H+ ion modulation of C-type inactivation of Shaker K+ channels

 The participation of an extracellular loop in C-type inactivation of voltage-gated K⁺ channels was investigated. A wild-type phenylalanine (at position 425) between the fifth putative transmembrane segment (S5) and the pore region of the Shaker K⁺ channel was mutated to a histidine and the functional consequences of protonating the imidizole group of the histidine were examined. C-type inactivation of both wild-type and mutant channels was sensitive to external pH over the range of 5.2–8. The pH dependence of wild-type channels was characterized by an apparent pK value of 5.0. The inactivation kinetics of F425H mutant channels had a pH dependence with a pK of 5.8 – in addition to the pH dependence of the wild-type channels. Moreover, at pH 7 and 8 the voltage dependence of C-type inactivation kinetics was manifest only in the F425H mutant channels. C-type inactivation in wild-type channels involves a chemical group with a low pK. Taken together, these results suggest that residues located in the extracellular S5-pore loop of the Shaker K⁺ channel participate in C-type inactivation. Received: 30 September 1998 / Received after revision: 8 January 1999 / Accepted: 18 January 1999     

Inactivation determined by a single site in K+ pores

An N-terminus peptide or a C-terminus mechanism involving a single residue in transmembrane segment 6 produces inactivation in voltage-dependent K⁺ channels. Here we show that a single position in the pore of K⁺ channels can produce inactivation having characteristics distinct from either N- or C-type inactivation. In a chimeric K⁺ channel (CHM), the point reversion CHM V 369I produced fast inactivation and CHM V 369S had the additional effect of halving K⁺ conductance consistent with a position in the pore. The result was not restricted to CHM; mutating position 369 in the naturally occurring channel Kv2.1 also produced fast inactivation. Like N- and C-types of inactivation, pore or P-type inactivation was characterized by short bursts terminated by rapid entry into the inactivated state. Unlike C-type inactivation, in which external tetraethylammonium (TEA) produced a simple blockade that slowed inactivation and reduced currents, in P-type inactivation external TEA increased currents. Unlike N-type inactivation, internal TEA produced a simple reduction in current and K⁺ occupancy of the pore had no effect. External TEA was not the only cation to increase current; external K⁺ enhanced channel availability and recovery from inactivation. Additional features of P-type inactivation were residue-specific effects on the extent of inactivation and removal of inactivation by a point reversion at position 374, which also regulates conductance. The demonstration of P-type inactivation indicates that pore residues in K⁺ channels may be part of the inactivation gating machinery.     

Modulation of volume-regulated anion channels by extra- and intracellular pH

 We have studied the modulation of the volume-regulated anion channel (VRAC) in cultured endothelial cells from bovine pulmonary artery (CPAE cells) by extra- and intracellular pH. The patch-clamp technique was used in combination with a fluorimetric measurement of intracellular pH using BCECF. Swelling of CPAE cells was accompanied by a slow acidification. The metabolites lactate and HCO₃ ^– both permeate through VRAC. The inactivation of VRAC currents at positive potentials is accelerated at a decreased extracellular pH and decelerated at alkaline pH. The instantaneous current amplitude is only slightly affected. Intracellular alkalization reduced whereas acidification enhanced the currents flowing through VRAC at all potentials. HCO₃ ^– and lactate permeation, as well as the up-regulation of VRAC at an acidic intracellular pH might be related to a possible role of this channel in cellular pH regulation. Received: 6 April 1998 / Received after revision: 11 May 1998 / Accepted: 12 May 1998     

Maxi K+ channels in the basolateral membrane of the exocrine frog skin gland regulated by intracellular calcium and pH

With the single-channel patch-clamp technique we have identified Ca²⁺-sensitive, high-conductance (maxi) K⁺ channels in the basolateral membrane (BLM) of exocrine gland cells in frog skin. Under resting conditions, maxi K⁺ channels were normally quiescent, but they were activated by muscarinic agonists or by high serosal K⁺. In excised inside-out patches and with symmetrical 140 mmol/l K⁺, single-channel conductance was 200 pS and the channel exhibited a high selectivity for K⁺ over Na⁺. Depolarization of the BLM increased maxi K⁺ channel activity. Increasing cytosolic free Ca²⁺ (by addition of 100 nmol/l thapsigargin to the bathing solution of cell-attached patches also increased channel activity, whereas thapsigargin had no effect when added to excised inside-out patches. An increase in cytosolic free Ca²⁺ directly activated channel activity in a voltage-dependent manner. Maxi K⁺ channel activity was sensitive to changes in intracellular pH, with maximal activity at pH 7.4 and decreasing activities following acidification and alkalinization. Maxi K⁺ channel outward current was reversibly blocked by micromolar concentrations of Ba²⁺ from the cytosolic and extracellular site, and was irreversibly blocked by micromolar concentrations of charybdotoxin and kaliotoxin from the extracellular site in outside-out patches.     

Two sets of amino acids of the domain I of Cav2.3 Ca(2+) channels contribute to their high sensitivity to extracellular protons

Extracellular acidification decreases Ca²⁺ current amplitude and produces a depolarizing shift in the activation potential (Va) of voltage-gated Ca²⁺ channels (VGCC). These effects are common to all VGCC, but differences exist between Ca²⁺ channel types and the underlying molecular mechanisms remain largely unknown. We report here that the changes in current amplitude induced by extracellular acidification or alkalinisation are more important for Cav2.3 R type than for Cav2.1 P/Q-type Ca²⁺ channels. This difference results from a higher shift of Va combined with a modification of channel conductance. Although involved in the sensitivity of channel conductance to extracellular protons, neither the EEEE locus nor the divalent cation selectivity locus could explain the specificity of the pH effects. We show that this specificity involves two separate sets of amino acids within domain I of the Cavα subunit. Residues of the voltage sensor domain and residues in the pore domain mediate the effects of extracellular protons on Va and on channel conductance, respectively. These new insights are important for elucidating the molecular mechanisms that control VGCC gating and conductance and for understanding the role of extracellular protons in other channels or membrane-tethered enzymes with similar pore and/or voltage sensor domains.     

Regulation of inwardly rectifying K+ channels by intracellular pH in opossum kidney cells

The effects of intracellular pH on an inwardly rectifying K⁺ channel (K_in channel) in opossum kidney (OK) cells were examined using the patch-clamp technique. Experiments with inside-out patches were first carried out in Mg²⁺-and adenosine triphosphate (ATP)-free conditions, where Mg²⁺-induced inactivation and ATP-induced reactivation of K_in channels were suppressed. When the bath (cytoplasmic side) pH was decreased from 7.3 to either 6.8 or 6.3, K_in channels were markedly inhibited. The effect of acid pH was not fully reversible. When the bath pH was increased from 7.3 to 7.8, 8.3 or 8.8, the channels were activated reversibly. The channel activity exhibited a sigmoidal pH dependence with a maximum sensitivity at pH 7.5. Inside-out experiments were also carried out with a solution containing 3 mM Mg-ATP and a similar pH sensitivity was observed. However, in contrast with the results obtained in the absence of Mg²⁺ and ATP, the effect of acid pH was fully reversible. Experiments with cell-attached patches demonstrated that changes in intracellular pH, which were induced by changing extracellular pH in the presence of an H⁺ ionophore, could influence the channel activity reversibly. It is concluded that the activity of K_in channels can be controlled by the intracellular pH under physiological conditions.     

Inactivation and recovery in Kv1.4 K+ channels: lipophilic interactions at the intracellular mouth of the pore

C-type inactivation is present in many voltage-gated potassium channels and is probably related to 'slow' inactivation in calcium and sodium channels. The mechanisms underlying C-type inactivation are unclear, but it is sensitive to mutations on both the extra- and intracellular sides of the channel. We used an N-terminal deleted channel with a valine to alanine point mutation at the intracellular side of S6 (fKv1.4[V561A]ΔN). This construct alters recovery from inactivation and inverts the relationship between C-type inactivation and [K⁺]_o. We used this inverted relationship to examine C-type inactivation and coupling mechanisms between N- and C-type inactivation. The valine to alanine mutation reduces the channel's affinity for both quinidine and the N-terminal domain. However, binding of the N-terminal or quinidine restores normal recovery from inactivation. This suggests that coupling between N- and C-type inactivation is dominated by allosteric mechanisms. The permeation mechanism, driven by a reduction in permeant [K⁺]_o following pore block (which would retard C-type inactivation), contributes minimally to coupling in these channels. We propose that the cytoplasmic half of S6 forms part of the N-terminal binding site, as previously predicted from X-ray crystallography studies in the distantly related KcsA channel. Binding of the N-terminal domain or a positively charged lipophilic compound such as quinidine interacts with the hydrophobic moieties on S6 in the bound state. This binding can orientate S6 into a conformation which resembles the normal C-type inactivated state. This is the probable mechanism by which drug or N-terminal binding increases the rate of C-type inactivation via an allosteric mechanism.     

Activation of K+ currents in cultured Schwann cells is controlled by extracellular pH

We analyzed the pH dependence of K⁺ currents recorded with the patch-clamp technique from cultured Schwann cells obtained from mouse dorsal root ganglia. Currents were activated at potentials more positive than –50 mV which was close to the resting membrane potential. Current amplitudes were affected by a change in extracellular pH (pH_o), being increased at alkaline, and decreased at acidic pH_o. The strongest effect of a pH_o change was observed on currents activated close to the resting membrane potential suggesting a functional role for the pH sensitivity of K⁺ currents. Analysis of the time course of current activation at different pH_o values led to the conclusion that the pH-sensitivity of K⁺ currents in Schwann cells is due to changes in surface charges shifting the potential sensed by the gating process of the channel. The reversal potential of the currents was not affected by a change in pH_o. This observation and the finding that even a strong acidification to a pH_o value of 5.0 did not lead to a blockade of the fully activated channel, indicate that the pH-sensitive charges are not located in the channel pore. Under the assumption that pH_o changes in a peripheral nerve are associated with nerve activity as in the optic nerve, the pH-sensitive K⁺ channel in Schwann cells could serve to facilitate the spatial buffering of extracellular K⁺.     

Intracellular and extracellular amino acids that influence C-type inactivation and its modulation in a voltage-dependent potassium channel

The rate of C-type inactivation of the cloned voltage-gated potassium channel, Kv1.3, measured in membrane patches from Xenopus oocytes, increases when the patch is detached from the cell; the structural basis for this on-cell/off-cell change was examined. First, four serine and threonine residues, that are putative sites for phosphorylation by protein kinases A and C, were mutated to alanines. Mutating any one of these residues, or two or three of them simultaneously, does not eliminate the change in C-type inactivation. However, the basal rate of C-type inactivation in the cell-attached patch is markedly slower in the triple phosphorylation site mutant. Second, a homologous potassium channel, Kv 1.6, does not exhibit the on-cell/off-cell change. When an extracellular histidine at position 401 of Kv1.3 is replaced with tyrosine, the residue at the equivalent position (430) in Kv1.6, the resulting Kv1.3 H401Y mutant channel does not undergo the on-cell/off-cell change. The results indicate that several potentially phosphorylatable intracellular amino acids influence the basal rate of C-type inactivation, but are not essential for the on-cell/off-cell change in inactivation kinetics. In contrast, an extracellular amino acid is critical for this on-cell/off-cell change.     

Activation of K+ currents in cultured Schwann cells is controlled by extracellular pH

We analyzed the pH dependence of K⁺ currents recorded with the patch-clamp technique from cultured Schwann cells obtained from mouse dorsal root ganglia. Currents were activated at potentials more positive than ?50 mV which was close to the resting membrane potential. Current amplitudes were affected by a change in extracellular pH (pH_o), being increased at alkaline, and decreased at acidic pH_o. The strongest effect of a pH_o change was observed on currents activated close to the resting membrane potential suggesting a functional role for the pH sensitivity of K⁺ currents. Analysis of the time course of current activation at different pH_o values led to the conclusion that the pH-sensitivity of K⁺ currents in Schwann cells is due to changes in surface charges shifting the potential sensed by the gating process of the channel. The reversal potential of the currents was not affected by a change in pH_o. This observation and the finding that even a strong acidification to a pH_o value of 5.0 did not lead to a blockade of the fully activated channel, indicate that the pH-sensitive charges are not located in the channel pore. Under the assumption that pH_o changes in a peripheral nerve are associated with nerve activity as in the optic nerve, the pH-sensitive K⁺ channel in Schwann cells could serve to facilitate the spatial buffering of extracellular K⁺.     

Purinergic activation of a leak potassium current in freshly dissociated myocytes from mouse thoracic aorta

Aim: Exogenous ATP elicits a delayed calcium‐independent K⁺ current on freshly isolated mouse thoracic aorta myocytes. We investigated the receptor, the intracellular pathway and the nature of this current. Methods: The patch‐clamp technique was used to record ATP‐elicited delayed K⁺ current in freshly dissociated myocytes. Results: ATP‐elicited delayed K⁺ current was not inhibited by a ‘cocktail’ of K⁺ channel blockers (4‐AP, TEA, apamin, charybdotoxin, glibenclamide). The amplitude of the delayed K⁺ current decreased after the reduction of extracellular pH from 7.4 to 6.5. These two characteristics suggest that this current could be carried by the TASK subfamily of ‘twin‐pore potassium channels’ (K2P). Purinergic agonists including dATP, but not ADP, activated the delayed K⁺ current, indicating that P2Y₁₁ is the likely receptor involved in its activation. The PKC activator phorbol ester 12,13‐didecanoate stimulated this current. In addition, the PKC inhibitor Gö 6850 partially inhibited it. Real‐time quantitative PCR showed that the genes encoding TASK‐1 and TASK‐2 are expressed. Conclusion: Our results indicate that blocker cocktail‐insensitive delayed K⁺ current in freshly dissociated aortic myocytes is probably carried by the TASK subfamily of twin‐pore channels.     

Modification of C-type inactivatingShaker potassium channels by chloramine-T

Shaker potassium channels undergo a slow C-type inactivation which can be hastened dramatically by single-point mutations in or near the pore region. We found that the oxidizing agent chloramine-T (Chl-T) causes an irreversible loss of current for those mutants which show C-type inactivation. For several mutants at position T449, which show a wide spectrum of inactivation time constants, the time constant of current rundown induced by Chl-T correlated with the speed of inactivation. Rundown was accelerated when the channels were in the inactivated state but rundown also occurred when channels were not opened or inactivated. Apparently, only those channels which can undergo C-type inactivation are accessible to Chl-T In order to gain information about the target amino-acid residue for the action of Chl-T and the structural rearrangements occurring during C-type inactivation, several mutant channel proteins were compared with respect to their response to Chl-T Since Chl-T can oxidize cysteine and methionine residues, we mutated the possible targets in and close to the pore region, namely C462 to A, and M440 and M448 to I. While the residues M440 and C462 were not important for channel rundown, mutation of M448 to I made the channels more resistant to Chl-T by about one order of magnitude. While inactivation was accelerated upon application of Chl-T in most mutants, mutation of M448 to I abolished this effect on the time course of inactivation, indicating that M448 is one of the target residues for Chl-T.     

Characterization of a whole-cell Ca2+-blockable monovalent cation current in isolated ectodermal cells of chick embryo

The presence of a Ca²⁺-blockable monovalent cation current is demonstrated in isolated ectodermal cells of the chick embryo using the whole-cell patch-clamp method. In the absence of any stimulation, the whole-cell current is time independent and rectifies outwardly at membrane potentials higher than +40 mV The outward current is neither carried by Cl^– channels nor by K⁺ channels. Application of a Ca²⁺-free solution containing 1 mmol/l ethylenediaminetetraacetic acid (EDTA) elicits a large inward current and increases the outward current. The inward current can be carried by extracellular Li⁺, Na⁺, K⁺ and Cs⁺, but notN-methyl-d-glucamine. The Ca²⁺-blockable monovalent cation channel discriminates very poorly among these cations. The estimated number of channels per cell is around 2000. Extracellular protons block the inward Na⁺ current in the absence of extracellular Ca²⁺. The apparent negative logarithm of the dissociation constant for proton (pK _H) at –100 mV is 5.8. Among 12 potential channel modulators, including verapamil and nifedipine, only quinine decreases the current. Quinine blocks this current with a dissociation constant,K _d, equal to 0.18 mmol/l, independent of the membrane potential. This study demonstrates the presence of a whole-cell Ca²⁺-blockade monovalent cation current in dissociated chick ectodermal cells with permeation properties similar to those observed at the single-channel level. Contrary to studies made of other tissues, we did not observe any blocking effect of verapamil and nifedipine on the Ca²⁺-blockable monovalent cation current.     

Modulation of jellyfish potassium channels by external potassium ions.

The amplitude of an A-like potassium current (I(Kfast)) in identified cultured motor neurons isolated from the jellyfish Polyorchis penicillatus was found to be strongly modulated by extracellular potassium ([K(+)](out)). When expressed in Xenopus oocytes, two jellyfish Shaker-like genes, jShak1 and jShak2, coding for potassium channels, exhibited similar modulation by [K(+)](out) over a range of concentrations from 0 to 100 mM. jShak2-encoded channels also showed a decreased rate of inactivation and an increased rate of recovery from inactivation at high [K(+)](out). Using site-directed mutagenesis we show that inactivation of jShak2 can be ascribed to an unusual combination of a weak "implicit" N-type inactivation mechanism and a strong, fast, potassium-sensitive C-type mechanism. Interaction between the two forms of inactivation is responsible for the potassium dependence of cumulative inactivation. Inactivation of jShak1 was determined primarily by a strong "ball and chain" mechanism similar to fruit fly Shaker channels. Experiments using fast perfusion of outside-out patches with jShak2 channels were used to establish that the effects of [K(+)](out) on the peak current amplitude and inactivation were due to processes occurring at either different sites located at the external channel mouth with different retention times for potassium ions, or at the same site(s) where retention time is determined by state-dependent conformations of the channel protein. The possible physiological implications of potassium sensitivity of high-threshold potassium A-like currents is discussed.     

Proton inhibition of GABA-activated current in rat primary sensory neurons

 The modulation of the Cl^– current activated by γ-aminobutyric acid (GABA) by changes in extracellular pH in freshly isolated rat dorsal root ganglia (DRG) neurons was studied using the whole-cell patch-clamp technique. In the pH range of 5.0–9.0, increased extracellular pH enhanced, and decreased extracellular pH suppressed, current activated by 10 μM GABA in a reversible and concentration-dependent manner with an IC₅₀ of pH 7.1 in these neurons. Acidification to pH 6.5 inhibited currents activated by the GABA_A-selective agonist muscimol in all neurons tested. The antagonism of GABA-activated current by lowering the pH was equivalent at holding potentials between –80 and +40 mV and did not involve a significant alteration in reversal potential. Acidification shifted the GABA concentration/response curve to the right, significantly increasing the EC₅₀ for GABA without appreciably changing the slope or maximal value of the curve. Inhibition of the GABA-activated current by protons was not significantly different when the patch-pipette solution was buffered at pH 7.4 or pH 6.5. These results suggest that extracellular protons inhibit GABA_A receptor channels in primary sensory neurons by decreasing the apparent affinity of the receptor for GABA. This represents a novel mechanism of inhibition by protons of a neurotransmitter-gated ion channel. Proton inhibition of GABA_A receptor channels may account in part for the modulation by protons of sensory information transmission under certain pathophysiological conditions. Received: 1 July 1997 / Received after revision: 29 September 1997 / Accepted: 15 October 1997     

Modulation of homomeric and heteromeric kcnq1 channels by external acidification

The I _KS K⁺ channel plays a major role in repolarizing the cardiac action potential. It consists of an assembly of two structurally distinct α and β subunits called KCNQ1 and KCNE1, respectively. Using two different expression systems, Xenopus oocytes and Chinese hamster ovary cells, we investigated the effects of external protons on homomeric and heteromeric KCNQ1 channels. External acidification (from pH 7.4 to pH 5.5) markedly decreased the homomeric KCNQ1 current amplitude and caused a positive shift (+25 mV) in the voltage dependence of activation. Low external pH (pH_o) also slowed down the activation and deactivation kinetics and strongly reduced the KCNQ1 inactivation process. In contrast, external acidification reduced the maximum conductance and the macroscopic inactivation of the KCNQ1 mutant L273F by only a small amount. The heteromeric I _KS channel complex was weakly affected by low pH_o, with minor effects on I _KS current amplitude. However, substantial current inhibition was produced by protons with the N-terminal KCNE1 deletion mutant Δ11-38. Low pH_o increased the current amplitude of the pore mutant V319C when co-expressed with KCNE1. The slowing of I _KS deactivation produced by low pH_o was absent in the KCNE1 mutant Δ39-43, suggesting that the residues lying at the N-terminal boundary of the transmembrane segment are involved in this process. In all, our results suggest that external acidification acts on homomeric and heteromeric KCNQ1 channels via multiple mechanisms to affect gating and maximum conductance. The external pH effects on I _Kr versus I _KS may be important determinants of arrhythmogenicity under conditions of cardiac ischaemia and reperfusion.     

Effects of intracellular pH and Ca2+ on the activity of stretch-sensitive cation channels in leech neurons

The effects of intracellular pH and calcium on the activity of the leech mechanosensitive cation channels have been studied. These channels exhibited two activity modes denoted as spike-like (SL) and multiconductance (MC). In the absence of mechanical stimulation, acidification of the intracellular side of membrane patches from 7.2 to 6.2 reversibly increased the mean channel open time as well as the opening frequency in the SL mode. Channels in MC mode were activated by a pH_i reduction from 7.2 to 6.2, but were inhibited at pH_i 5.5. Unlike MC mode, SL mode was strongly activated by intracellular Ca²⁺. Fura-2 imaging experiments showed that intracellular calcium was induced to increase by hypotonic cell swelling. The major component of this response did not require extracellular calcium. A component of the swelling-induced calcium response was sensitive to blockers of stretch-sensitive cation channels. The results indicate that the two activity modes of mechanosensitive channels of leech neurons respond differently to changes of intracellular pH and calcium. The sensitivity of the channel to micromolar concentrations of internal free calcium, along with its permeability to this ion, is consistent with a role in the amplification of mechanically induced Ca²⁺ signals in leech neurons.     

Separation of potassium and calcium channels in the nerve cell soma membranes

Investigation of isolated neurons ofHelix pomatia during intracellular dialysis revealed differences in the sensitivity of the channels for the outward potassium and inward calcium currents to changes in pH of the external medium. As a result of this difference, considerable separation of the regions of activation of the currents was obtained along the potential axis in solutions with low pH and the characteristics of the inward and outward currents could be studied during their minimal application. Channels for the outward current were shown to have some permeability for tris ions (P_Tris: p_K = 0.05), which is the reason why it is impossible to block this current completely by replacing the intracellular potassium by Tris. Channels for the inward calcium current are characterized by slow inactivation, with a first-order kinetics; their momentary voltage-current characteristic curve reveals significant Goldman's rectification. The selectivity of the calcium channels for other bivalent cations is: Ba:Sr:Ca:Mg = 2.8:2.6:1.0:0.2.Translated from Neirofiziologiya, Vol. 10, No. 6, pp. 645–653, November–December, 1978.     

Functional expression of a large-conductance Ca-activated K channel in mouse substantia nigra pars compacta dopaminergic neurons

The existence of large-conductance Ca²⁺-activated K⁺ (BK) channels in substantia nigra pars compacta (SNc) has been a matter of debate. Using the patch-clamp technique in the inside-out configuration, we have recorded BK channel currents in SNc dopaminergic neurons. The channel has a conductance of 301 pS with a slight inward rectification and is both voltage- and calcium-dependent. Paxilline, a specific BK channel blocker, can completely block the channel, while tetraethylammonium (TEA), a nonspecific blocker of voltage-gated potassium channels, reduces its conductance and a high concentration of TEA (30 mM) inhibits its activity. ATP and GTP reduce the channel activity, while ADP is less potent, and AMP has no effect. The channel is also sensitive to changes in intracellular pH. Our results indicate that functional BK channels are expressed in SNc and suggest the possibility that the BK channel may be involved in the response of SNc dopaminergic neurons to metabolic stress.     

The effect of external pH on the delayed rectifying K+ current in cardiac ventricular myocytes

The effects of extracellular pH (pHe) on the delayed rectifying K+ current iKr in rabbit ventricular myocytes were studied using the whole-cell-clamp technique. Since a variety of results have been reported on the effect of pH on expressed hERG channels, our aim was to investigate the effects of pH on iKr channels in their native environment. iKr is reduced by extracellular acidification and its deactivation is faster. Extracellular acidification results in a marked shift of the steady-state activation curve to more positive potentials, while alkalinization does not produce a significant shift. E1/2= - 11.3 mV, -20.2 mV, -21.4 mV at pHe 6.5, 7.4, 8.5 respectively; the slope factor is 6.2 mV, and is not affected by pHe. Deactivation of iKr is biexponential, with time constants of the order of 0.5 s and 10 s at -50 mV. Both time constants decrease with external acidification. Also the contribution of the fast component to the total amplitude becomes larger with acidification. Acidification also decreases the fully activated iKr current. Our experiments demonstrate that extracellular acidification reduces iKr by increasing the rate of deactivation, causing a shift of the voltage dependence of activation and producing a voltage-dependent block of the fully activated iKr current.