KChannelDB: Extraction of mutation data from the literature

2005, KChannelDB.

This data was extracted from Medline abstracts and full texts (when available) in an automated manner.

The table below describes the selected point mutation and provides links to other documents. The sentence(s) where the point mutation T256Q was found are listed after the table.

The mutated residues are also indicated in the family sequence alignments and hyperlinked to the corresponding mutation pages.

Point mutation T256Q in KAT1_ARATH

Point mutation:	T256Q
Domain:	Loop 5-6
General numbering (KChannelDB):	-
Protein:	KAT1_ARATH (KAT1,At5g46240,MPL12.)	Swiss-Prot Cross-reference table Family page
Other point mutations / same protein / same position	Point mutations at position 256 in KAT1_ARATH
Other point mutations / same protein	List of mutations in KAT1_ARATH
Family alignments	AKT-like Inward rectifiers 6TMs Plant potassium channels
Other point mutations / same position	Position 258 in AKT-like Inward rectifiers 6TMs family Position 258 in Plant potassium channels family
Reference:	Mutation in pore domain uncovers cation- and voltage-sensitive recovery from inactivation in KAT1 channel. Moroni A, Gazzarrini S, Cerana R, Colombo R, Sutter JU, DiFrancesco D, Gradmann D, Thiel G Biophys J 2000 Apr;78(4):1862-71.	Medline
Other point mutations / same article	List
Text source	HTML full text
Validation status	Not yet checked

Relevant sentences:

T256Q

The major discrepancy in T256Q from the wild-type channel (wt) was cation specific
The same effects could be induced in wt , or intensified in T256Q , by addition of the impermeant cation methylammonium (MA +) to the bath
Assuming that current inhibition could be described as entry of the channel into an inactive state , we modeled in both wt and in T256Q the relaxation kinetics of the clamp currents by a C-O-I gating scheme , where C (closed) and I (inactivated) are nonconductive states , and O is an open state allowing K+ and NH4+ passage
This cation-sensitive transition step ensures release of the channel from the inactive state and is ~30 times smaller in T256Q compared to wt
In the context of the aforementioned inhibition of the current by MA + in the wt channel , it is interesting to note that the mutation T256Q also affected the macroscopic conductance , depending on the transportable ion
MA + in wt and T256Q similarly caused a weak reduction of the K + current , with no obvious voltage dependency (Uozumi et al. , 1995(image) ; Moroni et al. , 1998(image) ) and a strong inhibition of the NH4+ current , in a way compatible with a voltage-dependent open channel block (Uozumi et al. , 1995(image) ; Moroni et al. , 1998(image) )
In this work we examine the electrophysiological properties of T256Q
Current responses in one oocyte expressing T256Q , evoked with similar clamp protocols , are illustrated in the right panel of Fig. 1
Current activation of T256Q was similar to that of the wt channel , while deactivation of the mutant was appreciably slower (Table 1 ; also see below)
In 10 oocytes the current through T256Q at -160 mV was 1.8 times smaller than in the wt
(image) View larger version (18K): [in this window] [in a new window] FIGURE 1 Hyperpolarization-induced activation of membrane currents in two Xenopus oocytes expressing the wt (left) and the mutant T256Q (right)
View this table: [in this window] [in a new window] TABLE 1 Time constants for activation , inactivation , recovery from inactivation , and deactivation , measured in oocytes expressing wt or T256Q (image) View larger version (14K): [in this window] [in a new window] FIGURE 2 Comparison of the mean normalized current / voltage relations between wt and T256Q in K + (A) and NH4+ (B)
Bars indicate ±SE from five oocytes for wt and 10 oocytes for T256Q
The response of T256Q to negative voltage steps was markedly different in the presence of NH4+
The data show that T256Q conducts more NH4+ current at moderate negative voltages compared to the wt channel
In 10 oocytes the NH4+ current at -160 mV was 9.8 times smaller in T256Q than in the wt
When present in control water-injected oocytes from the same batch , this current was more than 50 times smaller than the current measured in T256Q-expressing oocytes
Hence the slow voltage-dependent current inhibition must be a property of T256Q
To further characterize this slow inhibition of the T256Q channel at large negative V with NH4+ as the transportable ion , we applied hyperpolarizing steps from a holding voltage of -60 to -180 mV to resolve the biphasic current activation / inhibition time course (Fig. 3 A)
It appears that the kinetics of activation of T256Q are voltage-dependent (Fig. 3 C) and substantially faster than those of the wt channel (Table 1 )
(image) View larger version (15K): [in this window] [in a new window] FIGURE 3 Kinetics of T256Q current relaxation in NH4+ at different voltages
Under this condition the current carried by T256Q relaxed in a largely monotonous fashion (Fig. 4 , inset)
(image) View larger version (18K): [in this window] [in a new window] FIGURE 4 Time constant of wt and T256Q deactivation in K +
Plot shows mean values (± SE) from four oocytes for wt and six oocytes for T256Q
Inset: Sample of deactivation currents in 20 mM K + from wt-KAT1 and T256Q obtained by stepping , from -160 mV to -20 mV , after normalization
NH4+ inhibits K+ current in T256Q It was shown previously that K + and NH4+ do not interact significantly when passing through wt channel
To examine the effect of the mutation on the interaction between transportable ions , oocytes expressing T256Q were investigated in the presence of K + only or NH4+ only , or in a mixture (1:1) of the two ions
(image) View larger version (10K): [in this window] [in a new window] FIGURE 5 T256Q conductance in mixed solutions with NH4+ and K+
Cation dependence of T256Q gating The inactivation of T256Q in the presence of NH4+ implies that the transported ion itself is responsible for the inactivation of the channel
In the context of the behavior of T256Q conductance in K + , this further implies that external K + prevents inactivation with a higher efficiency than NH4+
To test this hypothesis we examined the activation of T256Q in a solution with a low concentration of external K +: Fig. 6 shows the current response of T256Q to a V step from -60 mV to -160 mV in 10 mM and 1 mM K +
With 10 mM K + the test voltage step evoked a monotonic activation of the T256Q conductance
The same biphasic current response to hyperpolarization of T256Q in 1 mM K + was found in six other oocytes
Thus , in low external K + , T256Q behaves as it does in NH4+
(image) View larger version (10K): [in this window] [in a new window] FIGURE 6 Effect of extracellular K + concentration on activation kinetics of T256Q
One oocyte was clamped from a holding voltage of -60 mV to -160 mV to activate the T256Q conductance
Kinetics of current activation , qualitatively similar to those in T256Q , can also be obtained with wt when oocytes are exposed to the nontransportable cation MA + (Moroni et al. , 1998(image) )
Fig. 7 illustrates the effect of MA + added to K +- or NH4+-containing solutions on inward currents carried by either wt (left) or T256Q (right)
The addition of 50 mM MA + to 50 mM K + in T256Q caused a stronger inhibition than in wt (upper right)
With NH4+ as the transportable ion , MA + completely and instantaneously inhibited in the same oocyte the NH4+ current through T256Q (lower right)
It is worth noting that the addition of MA + on wt and the mutation in T256Q acted similarly in the sense that they induced a reduction in current with no inactivation in K + (upper right and Fig. 2 A) and with inactivation in NH4+ (lower right)
(image) View larger version (19K): [in this window] [in a new window] FIGURE 7 Effect of methylammonium (MA +) on K+ and NH4+ conductance of wt (left) and of T256Q (right)
Inset: Currents normalized to the value reached after 200 ms to show the biphasic kinetics observed upon addition of MA + on K + in T256Q
Depletion of cations from external medium causes inactivation of wt The dependence of the wt and of the T256Q conductance on extracellular cations is consistent with a model in which the channel requires cations on the extrafacial side to release the channel from an inactive state into the open state (for details see the Discussion)
The prominent kinetic feature of T256Q is that in the presence of NH4+ or low K+ , the conductance responds to negative voltage steps in a biphasic manner
In the work by Dreyer and co-workers (1998)(image) , however , no evidence was found for Ca2+ blockage in T256Q , so that this mechanism can be ruled out as an explanation for the slow inactivation
First , replacement of LiCl with mannitol did not prevent the slow inactivation of T256Q (data not shown)
Hence the observed biphasic current response of T256Q in NH4+ and low K + indicates the presence of a slow inactivation of the channel directly related to the transportable ion itself
If that were the case , in fact , the onset of the inhibition should occur in the time range of ion transition and should therefore be orders of magnitude faster than the one observed with T256Q (e.g. , Hille , 1992(image) )
This is illustrated in detail in Fig. 9 A: the change from biphasic to monotonic responses in T256Q can be simulated by a ~30-fold increase in the transition I -->O (curve 1 compared to curve 32 , Fig. 9 )
Similarly , the higher steady-state current of wt compared to T256Q at large negative voltages in K + (>10 mM ; Fig. 2 ) can be explained by an approximately threefold faster transition I -->O (compare curves 32 , 100 , Fig. 9 A)
Curve 1 in Fig. 9 B resembles the biphasic tail current of T256Q in NH4+ (Fig. 3 )
The difference in decay velocity of the tail currents (curves 10 , 32 , and 100 , Fig. 9 B) corresponds to the slower time constants for T256Q compared to wt (Fig. 4 )
Nonetheless , the latter is unable to describe the experimental data for the following reason: quasimonotonic current activation kinetics upon negative voltage steps (i.e. , similar to wt or T256Q in K + , Fig. 1 ) can only be achieved with this scheme by a >300-fold inhibition in k i2 ,
However , the experimental data show that the mutation T256Q or the inhibition of the wt channel by MA + causes , at large negative voltages , a decrease of ~50% in steady-state K + current without perceivable biphasic kinetics (see Figs
From the difference between K+ and NH4+ currents in T256Q it can be estimated , based on the simulation , that the positive effect of K + must be >30 times stronger than that of NH4+
From the difference between the K + currents in wt and T256Q it can be estimated , based on the simulation , that the mutation T256Q decreases the K +-driven transition step by a factor greater than 2
Following the same gating scheme , the effect of MA + on K + and NH4+ currents in wt and T256Q can also be attributed to an effect of MA + on the transition step I -->O
The effects of MA + on T256Q are a direct consequence of the aforementioned interpretation
T256Q has features similar to those of the wt channel under inhibition with MA +
Based on the analysis of the mutant T256Q , we believe that the threonine at position 256 in the pore of KAT1 is an

F.Horn (kchanneldbcmbi.ru.nl), 17-Aug-2005