Biomedical subjects

R Koopmann

Publications and source records attributed to R Koopmann.

8 recordsLinked to original sources

PubMed2000

Structural elements determining activation kinetics in Kv2.1.

Voltage-dependent K+ channels open when depolarizing the membrane voltage. Among the different alpha-subunits, the time course of current activation spreads over a wide range. The structural basis underlying this diversity is not known. We constructed multiple chimeras between two voltage-dependent K+ channels, the rapidly activating Kv1.2 and the slowly activating Kv2.1, and we focused on the C-terminal half of the core region. The general strategy was to substitute parts of Kv2.1 by corresponding parts of Kv1.2 and to test for an acceleration of activation. We identified three regions which contribute to the determination of the activation kinetics: the S5-pore linker, the deep pore, and the S4-segment.

Amino Acid Sequence↗

PubMed1999

Anoxia generates rapid and massive opening of KATP channels in ventricular cardiac myocytes.

OBJECTIVE: The aim was to improve the measurement of both the time course and amplitude of anoxia-induced KATP-channel current (IKATP) in isolated heart cells to specify the role of these channels in the time course of K+ accumulation in the ischemic myocardium. METHODS: Ionic currents in isolated ventricular heart cells of the mouse were measured with a patch clamp technique under normoxic conditions (atmospheric pO2), during wash-out of oxygen, and under anoxic conditions (pO2 < 0.2 mmHg). During the measurement, the actual pO2 in the close proximity of the cell was determined with an optical technique by exciting Pd-meso-tetra(4-carboxyphenyl)porphin with light flashes of 508-570 nm and evaluating the quenching kinetics of the emitted phosphorescence signal at 630-700 nm. These quenching kinetics steeply depend on pO2 and can be evaluated best at pO2 values near 0 mmHg. RESULTS: Out of 28 cells, 23 cells started to develop IKATP at pO2 values between 0 and 0.4 mmHg, i.e. in the range of the level of half maximum activity of the cytochrome oxidase. The remaining five cells developed IKATP between 0.4 and 1.8 mmHg. With respect to the time course, 18 out of 27 cells started to develop IKATP within the first minute after pO2 had decreased to values below 0.2 mmHg. The amplitude of IKATP induced by anoxia and various metabolic inhibitors was large, 29 +/- 12 and 48 +/- 21 nA (+40 mV), respectively. The anoxia-induced IKATP was significantly smaller than IKATP induced by metabolic inhibitors. During the pulses of 50 ms duration to +40 mV, the amplitude of IKATP decayed and, after clamping back to -80 mV, IKATP generated large tail currents. This suggests a notable change in the concentration gradient of K+ ions in the time range of tens of milliseconds. CONCLUSIONS: The results in isolated myocytes indicate that KATP channels open sufficiently rapidly after starting anoxia and generate sufficiently large conductance at maintained anoxia to explain both the time course and magnitude of the ischemic K+ accumulation if an appropriate counter-ion flux is available.

Animals↗

PubMed1999

Gating by cyclic GMP and voltage in the alpha subunit of the cyclic GMP-gated channel from rod photoreceptors.

Gating by cGMP and voltage of the alpha subunit of the cGMP-gated channel from rod photoreceptor was examined with a patch-clamp technique. The channels were expressed in Xenopus oocytes. At low [cGMP] (<20 microM), the current displayed strong outward rectification. At low and high (700 microM) [cGMP], the channel activity was dominated by only one conductance level. Therefore, the outward rectification at low [cGMP] results solely from an increase in the open probability, P(o). Kinetic analysis of single-channel openings revealed two exponential distributions. At low [cGMP], the larger P(o) at positive voltages with respect to negative voltages is caused by an increased frequency of openings in both components of the open-time distribution. In macroscopic currents, depolarizing voltage steps, starting from -100 mV, generated a time-dependent current that increased with the step size (activation). At low [cGMP] (20 microM), the degree of activation was large and the time course was slow, whereas at saturating [cGMP] (7 mM) the respective changes were small and fast. The dose-response relation at -100 mV was shifted to the right and saturated at significantly lower P(o) values with respect to that at +100 mV (0.77 vs. 0.96). P(o) was determined as function of the [cGMP] (at +100 and -100 mV) and voltage (at 20, 70, and 700 microM, and 7 mM cGMP). Both relations could be fitted with an allosteric state model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric opening reaction. At saturating [cGMP] (7 mM), the activation time course was monoexponential, which allowed us to determine the individual rate constants for the allosteric reaction. For the rapid rate constants of cGMP binding and unbinding, lower limits are determined. It is concluded that an allosteric model consisting of four independent cGMP-binding reactions and one voltage-dependent allosteric reaction, describes the cGMP- and voltage-dependent gating of cGMP-gated channels adequately.

Animals↗

PubMed1998

Inactivation of single cardiac Na+ channels in three different gating modes.

In small cell-attached patches containing one and only one Na+ channel, inactivation was studied in three different gating modes, namely, the fast-inactivating F mode and the more slowly inactivating S mode and P mode with similar inactivation kinetics. In each of these modes, ensemble-averaged currents could be fitted with a Hodgkin-Huxley-type model with a single exponential for inactivation (tauh). tauh declined from 1.0 ms at -60 mV to 0.1 ms at 0 mV in the F mode, from 4.6 ms at -40 mV to 1.1 ms at 0 mV in the S mode, and from 4.5 ms at -40 mV to 0.8 ms at +20 mV in the P mode, respectively. The probability of non-empty traces (net), the mean number of openings per non-empty trace (op/tr), and the mean open probability per trace (popen) were evaluated at 4-ms test pulses. net inclined from 30% at -60 mV to 63% at 0 mV in the F mode, from 4% at -90 mV to 90% at 0 mV in the S mode, and from 2% at -60 mV to 79% at +20 mV in the P mode. op/tr declined from 1.4 at -60 mV to 1.1 at 0 mV in the F mode, from 4.0 at -60 mV to 1.2 at 0 mV in the S mode, and from 2.9 at -40 mV to 1.6 at +20 mV in the P mode. popen was bell-shaped with a maximum of 5% at -30 mV in the F mode, 48% at -50 mV in the S mode, and 16% at 0 mV in the P mode. It is concluded that 1) a switch between F and S modes may reflect a functional change of inactivation, 2) a switch between S and P modes may reflect a functional change of activation, 3) tauh is mainly determined by the latency until the first channel opening in the F mode and by the number of reopenings in the S and P modes, 4) at least in the S and P modes, inactivation is independent of pore opening, and 5) in the S mode, mainly open channels inactivate, and in the P mode, mainly closed channels inactivate.

Animals↗

PubMed1997

Functional differences of a Kv2.1 channel and a Kv2.1/Kv1.2S4-chimera are confined to a concerted voltage shift of various gating parameters.

When expressed in Xenopus oocytes, the voltage-dependent K+ channels Kv1.2 and Kv2.1 have similar steady state parameters of activation but the kinetics of activation is significantly faster in the Kv1.2 channels. Activation results from intramolecular arrangements which start with the movement of the voltage sensor and end with the opening of the pore. The S4-segment and the H5-loop comprise at least part of the respective involved structural elements. The molecular mechanism of coupling between sensing of voltage and opening of the pore is less well understood. We have measured whole cell and single channel ionic currents in the rapidly activating Kv1.2 channel of the rat, the slowly activating Kv2.1 channel of the human, and in an S4-chimera Kv2.1/Kv1.2S4. With respect to the Kv2.1 channel, steady state activation and steady state C-type inactivation of the chimeric channel are shifted by more than 50 mV in the depolarizing direction. The property of rapid activation in Kv1.2 channels was not transferred to the Kv2.1 channels with the transplanted S4-region. Instead, the kinetics of activation, deactivation, and recovery from C-type inactivation as well as the voltage sensitivity of the 4-aminopyridine block are similar to the corresponding processes in Kv2.1 channels if they are related to the steady state activation and inactivation, respectively. The unitary current and the mean open time of single channel openings of the S4-chimeric channels resemble the respective values of Kv2.1 channels. It is concluded that the insertion of the S4-segment of Kv1.2 channels into Kv2.1 channels modifies the gating at the early steps of activation leaving all properties associated with the open state(s) of the Kv2.1 channels unaffected.

4-Aminopyridine↗

PubMed1994

Gating and conductance properties of a human delayed rectifier K+ channel expressed in frog oocytes.

1. The human delayed rectifier K+ channel h-DRK1, a homologue to the DRK1 channel in the rat, was expressed in Xenopus oocytes. Single-channel currents were measured in micropatches; macroscopic currents were measured either in macropatches, giant patches, or whole oocytes. 2. Macroscopic currents activated at -20 mV and more positive. The instantaneous current-voltage relationship rectified outwardly to a higher degree than predicted by the Goldman-Hodgkin-Katz equation. 3. With the giant patch technique, ionic and putative on- and off-gating currents were recorded simultaneously. The large ratio of the moved gating charges to the amplitude of the ionic current indicated that less than 1% of the gating channels actually opened. 4. The single-channel conductance between 0 and +80 mV was calculated to be 9.4 pS. The channels opened with sublevels which appeared either independently of the fully open level as separate openings, in conjunction with the opening to and closing from the fully open level, or by starting from and ending at the fully open level. 5. The channels opened with two voltage-independent open time constants in the range 1-10 ms (filter 1 kHz). The burst open probability was fitted monoexponentially with time constants in the range of tens of milliseconds. 6. Assuming a sequential Markovian model with four independent voltage-controlled transitions, fit of the steady-state open probability of macroscopic currents showed two components of activation differing in their half-maximal value. 7. The fit of time courses of cumulative first latency and ensemble-averaged currents in single-channel patches suggested that even a single channel may operate with the two different components of activation. 8. It is concluded that h-DRK1 channels considerably rectify in an outward direction and that an apparently flat voltage dependence of activation may be explained by the overlap of two different components.

Animals↗

PubMed1994

Expression of integrins and CD44 isoforms in non-Hodgkin's lymphomas: CD44 variant isoforms are preferentially expressed in high-grade malignant lymphomas.

Low- and high-grade malignant non-Hodgkin's lymphomas have been investigated by immunohistochemistry for their expression of various integrins and CD44 isoforms. Comparison with the expression patterns obtained in non-malignant adult lymph nodes revealed the following differences: alpha 6 and beta 4 integrins were expressed in several high-grade malignant lymphomas to a lower degree than in both the low-grade malignant lymphomas and the normal lymph nodes; all other integrins (alpha 2, alpha 4, alpha 5, alpha v, beta 1, beta 2, beta 3, and beta 7) did not exhibit significant differences in the expression levels between malignant and non-malignant tissues. The standard isoform of CD44 (CD44s) was highly expressed in all lymphoid tissues. Using CD44 exons-specific monoclonal antibodies, CD44 variant isoforms were not detected in non-malignant lymph nodes and were detected only rarely in low-grade malignant lymphomas. In contrast, high-grade malignant lymphomas expressed several CD44 variant isoforms, which included the products from the variant exons 3v, 6v, and 9v, but not 4v. Specifically, detection of exon 3v and 6v products indicates a more aggressive phenotype.

Adult↗

PubMed1993

Thermodynamic entropy of two conformational transitions of single Na+ channel molecules.

Single cardiac Na+ channel currents were recorded with improved resolution (bandwidth up to 20 kHz) at two temperatures, 10 and 25 degrees C. The mean open time was determined at voltages between -50 and 0 mV by evaluation of the distribution of the event-related gaps in the center of the baseline noise. Fit of the voltage-dependent reciprocal mean open times at both temperatures allowed even for a single channel molecule to separate an entropic from an enthalpic part of activation energy for both deactivation and inactivation. Both entropies are positive and the entropy of deactivation exceeds that of inactivation by more than twice.

Animals↗