels, the mean open time of DPI 201‐106‐modified channels proved extremely voltage and Ca2+ dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of lidocaine on single cardiac sodium channels

Lidocaine block of single cardiac sodium channels was studied in cell free inside-out patches of ventricular cells isolated from guinea-pig hearts. When applied to the inner surface of the membrane lidocaine depressed Na channel currents by decreasing the probability P of the channels to open measured from the peaks of the averaged currents. In parallel to the decrease in P the relative number of empty sweeps (nulls) was increased. Half maximum block of the activity of single Na channels was observed at 2.9 microM. Lidocaine affected the gating behaviour of Na channels by shortening of the mean open time tau 0 from 0.44 +/- 0.17 (control) to 0.19 +/- 0.13 (5 microM lidocaine, holding potential-120 mV, test potential -60 mV). Five micromolar lidocaine completely suppressed burst-like openings of Na channels and abolished the slow decaying phase of the averaged currents. A shift from - 120 towards - 160 mV exerted relief from the effect of both P and tau 0.

Two types of transient outward currents in cardiac ventricular cells of mice

Ventricular cells of adult mice were prepared by an enzyme digestion procedure. Single channel currents were recorded by a conventional patch clamp technique from cell attached patches. Voltage steps from the holding potential of −80 mV to test potentials between −35 and +50 mV caused openings of two types of outward currents through single channels with the conductances of 27 and 12 pS, respectively. The averaged currents reveal transient time courses for both channel types. The current-voltage relations of both single channel currents were linear over the tested voltage range and intersected the voltage axis at −70 mV. This indicates that both single channel currents are mainly carried by potassium ions. All open and closed times were found to be voltage independent. The 27 pS channel had a mean open time of 3.9±1.0 ms (n=8). The closed time consisted of two components with τ1 = 2.1 ± 0.2 ms and τ2 = 50 ± 19 ms (n=8). The 12 pS channel had a mean open time of 34.0±5.2 ms (n=3) and the two components of the mean closed time have been calculated as τ1 = 8.3 ± 2.1 ms and τ2 = 120 ± 50 ms (n=3; all mean ±SD).

Inactivation of sodium channels in isolated myocardial mouse cells

Inactivation of sodium currents is investigated in single myocardial mouse cells by the use of a one suction pipette voltage clamp technique. Semilogarithmic plots of the decay phase of sodium currents show two phases and analysis yields a fast (τh1) and a slow (τh2) time constant. At increased depolarization the contribution of τh2 is decreased. Increasing the temperature from 22.0±0.5°C to 36.5±0.5°C influences τh1 and τh2 to the same extent. The Q10 values were 2.28±0.32 for τh1 and 2.46±0.35 (mean±SD) for τh2. Between-50 and-20 mV the time course of current decay is substantially faster than inactivation induced by prepulses. Onset of inactivation by the prepulse protocol needs two time constants (τc1τc2) for a satisfactory description. Both decrease steeply with increasing depolarization. For small depolarizations, τc2 values are in the range of several seconds. Recovery from inactivation by short prepulses (40 ms) could be described by two exponentials (τr1τr2). For longer prepulses (1.000 ms) a very slow component, τr3, was observed indicating a history dependence of inactivation. Delay time constants for the onset of inactivation (τd2) are determined between-50 and-20 mV. The more depolarizing voltages generate smaller delay times (0.55±0.10 ms at-20 mV, mean±SD) but larger deviations from the exponential time course. Delay in recovery from inactivation (time constant τd2) has the value 2.0±0.7 ms (mean±SD) at-80 mV and decreases at more hyperpolarizing potentials. The remaining series resistance at maximum compensation was calculated as 80 kΩ. Its influence on the sensitive delay in the double pulse inactivation is discussed.

A study of dynamic properties in isolated myocardial cells by the laser diffraction method

Laser diffraction patterns were investigated from enzymatically isolated, unattached myocardial cells of guinea-pigs and mice. Experiments were performed at 2.5 mM Ca2+ and room temperature. The mean sarcomere length of resting guinea-pig and mouse myocardial cells amounted to about 1.83 micron and 1.75 micron, respectively. When paced with alternating intervals by field stimulation carefully selected ventricular cells showed transient phenomena. (1) The staircase following a rested state contraction was positive in the case of guinea-pig and negative in the case of mouse myocardial cells; (2) The rested state as compared to the steady state sarcomere shortening of guinea-pig and mouse cardiac myocytes amounted to 35% and 600%, respectively; (3) The interval strength curve of guinea-pig myocardial cells passed through a maximum which was 0.26 +/- 0.06 micron (mean +/- S.D.) at a pacing interval of 2 s whereas myocardial cells of mice showed a rise of shortening with increasing intervals reaching a maximum at the rested state (0.24 +/- 0.08 micron). Results were similar to those obtained from multicellular preparations. We conclude therefore, dynamic properties of multicellular preparations are nicely reflected at the sarcomere level.