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Papers by Daniel Kwan

Research paper thumbnail of Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K + reduction and acidification

Cell Biochemistry and Biophysics, 2005

Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the function... more Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studies and are known to play essential roles in excitable cell function, providing an essential counterpoin to the various inward currents that trigger excitability. The serum potassium concentration [K o+] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K+ concentration. As well as changes in serum [K o+], tissue acification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K o+-dependent, and the suggestion has been made that pH and K o+ may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K o+. It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K+ ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K+ channels in the open vs inactivated states of the channels by physiological H+ and K+ concentrations represents an important pathway to control Kv channel function in health and disease.

Research paper thumbnail of Single Channel Analysis Reveals Different Modes of Kv1.5 Gating Behavior Regulated by Changes of External pH

Biophysical Journal, 2006

In the voltage-gated potassium channel Kv1.5, extracellular acidification decreases the peak macr... more In the voltage-gated potassium channel Kv1.5, extracellular acidification decreases the peak macroscopic conductance and accelerates slow inactivation. To better understand the mechanistic basis for these two effects, we recorded unitary currents of Kv1.5 expressed in a mouse cell line (ltk−) using the voltage clamp technique both in cell-attached and excised outside-out patches. Single channel current amplitude at 100 mV (1.7 ± 0.2 pA at pH 7.4, 1.7 ± 0.2 pA at pH 6.4) and the single channel conductance between 0 and 100 mV (11.8 ± 0.6 pS at pH 7.4 and 11.3 ± 0.8 pS at pH 6.4) did not change significantly with pH. External acidification significantly decreased the number of active sweeps, and this reduction in channel availability accounted for most of the reduction of the peak macroscopic current. The results of runs analyses suggested the null sweeps occur in clusters, and the rate constants for the transition between clusters of null and active sweeps at pH 6.4 were slow (0.12 and 0.18 s−1, to and from the active clusters, respectively). We propose that low pH facilitates a shift from an available mode (mode A) into an unavailable mode of gating (mode U). In addition to promoting mode U gating, external acidification accelerates depolarization-induced inactivation, which is manifest at the single channel level as a reduction of the mean burst length and an apparent increase of the interburst interval. These effects of external acidification, which are thought to reflect the protonation of a histidine residue in the turret (H-463), point to an important role for the turret in the regulation of channel availability and inactivation.

Research paper thumbnail of A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

Journal of General Physiology, 2007

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels... more Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltageclamp fl uorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fl uorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fl uorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fl uorescence demonstrated slow unquenching that also refl ects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fl uorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fl uorescence at low pH refl ects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K + or the mutation T449V) prevented fl uorescence loss at low pH, and the fl uorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fl uorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fl uorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

Research paper thumbnail of Block by internal Mg 2+ causes voltage-dependent inactivation of Kv1.5

European Biophysics Journal With Biophysics Letters, 2006

Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ bl... more Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ block of Kv1.5 induces voltage-dependent current decay at strongly depolarised potentials that contains a component due to acceleration of C-type inactivation after pore block. The voltage-dependent current decay was fitted to a bi-exponential function (τfast and τslow). Without Mg2+, τfast and τslow were voltage-independent, but with 10 mM Mg2+, τfast decreased from 156 ms at +40 mV to 5 ms at +140 mV and τslow decreased from 2.3 s to 206 ms. With Mg2+, tail currents after short pulses that allowed only the fast phase of decay showed a rising phase that reflected voltage-dependent unbinding. This suggested that the fast phase of voltage-dependent current decay was due to Mg2+ pore block. In contrast, tail currents after longer pulses that allowed the slow phase of decay were reduced to almost zero suggesting that the slow phase was due to channel inactivation. Consistent with this, the mutation R487V (equivalent to T449V in Shaker) or increasing external K+, both of which reduce C-type inactivation, prevented the slow phase of decay. These results are consistent with voltage-dependent open-channel block of Kv1.5 by internal Mg2+ that subsequently induces C-type inactivation by restricting K+ filling of the selectivity filter from the internal solution.

Research paper thumbnail of Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K + -free solutions at physiological pH

Cell Biochemistry and Biophysics, 2005

Extracellular acidification and reduction of extracellular K+ are known to decrease the currents ... more Extracellular acidification and reduction of extracellular K+ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K+]o at pH 7.4, it is very sensitive to [K+]o at pH 6.4, and in the mutant, H463G, the removal of K+o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K+o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na+ permeation through inactivated channels. Although the H463G currents were abolished in zero [K+]o, robust Na+ tail currents through inactivated channels were observed. The appearnnce of H463G Na+ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K+ was replaced by NMG+ and the inward Na+ current was recorded, addition of 1 mM K+ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K+]o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).

Research paper thumbnail of External K+ relieves the block but not the gating shift caused by Zn2+ in human Kv1.5 potassium channels

Journal of Physiology-london, 2001

Changing the extracellular concentration of divalent cations can have dramatic effects on the beh... more Changing the extracellular concentration of divalent cations can have dramatic effects on the behaviour of electrically excitable membranes as illustrated by the lowering or raising of the threshold for cell firing caused by hypo-or hypercalcaemia, respectively . Voltage-clamp analyses have shown that the divalent cation-induced change of excitability can be linked to a shift of the voltage dependence of channel gating. For example, an elevation of the external concentration of Ca 2+ ([Ca 2+ ] o ) produces a depolarizing shift of the midpoint (V 1/2 ) of the activation curves of both voltage-gated Na + and K + channels of the squid giant axon . Decreasing [Ca 2+ ] o produces the converse effect.

Research paper thumbnail of The External K + Concentration and Mutations in the Outer Pore Mouth Affect the Inhibition of Kv1.5 Current by Ni 2

Biophysical Journal, 2004

By examining the consequences both of changes of [K+]o and of point mutations in the outer pore m... more By examining the consequences both of changes of [K+]o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni2+ is similar to that for proton block. Ni2+ block is inhibited by increasing [K+]o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni2+ had no effect on gating currents. We propose that, as with Ho+, Ni2+ binding to H463 facilitates an outer pore inactivation process that is antagonized by Ko+ and that requires R487. However, whereas Ho+ substantially accelerates inactivation of residual currents, Ni2+ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co2+ and Mn2+, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM Ko+ is Zn2+ (KD ∼ 0.07 mM) ≥ Ni2+ (KD ∼ 0.15 mM) > Co2+ (KD ∼ 1.4 mM) > Mn2+ (KD > 10 mM).

Research paper thumbnail of Synergistic inhibition of the maximum conductance of Kv1.5 channels by extracellular K + reduction and acidification

Cell Biochemistry and Biophysics, 2005

Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the function... more Voltage-gated potassium (Kv) channels exist in the membranes of all living cells. Of the functional classes of Kv channels, the Kv1 channels are the largest and the best studies and are known to play essential roles in excitable cell function, providing an essential counterpoin to the various inward currents that trigger excitability. The serum potassium concentration [K o+] is tightly regulated in mammals and disturbances can cause significant functional alterations in the electrical behavior of excitable tissues in the nervous system and the heart. At least some of these changes may be mediated by Kv channels that are regulated by changes in the extracellular K+ concentration. As well as changes in serum [K o+], tissue acification is a frequent pathological condition known to inhibit Shaker and Kv1 voltage-gated potassium channels. In recent studies, it has become recognized that the acidification-induced inhibition of some Kv1 channels is K o+-dependent, and the suggestion has been made that pH and K o+ may regulate the channels via a common mechanism. Here we discuss P/C type inactivation as the common pathway by which some Kv channels become unavailable at acid pH and lowered K o+. It is suggested that binding of protons to a regulatory site in the outer pore mouth of some Kv channels favors transitions to the inactivated state, whereas K+ ions exert countereffects. We suggest that modulation of the number of excitable voltage-gated K+ channels in the open vs inactivated states of the channels by physiological H+ and K+ concentrations represents an important pathway to control Kv channel function in health and disease.

Research paper thumbnail of Single Channel Analysis Reveals Different Modes of Kv1.5 Gating Behavior Regulated by Changes of External pH

Biophysical Journal, 2006

In the voltage-gated potassium channel Kv1.5, extracellular acidification decreases the peak macr... more In the voltage-gated potassium channel Kv1.5, extracellular acidification decreases the peak macroscopic conductance and accelerates slow inactivation. To better understand the mechanistic basis for these two effects, we recorded unitary currents of Kv1.5 expressed in a mouse cell line (ltk−) using the voltage clamp technique both in cell-attached and excised outside-out patches. Single channel current amplitude at 100 mV (1.7 ± 0.2 pA at pH 7.4, 1.7 ± 0.2 pA at pH 6.4) and the single channel conductance between 0 and 100 mV (11.8 ± 0.6 pS at pH 7.4 and 11.3 ± 0.8 pS at pH 6.4) did not change significantly with pH. External acidification significantly decreased the number of active sweeps, and this reduction in channel availability accounted for most of the reduction of the peak macroscopic current. The results of runs analyses suggested the null sweeps occur in clusters, and the rate constants for the transition between clusters of null and active sweeps at pH 6.4 were slow (0.12 and 0.18 s−1, to and from the active clusters, respectively). We propose that low pH facilitates a shift from an available mode (mode A) into an unavailable mode of gating (mode U). In addition to promoting mode U gating, external acidification accelerates depolarization-induced inactivation, which is manifest at the single channel level as a reduction of the mean burst length and an apparent increase of the interburst interval. These effects of external acidification, which are thought to reflect the protonation of a histidine residue in the turret (H-463), point to an important role for the turret in the regulation of channel availability and inactivation.

Research paper thumbnail of A Direct Demonstration of Closed-State Inactivation of K+ Channels at Low pH

Journal of General Physiology, 2007

Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels... more Lowering external pH reduces peak current and enhances current decay in Kv and Shaker-IR channels. Using voltageclamp fl uorimetry we directly determined the fate of Shaker-IR channels at low pH by measuring fl uorescence emission from tetramethylrhodamine-5-maleimide attached to substituted cysteine residues in the voltage sensor domain (M356C to R362C) or S5-P linker (S424C). One aspect of the distal S3-S4 linker α-helix (A359C and R362C) reported a pH-induced acceleration of the slow phase of fl uorescence quenching that represents P/C-type inactivation, but neither site reported a change in the total charge movement at low pH. Shaker S424C fl uorescence demonstrated slow unquenching that also refl ects channel inactivation and this too was accelerated at low pH. In addition, however, acidic pH caused a reversible loss of the fl uorescence signal (pKa = 5.1) that paralleled the reduction of peak current amplitude (pKa = 5.2). Protons decreased single channel open probability, suggesting that the loss of fl uorescence at low pH refl ects a decreased channel availability that is responsible for the reduced macroscopic conductance. Inhibition of inactivation in Shaker S424C (by raising external K + or the mutation T449V) prevented fl uorescence loss at low pH, and the fl uorescence report from closed Shaker ILT S424C channels implied that protons stabilized a W434F-like inactivated state. Furthermore, acidic pH changed the fl uorescence amplitude (pKa = 5.9) in channels held continuously at −80 mV. This suggests that low pH stabilizes closed-inactivated states. Thus, fl uorescence experiments suggest the major mechanism of pH-induced peak current reduction is inactivation of channels from closed states from which they can activate, but not open; this occurs in addition to acceleration of P/C-type inactivation from the open state.

Research paper thumbnail of Block by internal Mg 2+ causes voltage-dependent inactivation of Kv1.5

European Biophysics Journal With Biophysics Letters, 2006

Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ bl... more Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ block of Kv1.5 induces voltage-dependent current decay at strongly depolarised potentials that contains a component due to acceleration of C-type inactivation after pore block. The voltage-dependent current decay was fitted to a bi-exponential function (τfast and τslow). Without Mg2+, τfast and τslow were voltage-independent, but with 10 mM Mg2+, τfast decreased from 156 ms at +40 mV to 5 ms at +140 mV and τslow decreased from 2.3 s to 206 ms. With Mg2+, tail currents after short pulses that allowed only the fast phase of decay showed a rising phase that reflected voltage-dependent unbinding. This suggested that the fast phase of voltage-dependent current decay was due to Mg2+ pore block. In contrast, tail currents after longer pulses that allowed the slow phase of decay were reduced to almost zero suggesting that the slow phase was due to channel inactivation. Consistent with this, the mutation R487V (equivalent to T449V in Shaker) or increasing external K+, both of which reduce C-type inactivation, prevented the slow phase of decay. These results are consistent with voltage-dependent open-channel block of Kv1.5 by internal Mg2+ that subsequently induces C-type inactivation by restricting K+ filling of the selectivity filter from the internal solution.

Research paper thumbnail of Constitutive inactivation of the hKv1.5 mutant channel, H463G, in K + -free solutions at physiological pH

Cell Biochemistry and Biophysics, 2005

Extracellular acidification and reduction of extracellular K+ are known to decrease the currents ... more Extracellular acidification and reduction of extracellular K+ are known to decrease the currents of some voltage-gated potassium channels. Although the macroscopic conductance of WT hKv1.5 channels is not very sensitive to [K+]o at pH 7.4, it is very sensitive to [K+]o at pH 6.4, and in the mutant, H463G, the removal of K+o virtually eliminates the current at pH 7.4. We investigated the mechanism of current regulation by K+o in the Kv1.5 H463G mutant channel at pH 7.4 and the wild-type channel at pH 6.4 by taking advantage of Na+ permeation through inactivated channels. Although the H463G currents were abolished in zero [K+]o, robust Na+ tail currents through inactivated channels were observed. The appearnnce of H463G Na+ currents with a slow rising phase on repolarization after a very brief depolarization (2 ms) suggests that channels could activate directly from closed-inactivated states. In wild-type channels, when intracellular K+ was replaced by NMG+ and the inward Na+ current was recorded, addition of 1 mM K+ prevented inactivation, but changing pH from 7.4 to 6.4 reversed this action. The data support the idea that C-type inactivation mediated at R487 in Kv1.5 channels is influenced by H463 in the outer pore. We conclude that both acidification and reduction of [K+]o inhibit Kv1.5 channels through a common mechananism (i.e., by increasing channel inactivation, which occurs in the resting state or develops very rapidly after activation).

Research paper thumbnail of External K+ relieves the block but not the gating shift caused by Zn2+ in human Kv1.5 potassium channels

Journal of Physiology-london, 2001

Changing the extracellular concentration of divalent cations can have dramatic effects on the beh... more Changing the extracellular concentration of divalent cations can have dramatic effects on the behaviour of electrically excitable membranes as illustrated by the lowering or raising of the threshold for cell firing caused by hypo-or hypercalcaemia, respectively . Voltage-clamp analyses have shown that the divalent cation-induced change of excitability can be linked to a shift of the voltage dependence of channel gating. For example, an elevation of the external concentration of Ca 2+ ([Ca 2+ ] o ) produces a depolarizing shift of the midpoint (V 1/2 ) of the activation curves of both voltage-gated Na + and K + channels of the squid giant axon . Decreasing [Ca 2+ ] o produces the converse effect.

Research paper thumbnail of The External K + Concentration and Mutations in the Outer Pore Mouth Affect the Inhibition of Kv1.5 Current by Ni 2

Biophysical Journal, 2004

By examining the consequences both of changes of [K+]o and of point mutations in the outer pore m... more By examining the consequences both of changes of [K+]o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni2+ is similar to that for proton block. Ni2+ block is inhibited by increasing [K+]o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni2+ had no effect on gating currents. We propose that, as with Ho+, Ni2+ binding to H463 facilitates an outer pore inactivation process that is antagonized by Ko+ and that requires R487. However, whereas Ho+ substantially accelerates inactivation of residual currents, Ni2+ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co2+ and Mn2+, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM Ko+ is Zn2+ (KD ∼ 0.07 mM) ≥ Ni2+ (KD ∼ 0.15 mM) > Co2+ (KD ∼ 1.4 mM) > Mn2+ (KD > 10 mM).