Carboxy-terminal Determinants of Conductance in Inward-rectifier K Channels

Previous studies suggested that the cytoplasmic COOH-terminal portions of inward rectifier K channels could contribute significant resistance barriers to ion flow. To explore this question further, we exchanged portions of the COOH termini of ROMK2 (Kir1.1b) and IRK1 (Kir2.1) and measured the resulting single-channel conductances. Replacing the entire COOH terminus of ROMK2 with that of IRK1 decreased the chord conductance at Vm = −100 mV from 34 to 21 pS. The slope conductance measured between −60 and −140 mV was also reduced from 43 to 31 pS. Analysis of chimeric channels suggested that a region between residues 232 and 275 of ROMK2 contributes to this effect. Within this region, the point mutant ROMK2 N240R, in which a single amino acid was exchanged for the corresponding residue of IRK1, reduced the slope conductance to 30 pS and the chord conductance to 22 pS, mimicking the effects of replacing the entire COOH terminus. This mutant had gating and rectification properties indistinguishable from those of the wild-type, suggesting that the structure of the protein was not grossly altered. The N240R mutation did not affect block of the channel by Ba2+, suggesting that the selectivity filter was not strongly affected by the mutation, nor did it change the sensitivity to intracellular pH. To test whether the decrease in conductance was independent of the selectivity filter we made the same mutation in the background of mutations in the pore region of the channel that increased single-channel conductance. The effects were similar to those predicted for two independent resistors arranged in series. The mutation increased conductance ratio for Tl+:K+, accounting for previous observations that the COOH terminus contributed to ion selectivity. Mapping the location onto the crystal structure of the cytoplasmic parts of GIRK1 indicated that position 240 lines the inner wall of this pore and affects the net charge on this surface. This provides a possible structural basis for the observed changes in conductance, and suggests that this element of the channel protein forms a rate-limiting barrier for K+ transport.

Biphasic Effects of Isoflurane on the Cardiac Action Potential

Background The mechanism underlying isoflurane modulation of cardiac electrophysiology is not well understood. In the present study, the authors investigated the effects of isoflurane on the cardiac action potential (AP) characteristics. The results were correlated to modulation of the L-type calcium (I(Ca,L)), the delayed-rectifier potassium (I(Kdr)), and the inward-rectifier potassium (I(Kir)) currents. Methods Single ventricular myocytes were enzymatically isolated from guinea pig hearts. The current clamp and whole cell voltage clamp configurations of the patch clamp technique were used to monitor the cardiac AP and ionic currents, respectively. A dynamic AP voltage protocol that mimicked changes in membrane potential during an AP was used to monitor the I(Ca,L), I(Kdr) and I(Kir). Results Isoflurane produced a concentration-dependent, biphasic effect on the AP duration (APD). At 0.6 mm (1.26 vol%), isoflurane significantly increased APD50 and APD90 by 50.0 +/- 7.6% and 48.9 +/- 7.2%, respectively (P < 0.05; n = 6). At 1.0 mm (2.09 vol%), isoflurane had no significant effect on APD (n = 6). In contrast, at 1.8 mm (3.77 vol%), isoflurane decreased APD50 and APD90 by 38.3 +/- 5.4% and 32.2 +/- 5.5%, respectively (P < 0.05; n = 7). The inhibitory effects of isoflurane on I(Kdr) chord conductance were greater than those on I(Ca,L) (P < 0.05; n = 6/group). Both I(Ca,L) inactivation and I(Kdr) activation kinetics were accelerated by isoflurane. Isoflurane had no significant effects on I(Kir) chord conductance (n = 6). Conclusion At the lower anesthetic concentration, the prolongation of the APD may be the result of the dominant inhibitory effects of isoflurane on I(Kdr). At the higher concentration, the shortening of the APD may be caused by the inhibitory effects on I (Ca,L) combined with the isoflurane-induced acceleration of I(Ca,L) inactivation kinetics. Because I(Kdr) is significantly inhibited by isoflurane, I(Kir) appears to be the major repolarizing current, which is minimally affected by isoflurane.

Permeation and block by internal and external divalent cations of the catfish cone photoreceptor cGMP-gated channel.

The ability of the divalent cations calcium, magnesium, and barium to permeate through the cGMP-gated channel of catfish cone outer segments was examined by measuring permeability and conductance ratios under biionic conditions and by measuring their ability to block current carried by sodium when presented on the cytoplasmic or extracellular side of the channel. Current carried by divalent cations in the absence of monovalent cations showed the typical rectification pattern observed from these channels under physiological conditions (an exponential increase in current at both positive and negative voltages). With calcium as the reference ion, the relative permeabilities were Ca > Ba > Mg, and the chord conductance ratios at +50 mV were in the order of Ca approximately Mg > Ba. With external sodium as the reference ion, the relative permeabilities were Ca > Mg > Ba > Na with chord conductance ratios at +30 mV in the order of Na > Ca = Mg > Ba. The ability of divalent cations presented on the intracellular side to block the sodium current was in the order Ca > Mg > Ba at +30 mV and Ca > Ba > Mg at -30 mV. Block by external divalent cations was also investigated. The current-voltage relations showed block by internal divalent cations reveal no anomalous mole fraction behavior, suggesting little ion-ion interaction within the pore. An Eyring rate theory model with two barriers and a single binding site is sufficient to explain both these observations and those for monovalent cations, predicting a single-channel conductance under physiological conditions of 2 pS and an inward current at -30 mV carried by 82% Na, 5% Mg, and 13% Ca.

Alteration of the sodium current in cat cardiac ventricular myocytes during primary culture

To determine the response of cardiac Na current (INa) in adult cardiac ventricular myocytes to culture, single isolated ventricular myocytes from collagenase-perfused adult cat hearts were placed in primary culture for up to 2 wk on a two-dimensional (2D) surface (laminin-coated coverslips), which allowed the morphology of the myocytes to change markedly, or in a three-dimensional matrix (3D) of alginate, in which cell shape changed only minimally. Action potentials and INa were recorded from groups of 1) freshly isolated myocytes serving as the control (day 0),2) cells maintained in 2D culture for 9-14 days (2D, day 9-14), and 3) cells cultured in alginate for 9-14 days (3D, day 9-14) with use of a conventional whole cell patch technique. Maximal upstroke velocity (Vmax) of the action potential was reduced by approximately 50% in 2D- and 3D-cultured cells relative to controls. INa in 2D- and 3D-cultured cells was strikingly different from that in control myocytes. Half-maximal voltage (V 1/2) for the chord conductance-voltage relationship was shifted approximately 15 mV negatively to that for controls in 2D- and 3D-cultured cells. INa steady-state availability curve also shifted negatively relative to controls in 2D- and 3D-cultured myocytes, but the magnitude of this shift (approximately 16-20 mV) was greater than that for the chord conductance-voltage curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Characteristics of Five Types of K+ Channel in Cultured Locust Muscle

K+ channel activity in cultured locust myofibres was investigated using gigaohm patch-clamp techniques. After 2 months in vitro the myofibres had a mean resting potential of −39 ± 7 mV (±S.D., 7V = 42). Five types of K+ channel were identified at this time. The majority of single-channel events recorded from cellattached patches were due to a small-conductance (type 1) and a largeconductance (type 2), inward rectifier, K+ channel. In cell-attached patches, with 180 mmoll−1 KC1 in the patch pipette, the type 1 channel had a chord conductance of 43 pS for inward currents and 8pS for outward currents; the type 2 channel had a chord conductance of 115 pS for inward currents and 29 pS for outward currents. The type 2 channel exhibited bursting kinetics, was ATP-sensitive and could be blocked by Ba2+. Two other channels (types 3 and 4) had linear conductances of 130pS and 207pS, respectively. The type 3 channel was Ca2+-sensitive. A further channel (type 5) appeared to be an inward rectifier with a conductance of 5pS. Openings of types 3, 4 and 5 channels occurred less frequently than openings of the other two channels. Types 1, 2, 3 and 4 channels possessed multiple closed and open states with non-linear gating mechanisms.

Nerve Stimulation and Electrical Properties of Frog Skin

The suitability of frog skin glands as a model for the study of secretory mechanisms in exocrine glands was explored. Periodic voltage clamp was used to determine continually the short-circuit current, chord conductance, and electromotive force of frog skin during neural and pharmacological activation of the skin glands. Both the chord conductance and the short-circuit current increased with glandular activation; the temporal dissociation of these increases suggests that there are at least two separate components to the secretory response. The sensitivity of the secretory electrical changes to changes in the ionic composition of the bathing solutions supports the notion of electrogenic chloride active transport as being basic to the activity of the exocrine glands.